Meningococcal protein-based vaccine formulations and methods for making same

Abstract

The present invention provides fusion proteins having the desire to reduce Factor H binding, in particular the present invention provides optimized processes for the manufacture of fusion proteins and formulations comprising fusion proteins. The present invention provides an effective platform process for the manufacture of an effective vaccine formulation against Neisseria meningitidis, which meets a variety of criteria, including improved immunogenicity, safety, stability, and burdenability.

Description

TECHNICAL FIELD

[0001] The present invention relates to the field of vaccine formulations and methods for manufacturing the same. In particular, the present invention relates to upstream, downstream and formulation development of recombinant / chimeric protein antigens based on Neisseria meningitidis (meningococcus) serogroup B, methods of manufacturing formulations based on such chimeric proteins and the use of these formulations for the prevention and / or treatment of subjects suffering from a Neisseria meningitidis (meningococcus) serogroup B infection. BACKGROUND

[0002] 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. The description herein includes information that can be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any specific or implicit publication is prior art.

[0003] Neisseria meningitidis is an important pathogen, particularly in children and young adults. Septicaemia and meningitis are the most life-threatening forms of invasive meningococcal disease (IMD). Case fatality rates remain at about 10% for disseminated disease, while one third of meningococcal disease survivors suffer significant debilitating and long-term sequelae. Because of its high morbidity and mortality, the disease has become a global health concern.

[0004] Toxoid-based vaccines have almost eliminated diphtheria and tetanus in affluent countries, while capsular-based vaccines have greatly reduced disease caused by Haemophilus influenzae, Streptococcus pneumoniae and some strains of Neisseria meningitidis. However, there remain challenges in vaccine development for pathogens for which toxoid- and capsular-based vaccines are not feasible. These pathogens include non-typeable strains of H. influenzae and S. pneumoniae, pathogens not encapsulated by a capsule such as Neisseria gonorrhoeae and Moraxella catarrhalis and the serogroup B N. meningitidis for which capsular-based vaccines are not feasible. Given the increase in the emergence of multi-drug resistant bacteria, new vaccine development approaches are needed. Development of a vaccine against serogroup B N. meningitidis presents particular difficulties because of the poor immunogenicity of the polysaccharide capsule due to its immunological similarity to human neural cell adhesion molecules. Furthermore, strategies to produce a successful vaccine are hampered by the pathogen diversity and difficulties associated with presenting epitopes from membrane-embedded surface proteins to the immune system.

[0005] Neisseria meningitidis is a gram-negative bacterium that colonizes the upper respiratory tract of humans and causes sporadic and periodic epidemic outbreaks of disease worldwide, most notably meningitis and septicemia. N. meningitidis typically has a cytoplasmic membrane, a peptidoglycan layer, an outer membrane that, together with a polysaccharide capsule, forms the bacterial wall, and pili that protrude into the external environment. Nm remains the leading cause of septicemia and bacterial meningitis in children and young adults. Approximately 500,000 cases of meningococcal disease occur each year, with about 50,000 deaths. In developed countries, the bacterium is a leading cause of childhood death, has important public health implications during outbreaks in schools and universities, and can cause severe disability in survivors. Capsule-encapsulated N. meningitidis strains are the leading cause of bacterial meningitis and septicemia in children and young adults. N. meningitidis can be classified into at least 12 serogroups (including serogroups A, B, C, H, I, K, L, 29E, W135, X, Y, and Z) based on chemically and antigenically distinct polysaccharide capsules. The most common serogroups are A, B, and C, which cause 90% of disease worldwide. Serogroup B is the most common cause of meningococcal disease in Europe, the United States, and several countries in Latin America, and causes epidemics in sub-Saharan Africa every 5-10 years. Despite the availability of antibiotics, meningococcus is a devastating disease that can kill children and young adults within hours. Thus, prophylactic immunization is the best way to protect individuals from meningococcal infection.

[0006] Vaccines based on the bacterial polysaccharide capsule have been developed, but the polysaccharide capsule of N. meningitidis serogroup B is poorly immunogenic because of its structural identity with human glycoproteins in neural tissue and can induce autoimmunity if used as a vaccine. Thus, serogroup B "humanization" halted early efforts toward developing a safe and immunogenic conjugate polysaccharide vaccine against group B, because the polysaccharide did not elicit serum bactericidal antibodies, and in vitro anti-capsular B antibodies recognized neural cell adhesion molecules in fetal brain tissue.

