Meningococcal hexavalent combination vaccine

Abstract

The present invention relates to combination vaccine comprising hexavalent serogroup of Neisseria meningitidis for prevention and prophylaxis of infection caused by meningococcal bacteria. The present invention relates to a hexavalent combination vaccine comprising serogroups A, B, C, Y, W, X of Neisseria meningitidis. The present invention relates to a hexavalent combination vaccine comprising polysaccharide- protein conjugate of serogroups A, C, Y, W, X and proteins of serogroup B of Neisseria meningitidis. The present invention specifically relates to a hexavalent combination vaccine comprising polysaccharide- protein conjugate of serogroup A, C, Y, W, X and mutated Factor H binding proteins (fHbp) of serogroup B of Neisseria meningitidis. The present invention further relates to the composition, use and process of preparation of hexavalent combination vaccine.

Description

Title: MENINGOCOCCAL HEXAVALENT COMBINATION VACCINEFIELD OF THE INVENTION

[0001] The present invention relates to biopharmaceuticals. The present invention relates to a combination vaccine comprising hexavalent serogroup of Neisseria meningitidis for prevention and prophylaxis of infection caused by meningococcal bacteria. The present invention relates to a hexavalent combination vaccine comprising serogroups A, B, C, Y, W- 135, X of Neisseria meningitidis. The present invention relates to a hexavalent combination vaccine comprising polysaccharide- protein conjugate of serogroups A, C, Y, W-135, X and recombinant proteins of serogroup B of Neisseria meningitidis. The present invention specifically relates to a hexavalent combination vaccine comprising polysaccharide- protein conjugate of serogroup A, C, Y, W-135, X and mutated Factor H binding proteins (fHbp) of serogroup B of Neisseria meningitidis. The present invention further relates to the composition, use and process of preparation of hexavalent combination vaccine.BACKGROUND OF THE INVENTION

[0002] Neisseria meningitidis is a Gram-negative bacterium that can cause severe infections, including sepsis and meningitis. This bacterium causes 10-15% of community acquired meningitis cases in adults, of which most cases occur in younger adults. The case fatality rate has been reported to be 3-4% (van Soest TM et.,al, Lancet Reg Health Eur. 2023 Apr 28;30: 100640).

[0003] Meningococcal meningitis is characterized by sudden onset of headache, fever, and neck stiffness, sometimes accompanied by nausea, vomiting, photophobia, or altered mental status. Meningococcal disease progresses rapidly and has a case-fatality rate of 10%— 15% , even with antimicrobial drug treatment. Without rapid treatment, fatality rates can be much higher. Approximately 30% of people with meningococcal disease present with meningococcal sepsis, known as meningococcemia. Symptoms of meningococcemia can include abrupt onset of fever, chills, vomiting, diarrhea, and a petechial or purpuric rash, which can progress to purpura fulminans. Meningococcemia often involves hypotension, acute adrenal hemorrhage, and multiorgan failure (https: / / wwwnc.cdc.gov / travel / yellowbook / 2024 / infections-diseases / meningococcal- disease#epi accessed on March 25, 2024)

[0004] To date, worldwide, there are 12 known distinct meningococcal serogroups designated based on the composition of CPS polymers (A, B, C, E, H, I, K, L, W, X, Y,and Z), with serogroups A, B, C, W, Y, and X, being responsible for the majority of disease. These 12 serogroups differ immunologically due to characteristic differences in their polysaccharide capsule; These polysaccharides form the basis of production for the current vaccines, however, for MenB, the vaccine's basis is on its outer membrane polysaccharides. (Daraghma R et.,al. StatPearls Publishing; 2024 Jan-).

[0005] Since the 1970s, meningococcal polysaccharide vaccinations have been available. Tetravalent serogroup A, C, W, and Y polysaccharide vaccines (Mencevax, GSK Vaccines; Menomune, Sanofi Pasteur) received their licence in the 1980s. Meningococcal carbohydrate-based vaccines commercialized or under development are made by conjugation of extracted polysaccharides to protein. Three carrier proteins (TT, Tetanus Toxoid; DT, Diphtheria Toxoid; CRM197, Cross Reacting Material 197) have been used for this type of vaccines. (Berti Fet et.,al Glycoconj J. 2021 Aug;38(4):401- 409)The meningococcal vaccine protects against infections caused by the bacteria Neisseria meningitidis (meningococci). Meningococcal infections can lead to meningitis (an infection of tissue covering the brain), dangerously low blood pressure (shock), and death. These bacteria are the leading cause of bacterial meningitis in children and the second leading cause of bacterial meningitis in adults. (https: / / www.msdmanuals.com / enin / home / infections / immunization / meningococcal- vaccine)

[0006] Invasive meningococcal disease (IMD), a serious disorder linked to high morbidity and mortality, can, however, occasionally result from nasopharynx colonisation. IMD is characterised by a wide range of clinical manifestations, including meningitis, meningococcaemia, or both, in addition to other clinical presentations, with a case fatality rate of roughly 6-10%. IMD can have long-term effects in survivors, including deafness, brain damage, and amputations of limbs or fingers. It is difficult to make an early diagnosis of IMD since at the time of the disease's onset, symptoms like sore throat may resemble those of common viral respiratory infections while rash and meningeal indications may not be evident. (Piccini, G et.al., Expert review of vaccines, 15(11), 1393-1407)

[0007] Serogroups A, B, C, X, Y, and W135 are mostly responsible for meningococcal disease. Regional differences affect each serogroup's relative importance. Humans are the only natural host of meningococci, and about 5-10% of adults are asymptomatic meningococcal carriers (Frasch, C et.al., Human vaccines & immunotherapeutics, 8(6), 715-724).

[0008] Vaccines containing recombinant proteins only (Trumenba, Pfizer), or polysaccharide-protein conjugates only (MenFive, Serum Institute of India), or recombinant proteins combined with OMVs (Bexsero, GSK Vaccines), or containing both meningococcal polysaccharide of serogroups C and OMPs of serogroup B (VA- Mengoc-BC, Finley Institute) have been developed and licensed. Meningococcal conjugate vaccines, in which the capsular polysaccharide is covalently linked to a carrier protein, were developed as a result of the shortcomings of the polysaccharide vaccines, which included poor immunogenicity in children under the age of two, a lack of immunological memory, and, in some instances, hypo responsiveness. (Berti, F et.al., Glycoconjugate Journal, 38(4), 401-409). The current conjugate vaccines include different carrier proteins as well as other elements including formulation technology and conjugation method. MenACWY-D and MenACWY-CRM197 were approved earlier than MenACWY-TT, which is the most recently licenced of the three MenACWY conjugate vaccines that are now on the market. (Tontini, M et.al., Vaccine, 31(42), 4827- 4833).

[0009] Using the current US schedule (i.e., MenACWY vaccine given at 11 and 16 years of age) and assuming current rates of vaccination uptake for MenACWY and MenB vaccines, a population-based dynamic model simulating transmission of meningococcal disease in the United States found that vaccination with 2 doses of each vaccine (total of 4 injections between 11 and 16 years of age) has the potential to avert 165 cases of IMD over a 10-year period compared with no vaccination. The same model estimated that a MenABCWY vaccine has the potential to prevent up to 256 cases of IMD in this population compared with no vaccine; the higher number of cases averted with the MenABCWY vaccine was predominantly attributed to the prevention of more serogroup B cases. With the existing routine recommendations for vaccination of adolescents, a large proportion of disease continues to occur, this is attributable to the other serogroups that are not present within the vaccine formulation (Gary S. Marshall et.al., Infect Dis Ther (2022) 11:937-951).

[0010] Six serogroups e ' Neisseria meningitidis (N. meningitidis), viz., A, B, C, W, X, and Y, are responsible for most of the infections. There are no vaccines that target all six of them. In the present invention, a hexavalent meningococcal vaccine combination targeting all six serogroups is disclosed.OBJECT OF THE INVENTION:

[0011] Objects of the present invention are to provide a combination vaccine comprising hexavalent serogroup of Neisseria meningitidis for prevention and prophylaxis of infection caused by meningococcal bacteria.

[0012] An object of the present invention is to provide a hexavalent combination vaccine comprising serogroup A, B, C, Y, W, X of Neisseria meningitidis.

[0013] An object of the present invention is to provide a hexavalent combination vaccine comprising polysaccharide- protein conjugate of serogroup A, C, Y, W, X and proteins of serogroup B of Neisseria meningitidis.

[0014] Another object of the present invention is to provide a hexavalent combination vaccine comprising polysaccharide-protein conjugate of serogroup A, C, Y, W, X and mutated Factor H binding proteins (fHbp) of serogroup B of Neisseria meningitidis.

[0015] Another object of the present invention is to provide a pharmaceutical composition of hexavalent combination vaccine comprising serogroup A, B, C, Y, W, X of Neisseria meningitidis.

[0016] Another object of the present invention is to provide process of preparation of hexavalent combination vaccine comprising serogroup A, B, C, Y, W, X of Neisseria meningitidis.

[0017] Yet another object of the present invention provides use of hexavalent combination vaccine comprising serogroup A, B, C, Y, W, X of Neisseria meningitidis for use in protecting a human against infection by N. meningitidis.SUMMARY OF INVENTION

[0018] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in detailed description section. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0019] Aspects of the present invention relates to a combination vaccine comprising hexavalent serogroup of Neisseria meningitidis for prevention and prophylaxis of infection caused by meningococcal bacteria. The present invention relates to a hexavalent combination vaccine comprising serogroups A, B, C, Y, W, X of Neisseria meningitidis. The present invention relates to a hexavalent combination vaccine comprising polysaccharide- protein conjugate of serogroups A, C, Y, W, X and recombinant proteins of serogroup B of Neisseria meningitidis. The present invention specifically relates to ahexavalent combination vaccine comprising polysaccharide- protein conjugate of serogroup A, C, Y, W, X and mutated Factor H binding proteins (fHbp) of serogroup B of Neisseria meningitidis. The present invention further relates to the composition, use and process of preparation of hexavalent combination vaccine.

[0020] Further aspect of the present invention is to provide a combination vaccine comprising of polysaccharide and carrier protein conjugation wherein the polysaccharides of serogroup A, C, Y, W and X are individually conjugated to carrier protein

[0021] In an aspect, the carrier protein is selected from diphtheria toxoid, CRM 197, tetanus toxoid, pertussis toxoid, E. coli LT, E. coli ST, and exotoxin A from Pseudomonas aeruginosa, Bacterial outer membrane proteins such as, outer membrane complex c (OMPC), porins, transferrin binding proteins, pneumolysis, pneumococcal surface protein A (PspA), or pneumococcal adhesion protein (PsaA).

[0022] In an aspect, the mutated fHbp is selected from the group consisting of (i) A02 comprising the amino acid sequence of SEQ ID NO: 1, or comprising at least 90% sequence identity to SEQ ID NO: 1; (ii) A42 comprising the amino acid sequence of SEQ ID NO: 2, or comprising at least 90% sequence identity to SEQ ID NO: 2; (iii) A46 comprising the amino acid sequence of SEQ ID NO: 3, or comprising at least 90% sequence identity to SEQ ID NO: 3; (iv) B44 comprising the amino acid sequence of SEQ ID NO: 4, or comprising at least 90% sequence identity to SEQ ID NO: 4; (v) B107 comprising the amino acid sequence of SEQ ID NO: 5, or comprising at least 90% sequence identity to SEQ ID NO: 5; and (iv) combinations thereof.

[0023] In another aspect, the present invention relates to a process of preparation of mutated factor H binding proteins (fHbp) of A02, A42, A46, B44 and B107 of N. meningitidis serogroup B fHbp comprising steps of: a. Cloning of the individual fHbp gene with the signal sequence into expression vector, b. Plasmid extraction, c. Transformation d. Induction of the respective fHbp’ s e. Purification of respective fHbp’s

[0024] In another aspect, the present invention relates to a pharmaceutical composition comprising the hexavalent combination vaccine, and a pharmaceutically acceptable excipient such as but not limited to a carrier, a diluent or an adjuvant.

[0025] In another aspect, the present invention relates to a pharmaceutical composition comprising, wherein based on the unit dose of administration each dose of the composition comprises of: a. polysaccharide-carrier protein glycoconjugates of serogroup A of N. meningitidis in an amount of 2pg-10pg per dose; b. polysaccharide-carrier protein glycoconjugates of serogroup C of N. meningitidis in an amount of 2pg-10pg per dose; c. polysaccharide-carrier protein glycoconjugates of serogroup Y of N. meningitidis in an amount of 2pg-10pg per dose ; d. polysaccharide-carrier protein glycoconjugates of serogroup W of N. meningitidis in an amount of 2pg-10pg per dose ; e. polysaccharide-carrier protein glycoconjugates of serogroup X of N. meningitidis in an amount of 2pg-10pg per dose ; f. one or more proteins of serogroup B of N. meningitides in an amount of 60 pg- 240 pg per dose g. an adjuvant in an amount of 150 pg- 500 pg per dose; h. sucrose in an amount of 10-30 mg per dose ; i. an emulsifier in an amount of 0.01 mg- 0.04 mg per dose - ; j. a -preservative in an amount of Ipl - 4 pl per dose ; k. a buffer to make the pH 6.8 ± 0.4; and

[0026] Another aspect of the present invention is to provide process of preparation of hexavalent combination vaccine comprising serogroup A, B, C, Y, W, X of Neisseria meningitidis.

[0027] In yet another aspect, the present invention relates to a method of using the hexavalent combination vaccine comprising serogroup A, B, C, Y, W, X of Neisseria meningitidis for use in protecting a subject against infection by N. meningitidis comprising administering to the subject a therapeutically effective amount of the hexavalent combination vaccine or a composition comprising the same according to the invention, wherein the method is preferably immunization.

[0028] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of theclaimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS

[0029] The following drawings form part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.

[0030] Figure 1 : HPLC results for Serotype A polysaccharide

[0031] Figure 2 : HPLC results for Serotype C polysaccharide

[0032] Figure 3 : HPLC results for Serotype W-135 polysaccharide

[0033] Figure 4 : HPLC results for Serotype X polysaccharide

[0034] Figure 5 : HPLC results for Serotype Y polysaccharide

[0035] Figure 6 : HPLC results for Serotype A - CRM197 conjugate

[0036] Figure 7 : HPLC results for Serotype C - CRM197 conjugate

[0037] Figure 8 : HPLC results for Serotype W135 - CRM197 conjugate

[0038] Figure 9 : HPLC results for Serotype X - CRM197 conjugate

[0039] Figure 10 : HPLC results for Serotype Y- CRM197 conjugate

[0040] Figure 11 : SDS PAGE Results of recombinant fHbp protein sequences for Serotype B

[0041] Figure 12 : Western Blot Results of recombinant fHbp protein sequences for Serotype B

[0042] DETAILED DESCRIPTION OF THE INVENTION

[0043] The following is a detailed description of embodiments of the disclosure. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.

[0044] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0045] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0046] In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term "about." Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0047] As used in the description herein and throughout the claims that follow, the meaning of "a," "an," and "the" includes plural reference unless the context clearly dictates otherwise.

[0048] Also, as used in the description herein, the meaning of "in" includes "in" and "on" unless the context clearly dictates otherwise.

[0049] Unless the context requires otherwise, throughout the specification which follow, the word "comprise" and variations thereof, such as, "comprises" and "comprising" are to be construed in an open, inclusive sense that is as "including, but not limited to."

[0050] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein canbe performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. "such as") provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any unclaimed element essential to the practice of the invention.

[0051] The description that follows, and the embodiments described therein, is provided by way of illustration of an example, or examples, of particular embodiments of the principles and aspects of the present disclosure. These examples are provided for the purposes of explanation, and not of limitation, of those principles and of the disclosure.

[0052] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.

[0053] Various terms as used herein are shown below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.

[0054] The term "protein” according to the present invention means, but not limited to, refers to large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. The chain amino acid residue may be of any length and may be linear or branched. This term encompasses all the naturally occurring or modified / recombinant proteins.

[0055] The term “ Neisseria meningitidis ” or “TV. meningitidis ' according to the present invention means, but not limited to, refers to diplococcal, Gram-negative, and human commensal bacterium of the upper respiratory tract. The pathogen can invade the mucosa and gain access to the bloodstream, resulting meningitis, severe sepsis, or localized infections in joints and heart.

[0056] The term “Adjuvant / adjuvant component” according to the present invention means, but not limited to, refers to an adjuvant or an adjuvant component in the broadest sense is typically a (e.g., pharmacological or immunological) agent or composition that may modify, e.g., enhance, the efficacy of other agents, such as a drug or vaccine. Conventionally the term refers in the context of the invention to a compound or composition that serves as a carrier or auxiliary substance for immunogens and / or other pharmaceutically active compounds. It is to be interpreted in a broad sense and refers toa broad spectrum of substances that are able to increase the immunogenicity of anti-gens incorporated into or co -administered with an adjuvant in question. In the context of the present invention an adjuvant will preferably enhance the specific immunogenic effect of the active agents of the present invention. Typically, “adjuvant” or “adjuvant component” has the same meaning and can be used mutually. Adjuvants may be divided, e.g., into immuno potentiators, antigenic delivery systems or even combinations thereof. The term “adjuvant” is typically understood not to comprise agents which confer immunity by themselves. An adjuvant assists the immune system unspecifically to enhance the antigen- specific immune response by e.g., promoting presentation of an antigen to the immune system or induction of an unspecific innate immune response. Furthermore, an adjuvant may preferably e.g., modulate the antigen- specific immune response by e.g., shifting the dominating Th2-based antigen specific response to a more Thl-based antigen specific response or vice versa and / or by inducing of mucosal immune responses and / or increased IgA titers. Accordingly, an adjuvant may favourably modulate cytokine ex-pression / secretion, antigen presentation, type of immune response etc.

[0057] Advantages of adjuvants include the enhancement of the immunogenicity of antigens, modification of the nature of the immune response, the reduction of the antigen amount needed for a successful immunization, the reduction of the frequency of booster immunizations needed and an improved immune response in elderly and immunocompromised vaccines. These may be co -administered by any route, e.g., intramuscularly, subcutaneous, IV or intradermal injections.

[0058] The term “therapeutically effective amount” according to the present invention means, but not limited to, refers to an amount of the active ingredient (i.e., therapeutic protein or antibody) sufficient to produce the desired therapeutic effect in a human or animal, e.g., the amount necessary to treat, cure, prevent, or inhibit development and progression of disease or the symptoms thereof and / or the amount necessary to ameliorate symptoms or cause regression of disease. Such a therapeutically effective amount may vary depending on the structure and potency of the active ingredient and the contemplated mode of administration.

[0059] The term “treatment” according to the present invention means, but not limited to, refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those individuals, such as humans and animals, already with the disorder or condition to be treated as well as those prone to have the disorder or thosein which the disorder is to be prevented. As used herein, “treatment” also includes reduction of the likelihood of obtaining the disorder, reduction of the severity of the disorder in those already afflicted, and the induction of regression of the disorder or symptoms thereof.

[0060] The term “pharmaceutically-acceptable carrier” according to the present invention means, but not limited to, refers to a liquid filler, diluent or encapsulating substance that may be safely used in systemic administration. Depending upon the particular route of administration, a variety of pharmaceutically acceptable carriers, well known in the art may be used. These carriers may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions including phosphate buffered saline, emulsifiers, isotonic saline, and pyrogen free water. In particular, pharmaceutically acceptable carriers may contain different components such as a buffer, sterile water for injection, normal saline or phosphate-buffered saline, sucrose, histidine, salts and polysorbate. Terms such as “physiologically acceptable”, “diluent” or “excipient” can be used interchangeably.

[0061] In an embodiment of the present invention is to provide a combination vaccine comprising hexavalent serogroup of Neisseria meningitidis for prevention and prophylaxis of infection caused by meningococcal bacteria.

[0062] In another embodiment of the present invention is to provide a hexavalent combination vaccine comprising polysaccharide- protein conjugate of serogroups A, C, Y, W, X and proteins of serogroup B of Neisseria meningitidis.

[0063] In further embodiment, the present invention provides a hexavalent combination vaccine comprising polysaccharide- protein conjugate of serogroups A, C, Y, W, X and mutated Factor H binding proteins (fHbp) of serogroup B of Neisseria meningitidis.

[0064] In another embodiment of the present invention is to provide a hexavalent combination vaccine comprising serogroups A, B, C, Y, W, X of Neisseria meningitidis In some embodiments, the ratio of glycoconjugate of serogroups A, C, Y, W, and X of Neisseria meningitidis is 1 : 1 : 1 : 1 : 1.

[0065] In another embodiments, the ratio of glycoconjugate of serogroups A, C, Y, W, and X and the proteins of serogroup B of Neisseria meningitidis is 1: 4.5 to 5.0.ACYWX polysaccharide

[0066] In further embodiment the present invention provides a combination vaccine comprising of polysaccharide and carrier protein conjugation wherein thepolysaccharides of serogroup A, C, Y, W and X are individually conjugated to carrier protein selected from diphtheria toxoid, CRM197, tetanus toxoid, pertussis toxoid, E. coli LT, E. coli ST, and exotoxin A from Pseudomonas aeruginosa, Bacterial outer membrane proteins such as, outer membrane complex c (OMPC), porins, transferrin binding proteins, pneumolysis, pneumococcal surface protein A (PspA), or pneumococcal adhesin protein (PsaA) and like thereof.

[0067] In further embodiment the present invention provides a combination vaccine comprising of polysaccharide and carrier protein conjugation wherein the polysaccharides of serogroups A, C, Y, W and X are individually conjugated to carrier protein selected from diphtheria toxoid, CRM197, tetanus toxoid, pertussis toxoid, and like thereof.

[0068] In another embodiment of the present invention, the hexavalent combination vaccine wherein the ratio of polysaccharide and carrier protein in each of the glycoconjugate is 1:1 to 3.0.

[0069] In another embodiment the present invention provides a hexavalent combination vaccine comprising polysaccharide-protein glycoconjugate of serogroups A, C, Y, W, X and mutated Factor H binding proteins (fHbp) of serogroup B of Neisseria meningitidis wherein the process for the preparation of polysaccharide- protein conjugate of serogroups A, C, Y, W, X comprises the steps of: a. Fermentation of polysaccharides b. Activation of Polysaccharides and carrier protein c. Polysaccharide Protein Conjugation d. Purification

[0070] In an embodiment, a method of preparing the polysaccharide-carrier protein glycoconjugates of serogroups A, C, Y, W, and X of Neisseria meningitidis as claimed in anyone of claims 1-5 comprises the steps of: a) cultivating meningitis serogroup strains A, C, Y, W and X in presence of l-Cyano-4-Dimethylaminopyridine Tetrafluoroborate (CDAP) for about 12-20 hours to produce meningococcal A, C, Y, W, and X polysaccharides; b) purifying the meningococcal A, C, Y, W, and X polysaccharides by processes selected from ultrafiltration, cetyl-trimethylammonium bromide (CTAB) precipitation, centrifugation, depth filtration, ethanol, and salt precipitation; c) activating each of the purified meningococcal A, C, Y, W, and X polysaccharides at an alkaline pH of 8.5-9.5 and at a temperature ranging from 8-15°C; d) activating or derivatizing the carrier protein by l-ethyl-3-(dimethylaminopropyl)-carbodiimide (E DAC) for about16-24 hours; and e) mixing and conjugating the activated A, C, Y, W, and X polysaccharides and the activated carrier proteins to obtain the polysaccharide-carrier protein glycoconjugates of serogroups A, C, Y, W, and X of Neisseria meningitidis.Proteins of sero group B

[0071] In an aspect of the present disclosure, there is provided a modified variant of proteins of serogroup B of Neisseria meningitidis. In some embodiments, the modified variant of proteins of serogroup B of Neisseria meningitidis is mutated factor H binding proteins (fHbp) of N. meningitidis serogroup B. In an embodiment the said variant polypeptide sequence is as set forth in at least one of the SEQ ID NOs. 1, 2, 3, 4 and 5. In an embodiment, the said variant is codon optimized for expression in a prokaryotic expression system. In some embodiments, the modified variant of proteins of serogroup B of Neisseria meningitidis is selected from modified variant of factor H binding proteins A02, A42, A46, B44 and B107 of A. meningitidis fHbp family.

[0072] In another aspect of the present disclosure, there is provided a DNA construct comprising a gene encoding a polypeptide having at least one of the amino acid SEQ ID NOs. 1, 2, 3, 4 and 5. In another aspect of the present disclosure, there is provided an expression vector comprising a DNA fragment capable of being transcribed into a molecule which can be translated by cellular machinery to produce a polypeptide comprising an amino acid sequence as set forth in at least one of the SEQ ID NOs. 1, 2, 3, 4 and 5.

[0073] In yet another aspect of the present disclosure, there is provided a recombinant host cell comprising an expression vector, wherein said expression vector comprises a DNA fragment capable of being transcribed into a molecule which can be translated by cellular machinery to produce a polypeptide comprising an amino acid sequence as set forth in at least one of SEQ ID NOs. 1, 2, 3, 4 and 5.

[0074] In still another aspect of the present disclosure, there is provided a method for production of a polypeptide of at least one of SEQ ID NOs. 1, 2, 3, 4 and 5, said method comprising: (a) obtaining a recombinant host cell comprising an expression vector, wherein said expression vector comprises a DNA fragment capable of being transcribed into a molecule which can be translated by cellular machinery to produce a polypeptide comprising an amino acid sequence as set forth in at least one of SEQ ID NOs. 1, 2, 3, 4 and 5; and (b) incubating said recombinant host cell in conditions suitable for expression and secretion of polypeptide of at least one of SEQ ID NOs. 1, 2, 3, 4 and 5.Hexavalent Composition

[0075] In another embodiment of the present invention is to provide a hexavalent combination vaccine comprising polysaccharide-protein glycoconjugate of serogroups A, C, Y, W, X and proteins of serogroup B of Neisseria meningitidis wherein proteins of sero group B is mutated factor H binding proteins (fHbp) of N. meningitidis serogroup B.

[0076] In some embodiments, the present invention provides a hexavalent combination vaccine comprising (a) a combination of polysaccharide-carrier protein glycoconjugates of serogroups A, C, Y, W, and X of Neisseria meningitidis', and (b) one or more proteins of serogroup B of N. meningitidis wherein the ratio of (a) and (b) is 1:4.5 to 5.0.

[0077] In still another embodiment of the present invention is to provide a hexavalent combination vaccine comprising polysaccharide-protein glycoconjugate of serogroups A, C, Y, W, X and proteins of serogroup B of Neisseria meningitidis wherein proteins of serogroup B is selected from mutated factor H binding proteins (fHbp) of A02, A42, A46, B44 and B107 of N. meningitidis fHbp family having at least 90% sequence identity to SEQ ID NOs. 1, 2, 3, 4 and 5 respectively.

[0078] In still another embodiment of the present invention is to provide a hexavalent combination vaccine comprising polysaccharide-protein glycoconjugate of serogroup A, C, Y, W, X and proteins of serogroups B of Neisseria meningitidis wherein proteins of serogroup B involves a process of preparation of mutated factor H binding proteins (fHbp) of A02, A42, A46, B44 and B107 of N. meningitidis serogroup B fHbp comprising steps of: a. Cloning of the individual fHbp gene with the signal sequence into expression vector, b. Plasmid extraction, c. Transformation d. Induction of the respective fHbp’ s e. Purification of respective fHbp’s

[0079] In still another embodiment of the present invention is to provide a hexavalent combination vaccine comprising polysaccharide-protein glycoconjugate of serogroups A, C, Y, W, X and proteins of serogroup B of Neisseria meningitidis wherein proteins of serogroup B comprise of at least two of the following: a) mutated protein of fHbp family A02 of N. meningitidis (SEQ ID NO: 1); b) mutated protein of fHbp family A42 of N. meningitidis (SEQ ID NO: 2);c) mutated protein of fHbp family A46 of N. meningitidis (SEQ ID NO: 3); d) mutated protein of fHbp family B44 of N. meningitidis (SEQ ID NO: 4); and e) mutated protein of fHbp family B107 of N. meningitidis (SEQ ID NO: 5).

[0080] Another embodiment of the present invention is to provide a composition of hexavalent combination vaccine comprising serogroups A, B, C, Y, W, X of Neisseria meningitidis.

[0081] Another embodiment of the present invention is to provide a composition of hexavalent combination vaccine comprising: a. serogroup A-protein conjugate of Neisseria meningitides', b. serogroup C-protein conjugate of Neisseria meningitidis', c. serogroup Y-protein conjugate of Neisseria meningitidis', d. serogroup W-protein conjugate of Neisseria meningitidis', e. serogroup X-protein conjugate of Neisseria meningitidis', f. mutated Factor H binding proteins (fHbp) of serogroup B of Neisseria meningitidis', and g. one or more pharmaceutically acceptable excipients.

[0082] In another embodiment of the present invention, the mutated Factor H binding proteins (fHbp) of serogroup B is selected from mutated factor H binding proteins (fHbp) of A02, A42, A46, B44 and B107 of N. meningitidis fHbp family having at least 90% sequence identity to sequence ID nos. 1, 2, 3, 4 and 5 respectively.

[0083] In yet another embodiment, the one or more pharmaceutically acceptable excipients include, but not limited to, adjuvant(s), emulsifier(s), preservative(s), chelating agent(s), pH modifying agent(s), antioxidant(s), buffer(s), stabilizing agent(s) or stabilizer, isotonic agent(s), chelating agent(s), and any mixtures thereof.

[0084] In an embodiment of the present invention, the composition can include one or more adjuvant(s). Suitable adjuvant(s) include but not limited to aluminum phosphate, Aluminium hydroxide, Potassium aluminium Sulfate, Squalene, AS03 and a combination thereof. Adjuvant(s) may be present at concentrations of from 0.1 to 1 mg of Aluminum content per dose.

[0085] In an embodiment of the present invention, the composition can include an emulsifier. Suitable emulsifier include but not limited to PS -80, and PS20 and a combination thereof. Emulsifier may be present at concentrations of from 0.010 to 0.1 mg per dose

[0086] In an embodiment of the present invention, the composition can include a stabilizer. Suitable stabilizer include but not limited to Sucrose, Lactorse, , mannitol, Glycine or Gutamic acid sodium salt and a combination thereof. Stabilizer may be present at concentrations of from 3 mg to 50 mg per dose.

[0087] Exemplary stabilizing agent or stabilizer suitable for use in the composition of the present disclosure includes, but not limited to, mannitol, sucrose, lactose, mannitol, histidine, glutamic acid and glycine.

[0088] In an embodiment of the present invention, the pH value of the composition may be adjusted if necessary to pH 6 to 7. The pH can be adjusted by adding a pH modifying agent. Non-limiting examples of pH modifying agents include sodium hydroxide, sodium bicarbonate, hydrochloric acid and a combination thereof.

[0089] In an embodiment of the present invention, the composition can include one or more preservatives. Suitable preservatives include but not limited to 2 phenoxy ethanol benzyl alcohol, methylparaben, propylparaben, phenol, cresol and a combination thereof. Preservative(s) may be present at concentrations of from 0.5 mg -2.5 mg per dose.

[0090] In an embodiment of the present invention, the composition can include a buffer and the buffer may also be used to adjust the pH of the composition. Suitable buffers range may be adjusted to pH 6-pH 7.2 using suitable buffers include but not limited to sodium and potassium phosphates, sodium and potassium citrates, mono-, di- and triethanolamines, bicarbonate buffer, carbonate buffer, histidine buffer, tartrate buffer, Tris buffer and mixtures thereof. Buffer(s) may be present at concentrations of from 0.05 to 2% w / v.

[0091] In an embodiment, the present invention provides a pharmaceutical composition comprising: polysaccharide-carrier protein glycoconjugates of serogroup A of N. meningitidis', polysaccharide-carrier protein glycoconjugates of serogroup C of N. meningitidis', polysaccharide-carrier protein glycoconjugates of serogroup Y of N. meningitidis', polysaccharide-carrier protein glycoconjugates of serogroup W of N. meningitidis', polysaccharide-carrier protein glycoconjugates of serogroup X of N. meningitidis', one or more proteins of serogroup B of N. meningitides; an adjuvant; sucrose; an emulsifier; a stabilizer; a buffer to make the pH 6-7.2;

[0092] In various embodiments of the present invention, the pharmaceutical composition may be presented in unit-dose or multi-dose forms. In some embodiments, the unit dose of the pharmaceutical composition formulated as a sterile solution suitable for administration is 0.5 ml. Each unit dose can containa. polysaccharide-carrier protein glycoconjugates of serogroup A of N. meningitidis in an amount of 2pg-10pg per dose; b. polysaccharide-carrier protein glycoconjugates of serogroup C of N. meningitidis in an amount of 2pg-10pg per dose; c. polysaccharide-carrier protein glycoconjugates of serogroup Y of N. meningitidis in an amount of 2pg-10pg per dose ; d. polysaccharide-carrier protein glycoconjugates of serogroup W of N. meningitidis in an amount of 2pg-10pg per dose ; e. polysaccharide-carrier protein glycoconjugates of serogroup X of N. meningitidis in an amount of 2pg-10pg per dose ; f. one or more proteins of serogroup B of N. meningitides in an amount of 60 pg- 240 pg per dose g. an adjuvant in an amount of 150 pg- 500 pg per dose; h. sucrose in an amount of 10-30 mg per dose ; i. an emulsifier in an amount of 0.01 mg- 0.04 mg per dose - ; j. a -preservative in an amount of Ipl - 4 pl per dose ; k. a buffer to make the pH 6.8 ± 0.4; and

[0093] In one of the embodiment, the present invention provides a hexavalent combination vaccine comprising polysaccharide- protein conjugate of serogroups A, C, Y, W, X and proteins of serogroup B of Neisseria meningitidis is in fully liquid form or lyophilized form or a combination of liquid and lyophilized form thereof.

[0094] In an embodiment of the present invention, the unit dose can be administered to a human subject once or twice. One of skill in that art will realize that the particular administration schedule will depend on the human subject and the dosage being used. The administration schedule can also be different for individual subjects or change during the course of the therapy depending on the subject's reaction. In one preferred embodiment, the unit dose is administered twice.

[0095] In an embodiment, the pharmaceutical composition of this invention is presented in liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation may also be emulsified or encapsulated in liposomes for enhanced adjuvant effect, as discussed above under pharmaceutically acceptable carriers.

[0096] In one of the embodiment, the present invention provides a hexavalent combination vaccine comprising polysaccharide- protein conjugate of serogroups A, C,Y, W, X and proteins of serogroup B of Neisseria meningitidis which is administered directly to a patient via pharmaceutical injection (e.g. Subcutaneously, intraperitoneally, intravenously, intramuscularly, or to the interstitial space of a tissue), or by rectal, oral, vaginal, topical, transdermal, intranasal, ocular, aural, pulmonary or other mucosal administration. Dosage treatment can be a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunisation schedule and / or in a booster immunisation schedule. A primary dose schedule may be followed by a booster dose schedule. Suitable timing between priming doses (e.g. between 4-16 weeks), and between priming and boosting, can be routinely determined.

[0097] In an embodiment, the pharmaceutical composition of this invention is presented in unit dose form in ampoules, vials or pre-filled syringes.

[0098] In an embodiment, the pharmaceutical composition of this invention has a long shelf-life. In an embodiment, the shelf-life may be about 12 to about 36 months. In an embodiment, the aqueous injectable composition has a shelf-life of 18 months.

[0099] In yet another embodiment, the present invention provides a process of preparing the pharmaceutical composition comprising the steps of: (a) Adding Adjuvant (b) providing a combination of polysaccharide-carrier protein glycoconjugates of serogroups A, C, Y, W, and X of Neisseria meningitidis; (c) adding one or more proteins of serogroup B of A. meningitidis ; (d) adding excipients, emulsifier, stabilizer, and buffer one by one.[000100] In yet another embodiment, the present invention provides a method of treating a subject, comprising administering to the patient a therapeutically effective amount of a composition according to the invention. The method is preferably immunization.EXAMPLES[000101] The present invention is further explained in the form of following examples. However, it is to be understood that the following examples are merely illustrative and are not to be taken as limitations upon the scope of the invention.[000102] Example 1- Determination of Sialic acid content in meningococcal polysaccharide of serotypes C, W135 and Y.The standard NANA (N- Acetylneuraminic acid) solution was diluted was demonstrated in the table above. Dilutions ranging from 20 - 100 pg / ml of the standard NANA solution was prepared using purified water. In case of dry polysaccharide, a solution containing about 1 mg / ml of polysaccharide in purified water was prepared; the test sample was diluted 1: 10-fold to get a concentration of 100 pg to be used as test sample. From the above standard and sample dilutions preparation, 100 pl of each standard and sample was taken in duplicates. 1 ml of 5.0 mg / ml resorcinol solution was added to each tube and the tube were shut tight with screw caps and incubated in water bath at 100°C for 30 mins. On completion of the incubation period, tubes were cooled in ice tray. Then 2.0 ml of Butanol: Butyl acetate solution was added to each tube. The tubes were mixed vigorously on the vortex mixture and allowed to separate into phases. The liquid from the upper phase was collected ensuring that the lower phase does not get mixed with the upper phase. Take absorbance of the sample at 580 nm against the blank using spectrophotometer.Results - The sialic acid content was identified to be 90.3% for type C, 69.4% for type W-135 and 60.4% for type Y.[000103] Example 2 - Estimation of phosphorus content of Meningococcal A polysaccharide[000104] Series of standard solution from 20|jg / ml Phosphorus standard solution were prepared. Purified water was added and to make final volume to 200 pl in 5 ml test tubes. In case of dry polysaccharide a solution was prepared containing about 1 mg / ml of polysaccharide in Purified water and was used as a sample. 50pL of Reagent A solution (2 ml of sulphuric acid and 1 ml perchloric acid) was added to each tube and vortex. The tubes were heated in a Furnace to 250°C for 41 / 2 hours. 50 pl of hydrogen peroxide was added if charring occurred and heated further. Thde tubes were cooled down to room temperature. 200pL of purified water was added to each tube. 1 ml of reagent E comprising (1 ml each of 3 M sulphuric acid, 2.5% ammonium molybdate, 10 % ascorbic acid and 2 ml of purified water) to each tube and vortex. The tubes were incubated in a 37 °C water bath for 2 hours. The absorbance was measured at 825 nm with a spectrophotometer.Results: The phosphorus content for serotype A and X was identified to be 8.39% and 8.55 % respectively[000105] Example 3- High Performance Size Exclusion Chromatography Analysis Of Meningococcal Polysaccharide Samples[000106] Shodex guard column, Shodex 805 column and Shodex 804 column in series were mounted on calibrated HPLC system. The purge valve of pump (by turning it counter clockwise) was opened and flow rate was set to 3-5ml / min. The vacuum degasser was flushed with lOmM PBS for ten minutes. The flow rate was then set to l.Oml / min and the reference cell of detector purged for 40 minutes with 10 mM phosphate buffer saline solution until a stable baseline is obtained which can be viewedon the chromatographic software. Then samples of meningococcal polysaccharide where then filtered 0.2 pm syringe filter in HPLC glass vials following to the injection of 50 pl of the sample on HPLC.Results: The percentage purity for serotypes was identified to be 90% for serotype A, 93% for serotype C, 96% for serotype Y, 90% for serotype W -135 and 91% for serotype X. The Kavg for all samples analysed was identified to be 0.15, 0.27, 0.18, 0.16 and 0.20 for serotypes A, C, Y, W-135 and X respectively (Fig 1-5).[000107] Example 4 - Estimation Of Protein Content In Samples Of Meningococcal Polysaccharide Conjugate Vaccine By Lowry Method[000108] 500 pl of the standard dilutions of the working stock solution was prepared in duplicates. In case of dry polysaccharide a solution containing about 1-5 mg / ml of polysaccharide in Purified water was prepared and used as test sample. 2.5 ml of Cupric-tartaric solution was added to each of the standard blank (0.1M Sodium hydroxide) and sample. The test tubes were vortexed and incubated in dark at room temperature for 10 min. 250 pl of 1 N Folin & Ciocalteu’s phenol reagent was added and mixed well. The test tubes were incubated at room temperature for 30 minutes in dark. The blue color was observed which varies with the concentration. Absorbance of the sample at 730nm against the blank using spectrophotometer was taken.Results - The protein content within the polysaccharide was identified to be 0.27% for serotype A, 0.34% for serotype C, 2.75% for serotype Y, 1.55% for serotype W-135 and 0.36% for serotype X.[000109] Example 5 - Estimation of the Nucleic acid content in Meningococcal polysaccharide samples.[000110] The absorbance of the sample at 260 nm against the blank (Purified Water) using spectrophotometer was taken. The sample was diluted appropriately with Purified water to bring the OD value below 1.0. In case of dry polysaccharide, a Img / ml solution of polysaccharide was prepared in Lal reagent water and use it as sampleResults: The nucleic acid content in polysaccharide was identified to be 0.19% and 0.15% for serotype A and X respectively. For serotype C, Y and W - 135 the nucleic acid content was identified to be 0.23%, 1.18% and 1.02% respectively[000111] Example 6 - Conjugation of Meningitis serogroup A, C, Y, W, X with rCRM197:[000112] The meningitis serogroup strains were first cultivated in a bioreactor at Separate fermentation runs for 12-20 hours to produce meningococcal A, C, Y, W, and X polysaccharides. Purified polysaccharide was activated using a cyanalating agent, in this case l-cyano-4- dimethylaminopyridine tetrafluoroborate (CDAP) was used, in an alkaline environment(pH=8.5 - 9.5) at a temperature ranging from 8-15°C, the hydroxyl group of PS reacts with the cyanogen group of CDAP. On the other hand, the carrier protein i.e., rCRM was simultaneously activated using Carbodiimide reagent such as ED AC which helps in coupling reactions as a catalyst (protein and ED AC ration 1:1). The reaction was carried out with an optimum reaction time of 16-24 hours. The optical density of ACYWX batches was achieved between 3.01 and 10.4 in aerobic conditions and feed addition as per requirement, with pH ranging from 6.5-7.4, temperature ranging from 35-37 °C, and RPM of 50-220. After individually activating PS and rCRM both of them were mixed in an alkaline environment (pH= 9-9.5) that was maintained using TEA buffer. This mixture was kept on mixing for Incubation 12-30 hrs preferably 16-24 hrs at 2-8°C for polysaccharide-protein conjugate formation to take place.Different ratio of protein was used for different polysaccharides (PS) of serotypes of Neisseria meningitidis i.e., A, C, Y, W, X (PS: protein ratio of 1: 1-2.5). After incubation period was completed this PS -Protein conjugate was concentrated and diafiltered (using 100 KDa cassette and sterile filtered using 0.2p filters. The amount of conjugate formation and free polysaccharide was verified by HPLC analysis.[000113] Example 7 - Determination Of Total and Unconjugated (Free) Polysaccharide of Meningococcal CRM Conjugate Using Resorcinol AssayCalibration Curve (2.5 - 25 pg / ml) is prepared in duplicates as follow:[000114] 1 ml of sample (dilute if required)was taken having about 100-150 pgPolysaccharide in a microfuge tube, 100 pl of 5% DOC was added and mixed and placed on ice bath for 30 minutes. 50 pl IM HC1 was added and mixed thoroughly. The mixture was centrifuged at 2-8°C for 15 minutes at 10000 RPM. 400pl supernatant volume was taken in separate test tube for free Polysaccharide estimation and 400pL of without treated sample for Total polysaccharide estimation. The test was performed in duplicate for each sample. 400 pL purified water was used as blank in thermoresistant test tube. 400 pL of Resorcinol solution was added to tubes of calibration curve, Blank and samples and vortexed. 2 mL of 75% H2SO4 was added to all test tubes and vortexed well. The tubes were covered with aluminium foil and placed in water bath at 90°C for 30 minutes. The tubes were then cooled and placed in dark for 30 minutes. The tubes were then allowed to come at room temperature and absorbance was measured at 430 nm against the blank.Results: Total polysaccharide content was identified to be 70.8 pg / ml for serotype A, 81.1 pg / ml for serotype C, 87.9 pg / ml for serotype Y, 69.4 pg / ml for serotype W- 135pg / ml and 75.6 pg / ml for serotype X.[000115] Example 8 - Protein Concentration In Active Raw Material Of CRM And Pneumococcal CRM Conjugate Vaccine By BCA Protein Assay Kit[000116] Preparation of diluted Bovine Serum Albumin (BSA) standards: Each 1 ml ampule of 2.0 mg / ml (stock) albumin standard is used to prepare the curve as follows in microcentrifuge tube:Prepare fresh working reagent by mixing 50 parts of BCA reagent A and 1 part of BCA reagent B.Pipette 0.1 ml of each standard, purified water (as standard blank), and sample (diluted in case the concentration falls outside the calibration curve) into an appropriately labeled test tube in duplicates. Add 2.0 ml of working reagent to each tube and vortex well. Cover the tubes with Aluminium foil and incubate at 60°C in a water bath for 30 minutes. Cool all the tubes to Room temperature. Set the wavelength in spectrophotometer at 562 nm.Results : The protein content was identified to be 132.1 pg / mL for serotype A, 51.9 pg / mL for serotype C, 248.0 pg / mL for serotype Y, 31.0 pg / mL for serotype W-135 and 82.7 pg / mL for serotype X.[000117] Example 9 - High performance size Exclusion Chromatography Analysis for Meningococcal polysaccharide and CRM197 conjugate[000118] 0. IM Sodium Phosphate buffer was prepared in 1000ml. In addition, Sodium azide 0.05% and 5mg of Salmon DNA in 5 ml of purified water was also prepared. All the solutions prepared for use are vacuum filtered using Nylon 6, 6 membrane 0.20 / 0.22 / 0.45 pm, depending upon the nature of solution to be filtered (filtration not necessary if the solution is prepared in 0.2p filtered purified water). For organic solvents filtration not necessary if the solution is HPLC grade. Set the flow rate to 0.5mL / min. Equilibrate the system and column (TSK gel G6000) with 0.1M sodium phosphate solution until a stable baseline for RID is obtained. 1 pl of diluted p-Aminobenzoic acid is injected 6 times for system suitability test. Load the required volume of sample as per the PS concentration equivalent to 2.5pg in the sample vial. Each sample run will be of 30 min and after running all the samples the sequence will automatically stop. The retention time of the unretained compound is determined with Salmon DNA. The Retention time of the component which has full access to the pores of stationary phase is determined with Sodium Azide. For purified polysaccharide samples the molecular size distribution should be less than 80% or KaVg= 0.50.Results : The percentage purity was identified to be 100% for serotype A and X, 99% for serotype Y, 93% for serotype C and 84% for serotype W-135. The KaVgwas identified tobe 0.33 for serotype A, 0.26 for serotype C, 0.10 for serotype Y, 0.19 for serotype W- 135 and 0.27 for serotype X (Fig 6- 10).[000119] Example 10 - Determination of Free carrier protein in Meningococcal polysaccharide and CRM197 conjugate by high performance size exclusion chromatography[000120] 0. IM Sodium Phosphate buffer was prepared in 1000ml. In addition, Sodium azide 0.05% and 1 mg / mL of p- Amino benzoic Acid Stock was also prepared. 1 mg / mL of the Aminobenzoic Acid Stock solution was diluted to 1 / 10 with purified water. All the solutions prepared for use are vacuum filtered using Nylon 6,6 membrane 0.20 pm or 0.22 pm , depending upon the nature of solution to be filtered. For organic solvents 0.45pm filters should be used. Prepare the HPLC column for run. Set the flow rate to 0.75 mL / min. Equilibrate the column (TSK gel 4000) with mobile 0.1 M sodium phosphate buffer at 280nm until a stable baseline is observed. 1 pl of diluted p-Aminobenzoic acid is injected 6 times for system suitability test. Load the required volume of sample as per the PS concentration equivalent to 2.5 pg in the sample vial. Each sample run will be of 20 min. and after running all the samples the sequence will automatically stop. Inject the eluent blank after every 20 injections. Area under curve for the standards / sample is analyzed by Chemstation software. The retention time for p-Aminobenzoic acid is calculated. Results of percentage of area of peak towards higher Molecular weights (Conjugated) and percentage of area of peak towards lower. Molecular weights (Unconjugated). The free protein identified shall be < 5% of total protein contentResults: The percentage of free carrier protein was identified to be 2.21% and 2.52% for serotype A and X, 1.85% for serotype Y, 2.22% for serotype C and 0.53% for serotype W -135[000121] Example 11 - Testing of Bacterial Endotoxin by LAL Gel Clot method.Using 1 EU / ml endotoxin solution, prepare a serial two-fold dilution series in LAL Reagent water till two points lower than the endpoint of the lysate is reached.[000122] The specification limit of endotoxin was 0.75 EU / pG of polysaccharide, therefore sample was diluted to 1 pg / ml using lai reagent water to its maximum valid dilution (MVD),. MVD for Ipg / ml purified polysaccharide sample was calculated to be 12 therefore it was diluted further to 12 fold, lysate sensitivity was identified to be 0.06 EU. Product positive control was prepared, by diluting the test samples according to MVD / 2 value with LAL Reagent Water. 0.2 ml of 4 standard endotoxin was added to 0.2 ml of this dilution. 0.1 ml of the LAL reagent was added to each tube beginning with the highest concentration of standard endotoxin followed by negative control, test samples and PPCs. LAL reagent water was taken as the negative control for the assay which was prepared by adding 100 pl of LRW in reaction tube and add 100 pl of 2 standard endotoxin.Following the addition of lysate, the tubes were incubated at 37+ 1°C for 60 + 2 minutes in a tube heating block. After 60 + 2 minutes of incubation each reaction tube was examined for gelation. A positive reaction was indicated by a firm gel that remains intact momentarily when tube is inverted at 180°. If clot formed in the test sample, it meant that it contains endotoxin equal to or greater than 0.06 EU / ml. If no clot formed, it meant that the test sample contains endotoxin less than 0.06 EU / ml.Results - No clot were observed in the test samples which indicate that the endotoxin content in the sample is less than the specified limits.[000123] Example 12- Process of preparation of mutated factor H binding proteins (fHbp) 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) of N. meningitidis serogroup B[000124] The cloning of the respective fHbp genes (having the P4 signal sequence at the start of N terminal) was done into expression vector pET3O3 (Thermo, USA). Further processes involved codon optimization, gene synthesis, sub cloning, transformation of E.coli DH5a (NEB, USA) strain, and the positive clone strain was extracted to obtainplasmid. All the five respective His-Tag [6 His-Tag] (at C terminal of) fHbp’s were expressed in E.coli BL21 DE3 [NEB, USA] cells in LB media (Sigma, USA) having lOOpg / ml ampicillin. The induction for all the five fHbp’s (of subfamily A and B) was performed (at an OD600nm between 0.6-0.8 using ImM IPTG (Sigma, USA) at 37°C, 250 rpm for 4 hours in the incubator shaker. Before induction, an uninduced sample was withdrawn and kept at the same conditions as described for induction. After induction, the total cell pellet was centrifuged at 6000rpm for 20 mins and the pellet was stored and supernatant discarded. The mutated protein were purified by Ni NT A chromatography. The purified fHbp proteins from both the subfamilies (A and B) were desalted and brought in phosphate buffer saline using PD 10 columns and analysed using SDS PAGE and Western blot for the purified fHbp (Fig 11 - 12).[000125] Example 13 - Hexavalent Combination Vaccine Composition[000126] Purified Bulk of conjugate is formulated along with other purified bulk conjugate in to desired doses form using appropriate buffer, Aluminum compound adjuvant, suitable excipients and preservative. Individual conjugates of ACYWX along with fHbp shall be formulated by adsorbing on alum to develop a fully liquid hexavalent vaccine. Formulation activity is performed at required temperature for desired time. Once tested and approved, formulated vaccine is filled of desired dose volume of either 0.5-1 ml ampules. The labelled vials were stored at about 2-8°C for optimum shelf life. The single dose formulation was without any preservative whereas multi dose formulation contained suitable stabilizer and preservative.[000127] Example 14 - Formulation of MenHexa:[000128] MenHexa vaccine was formulated using six serotypes of Neisseria meningitis out of which five i.e, A, C, Y, W, X were glycoconjugates and one serotype i.e., Serogroup B was a recombinant fHBP protein which was further composed of two subfamilies A and B. For formulation all the serotypes were mixed wherein 25pg per dose of polysaccharides of serotypes A, C, Y, W and X were taken and 120 pg (60 pg subfamily A and 60 pg subfamily B) per dose of fhbp protein of serotype B was taken. All these serotypes were mixed with 250 pg per dose of adjuvant, 20 mg Sucrose per dose, 0.2 mg PS-80 per dose, 2 PE (2 mg per dose) and Phosphate buffer saline at 6.8 pH.[000129] Example 15 - In vitro Study[000130] The hexavalent composition comprising fHbp’s of Neisseria meningitidis serogroup B and conjugate of serogroups A, C, Y, W and X with CRM197 were 1administered into animal models. The study involved female BALB / c mice for vaccine testing. Mice were selected due to their relevance in immunological research, sourced from Hylasco Biotechnology, and included nulliparous, non-pregnant females aged 9-10 weeks. Before the study, the mice underwent veterinary examinations to ensure good health. They were housed in groups of up to five per cage in polypropylene cages with stainless steel grills, under controlled environmental conditions, including a temperature range of 19-25°C, humidity of 30-70%, and a 12-hour light / dark cycle. The animals were provided with ad libitum 'Altromin' pelleted rodent feed and reverse osmosis water, both tested for contaminants. The test vaccine was administered subcutaneously, as it is a standard method for vaccine delivery in mice. According to WHO guidelines, the maximum human dose was adjusted for mice, with a total volume of 0.5 mL per mouse divided into two 0.25 mL injections at different sites. Group G2 received a smaller dose of 0.1 mL. The injections were administered on days 0 and 14, using sterile stainless steel needles (26G) and appropriate syringes. Buffer saline was administered similarly for the control group. This design and dosing were aimed at ensuring an appropriate immune response while adhering to established animal care protocols.This is to check the effect of combination of sero group together in a composition and also to check the biological activity produced. Indirect ELISA was performed. Plates were coated with serogroups and then the samples of animal models were added and incubated. The plates were then washed with wash buffer and then with blocking buffer. The plates were incubated for 1 hour at RT with secondary antibody (anti mouse conjugated with horseradish peroxidase (HRP). After the incubation was complete, the plates were washed and the TMB substrate (3,3',5,5'-Tetramethylbenzidine). After 10 mins, H2SO4 was added to end the reaction and the absorbance of the respective plates was read. The QC serum prepared was given an arbitrary value and used as a standard in each plate. The standard of each plate was utilized to generate a standard ELISA curve facilitating in the estimating of IgG concentrations of the corresponding samples / groups.ELISA buffers and reagents:1. Phosphate buffer saline (PBS) [10X]: Weigh 2.6 gram (gm) KH2PO4, 11.5 gm Na2HPO4 and 87.1 gm NaCl in 500-6000ml Milli Q water (MQW). Adjust pH at 7.3 ± 0.1 with HC1 or NaOH and make up volume up to 1000ml. It is advisable to filter the buffer through 0.2p filter assembly before use. After the buffer is made it can be kept atroom temperature (RT) [25±2°C] for one month, however discard it, if any clumps, aggregation or any growth or contamination is observed.2. Coating buffer (IX PBS): This has to be made as IX PBS, pH 7.3+0.1, i.e Dilute the 10X PBS to make it IX in MQW. Mix gently. Prepare as required and to be made fresh while performing the ELISA. Volume to be made as per requirement.3. Wash Buffer: Wash buffer comprises IX PBS, pH 7.3+0.1 containing 0.1 % Tween 20. The 100 ml wash buffer contains 0.1 ml [lOOpl] Tween 20 and 99.9 ml of IX PBS. Mix gently. Prepare as required and to be made fresh while performing the ELISA. Volume to be made as per requirement.4. Blocking Buffer / Assay buffer: Blocking Buffer / Assay buffer comprises IX PBS, pH 7.3+0.1, 0.1% tween 20 and 5% fetal bovine serum (FBS). The 100ml blocking buffer / assay buffer will have 5 ml FBS and 0.1 ml [lOOpl] Tween 20 solution to 94.9 ml IX PBS. Prepare as required and to be made fresh while performing the ELISA. Volume to be made as per requirement.5. Coating antigen: The coating antigen will be respective polysaccharide [PS] for which the ELISA is to be performed. Whichever antigen is to be coated on the 96 well plate. Mix 50 pl mHS A (from 1 mg / ml stock prepared in MQW and stored at -200C) and 50 pl N. meningitidis PS of respective serogroup (from 1 mg / ml stock prepared in MQW and stored at -200C) in 9.9 ml coating buffer. Prepare fresh every day.6. Secondary antibody (2° Ab): Anti- Mouse IgG (whole molecule)-Peroxidase antibody (Cat A4416, Sigma).7. Substrate: Readymade 3, 3 ',5, 5 '-Tetramethylbenzidine (TMB) [Cat T0440, Sigma], which was cooled to room temperature before adding to the plate during the last incubation step.8. Stop solution (2 M H2SO4)9. Test samples and the adjuvant control and buffer / saline as negative control to be stored -20°C. Test samples are the serum samples from several groups from the proof of concept study performed using female swiss albino or BALB / c mice.10. Quality control (QC) sera composition: The QC sera is to be prepared by pooling approx. 30pl or more and equal volume of serum from each mice from the groups which are assumed to have generated high IgG concentrations. After pooling of the serum, designate appropriate name to the QC sera and assign an arbitrary value such as 6000 EU / ml or 5000 EU / ml for the evaluation and quantification of anti-meningococcal IgG concentrations in the test sera samples. The QC sera was stored at -20°C.For each serogroup such as ABCYWX QC sera to be optimized for predilutions of both primary (l°Ab) and secondary (2°Ab) antibodies.The primary (l°Ab) antibodies are serum samples and secondary (2°Ab) antibody is the commercially available antibody A4416 from Sigma.ELISA procedure:1. Coating: 50 pl mHSA (from 1 mg / ml stock prepared in MQW and stored at - 20°C) and 50 pl N. meningitidis PS of respective serogroup (from 1 mg / ml stock prepared in MQW and stored at -20°C) were mixed in 9.9 ml coating buffer and were prepared fresh every day. Afterwards, 100 pl coating buffer was added per well of the 96-well ELISA plate having 500ng of polysaccharide.2. The coated plates were incubated overnight at 2-8°C.3. The plates were taken out next day and washed with wash buffer (PBS, 7.3+0.1 + 0.1% tween 20), followed by blocking of the plate with 200 pl of blocking buffer / well for 60 minutes (mins) at RT.4. In the meantime, the pre-dilutions of QC sera were prepared. It is important to optimize the predilutions of QC sera for each of the serogroups i.e. ACYWX using a checker board having several dilutions of both primary antibody = l°Ab as well as secondary antibody (A 4416) = 2°Ab as mentioned below (Table-1), however several other predilutions of both l°Ab as well as 2° Ab were also tried and checked.No primary antibody (test sera samples) control = No l°Ab C should not give an OD >0.1No secondary antibody (A 4416) control = No 2° Ab should not give anOD >0.1The predilutions and combination of l°Ab and 2°Ab gave OD values between 1.5+0.5, however more preferably an OD of 2 was considered for each of the serogroups i.e. ACYWX for the QC sera to be used in the successive ELISA experiments. Also each time ELISA was performed for each of the serogroups i.e. ACYWX, the optimized QC sera had to be put in duplicate along with the test samples (in duplicate). After ELISA, QC sera OD values were mapped along with test sample OD values obtained, in the combitats software, which followed a four parametric logistic curve model (4PL) for the estimation IgG of all the samples in the study design.After blocking was over from step 3, the plates were washed thrice as mentioned in step B3. 200 pl of pre-diluted QC sera were added in the respective well of the row A. 100 pl assay buffer was added in all the other wells. 6 two-fold serial dilutions of QC sera were performed in the respective wells by adding transferring 100 pl from row A to row B and so on till row G for the QC sera samples. 100 pl discarded from last serum dilution well used so that each well had 100 pl diluted serum with buffer in it. The plates were put for at RT with 2 hours of incubation time.5. The plates were washed thrice wash buffer as mentioned in step B3.6. Secondary antibody [Anti-Mouse IgG (whole molecule)-Peroxidase antibody, A4416] was prepared in assay buffer and 100 pl / well was added with 60 mins incubation at RT. ELISA plates were covered and placed in a dark place.7. The plates were washed as mentioned in step B3 and 100 pl of substrate solution (TMB) / well was added, followed by incubating the plate for 10+1 min at RT in a dark place to protect the ELISA reaction, blue color developed after the reaction of HRP with substrate.8. The reaction is stopped by adding 50 pl of 2 M H2SO4 per well. The color changed from blue to Yellow.9. The absorbance was measured at 450 / 630nm ELISA reader & data was transferred to an excel file for analysis.10. After ELISA QC sera OD values were compared with sample OD values obtained in the combistats software, which followed a four parametric logistic curve model (4PL) for the estimation IgG of all the samples in the study design.Note:For each of the serogroups i.e. ACYWX, the test sample may have the l°Ab predilution as half of the QC sera dilution whereas for 2°Ab, the predilution may be the same as optimized for the QC sera for each serogroup of ACYWX.For each serogroup of ACYWX the steps of ELISA as mentioned above will remain the same for the test samples.[000131] SEQUENCE LISTING[000132] ADVANTAGES OF THE PRESENT INVENTION[000133] The present invention provides a hexavalent combination vaccine comprising polysaccharide- protein conjugate of serogroups A, C, Y, W, X and mutated Factor H binding proteins (fHbp) of serogroup B of Neisseria meningitidis.[000134] The present invention provides the composition, process of preparation of hexavalent combination vaccine.[000135] The present invention provides a novel, specific and industrially advantageous composition of hexavalent combination vaccine that are capable of complete prophylaxis against meningococcal diseases. These compositions shall be fulfilling the unmet medical need for Meningitis prophylaxis and treatment.

Claims

We Claim:

1. A hexavalent combination vaccine comprising (a) a combination of polysaccharide- carrier protein glycoconjugates of serogroups A, C, Y, W, and X of Neisseria meningitidis', and (b) one or more proteins of serogroup B of N. meningitidis.

2. The hexavalent combination vaccine as claimed in claim 1, wherein the carrier protein selected from diphtheria toxoid, CRM197, tetanus toxoid, pertussis toxoid, E. coli LT, E. coli ST, and exotoxin A from Pseudomonas aeruginosa, Bacterial outer membrane proteins such as, outer membrane complex c (OMPC), porins, transferrin binding proteins, pneumolysis, pneumococcal surface protein A (PspA), or pneumococcal adhesin protein (PsaA).

3. The hexavalent combination vaccine as claimed in claim 1, wherein the carrier protein is individually conjugated to the polysaccharides of serogroups A, C, Y, W and X.

4. A method of preparing the polysaccharide-carrier protein glycoconjugates of serogroups A, C, Y, W, and X of Neisseria meningitidis as claimed in anyone of claims 1-3 comprises the steps of: a) cultivating meningitis serogroup strains A, C, Y, W and X in presence of complex media for about 12-20 hours to produce meningococcal A, C, Y, W, and X polysaccharides; b) purifying the meningococcal A, C, Y, W, and X polysaccharides by processes selected from ultrafiltration, cetyl-trimethylammonium bromide (CTAB) precipitation, centrifugation, depth filtration, ethanol, and salt precipitation; c) activating each of the purified meningococcal A, C, Y, W, and X polysaccharides at an alkaline pH of 8.5-9.5 in the presence of 1 -Cyano -4 Dimethylaminopyridine Tetrafluoroborate (CDAP) and at a temperature ranging from 8-15 °C; d) activating or derivatizing the carrier protein by 1- ethyl- 3- (dimethylaminopropyl) - carbodiimide (ED AC) for about 16-24 hours; e) mixing and conjugating the activated A, C, Y, W, and X polysaccharides and the activated carrier proteins to obtain the polysaccharide-carrier protein glycoconjugates of serogroups A, C, Y, W, and X of Neisseria meningitidis.

5. The hexavalent combination vaccine as claimed in claim 1, wherein the one or more proteins of serogroup B are selected from recombinant Factor H binding proteins (fHbp) of serogroup B.

6. The hexavalent combination vaccine as claimed in claim 1, wherein the recombinant fHbp of serogroup B is a mutated fHbp.

7. The hexavalent combination vaccine as claimed in claim 1, wherein the mutated fHbp of serogroup B has at least 90% sequence identity to SEQ ID NO: 1, 2, 3, 4 and 5.

8. A pharmaceutical composition wherein based on the unit dose of the composition administered, each unit dose comprises of : a. polysaccharide-carrier protein glycoconjugates of serogroup A of N. meningitidis in an amount of 2pg-10pg per dose; b. polysaccharide-carrier protein glycoconjugates of serogroup C of N. meningitidis in an amount of 2pg-10pg per dose; c. polysaccharide-carrier protein glycoconjugates of serogroup Y of N. meningitidis in an amount of 2pg-10pg per dose; d. polysaccharide-carrier protein glycoconjugates of serogroup W of N. meningitidis in an amount of 2pg-10pg per dose ; e. polysaccharide-carrier protein glycoconjugates of serogroup X of N. meningitidis in an amount of 2pg-10pg per dose ; f. one or more proteins of serogroup B of N. meningitides in an amount of 60 pg-240 pg per dose g. an adjuvant in an amount of 150 pg- 500 pg per dose; h. sucrose in an amount of 10 -30 mg per dose ; i. an emulsifier in an amount of 0.01 - 0.04 ug per dose - ; j. a -preservative in an amount of Ipl - 4 pl per dose ; k. a buffer to make the pH 6.8 ± 0.4; and9. The pharmaceutical composition as claimed in claim 8 wherein the unit dose administered is 0.5 ml.

10. The hexavalent combination vaccine claimed in anyone of claims 1- 9 is in fully liquid form and or lyophilized form.

11. The hexavalent combination vaccine claimed in anyone of claims 1-9 is in a combination of liquid and lyophilized form wherein one or more serotypes from A, C, Y, W and X and serotype B is either in liquid or lyophilized form.

12. A pharmaceutical composition comprising the hexavalent combination vaccine as claimed in anyone of claims 1-11, and a pharmaceutically acceptable carrier, diluent or adjuvant.

13. The pharmaceutical composition as claimed in claim 10, wherein the pharmaceutically acceptable excipient is selected from the group consisting of adjuvant(s), emulsifier(s), preservative(s), chelating agent(s), pH modifying agent(s), antioxidant(s), buffer(s), stabilizing agent(s), isotonic agent(s), chelating agent(s), and any mixtures thereof.

14. The pharmaceutical composition as claimed in claim 1- 13 is administered once or twice.

15. The pharmaceutical composition as claimed in any of the preceding claim 1- 14 useful for the treatment, prevention or diagnosis of meningitis.

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