[0007] Two main approaches have been used to develop vaccines against serogroup B N. meningitidis; outer membrane vesicle vaccines (OMVV) and recombinant protein subunit vaccines. OMVVs were first developed in the 1980s. The immunodominant antigen in meningococcal OMVVs is PorA, a major outer membrane porin with eight surface-exposed loops. Loops one and four are known as variable regions 1 and 2 (VR1 and VR2) because they generate immune responses and have a propensity for antigenic variation. The VR2 loop dominates PorA-specific immunity elicited by OMVVs, which provides limited or no cross-protection against strains expressing PorA with different VR2. When longer follow-up periods were assessed, including the OMV vaccine studied for 20 months in Chile, efficacy dropped to 50%, indicating poor duration of response (A.L. Wilkins, M.D. Snape et al 2017).

[0008] To expand coverage, OMVVs containing multiple PorAs have been developed and selected for prevalence of PorA sequences in circulating strains. However, OMVVs present complex manufacturing and regulatory issues. Prior art vaccines often utilize purification of so-called "blebs", which represent vesicles shed from the cell surface of the particular organism of interest. However, this crude product presents many problems. For example, there is great variability in the composition of these vesicles. There is no reliable way to control which proteins are included or excluded from these vesicles. These vesicles can or can not include polysaccharide-coated elements of the organism of interest. The proportions of the various components of the vesicles relative to each other cannot be reliably determined. The composition of these vesicles cannot be easily determined or controlled.

[0009] The OMV in Bexsero® (GSK Vaccines) is highly reactogenic, so the vaccine is routinely administered with paracetamol due to parental concerns about adverse reactions (Prymula, R. 2014). Bexsero® provides uncertain coverage because 1) antigens are derived from a single meningococcal strain (Tan, L., et al. 2010), 2) most immunogenicity studies were performed using the 3+1 schedule rather than the 2+1 schedule planned for the UK (dosing at 2, 4, and 12 months), 3) indirect rather than direct association with protection was measured primarily in 13-month-olds rather than in the most at-risk infants.

[0010] Meningococcal serogroup B vaccines based on outer membrane vesicles (OMV) have been successfully used to prevent epidemics (most recently in New Zealand), but only provide protection against strains expressing the same PorA (outer membrane porin) variants as in the OMV. OMVVs are not favored as immunogens because consistency and toxicity can be issues in the manufacturing process. For example, OMVVs can contain toxic lipopolysaccharide (LPS).

[0011] PorA is the most abundant meningococcal outer membrane protein (OMP) that also elicits SBA and is the major target of the immune response elicited by OMV vaccines that have been successfully used in outbreaks. PorA variants differ in their variable regions (VRs), surface-exposed loops, that are the targets of the immune response. Notably, VR2 is responsible for most of the SBA elicited by OMV vaccines. However, PorA is a complete OMP with multiple hydrophobic domains. This makes it difficult to produce PorA as a recombinant protein in its native conformation, limiting its use as an antigen in subunit vaccines.

[0012] Factor H binding protein (fHbp) is also an antigen that elicits serum bactericidal antibody responses in immunized individuals and is a key component of investigational vaccines currently being evaluated in clinical trials for the prevention of meningococcal disease, particularly serogroup B. Factor H binding protein (fHbp, also known in the art as lipoprotein 2086 (Fletcher et al (2004) Infect Immun 72: 2088-2100), genome-derived Neisserial antigen (GNA) 1870 (Masignani et al. (2003) J Exp Med 197: 789-99) or "741") is a surface-exposed lipoprotein expressed in Neisseria meningitidis bacteria. Based on differences in nucleotide and predicted amino acid sequences, fHbp from different N. meningitidis strains is classified using several schemes. These include two subfamilies (A and B) (Murphy E, et al. (2009) The Journal of Infectious Diseases 200: 379- 389) or three variant groups (V1, V2 and V3) (Masignani V, et al. (2003) The Journal of Experimental Medicine 197: 789-799), with subfamily A corresponding to V2 and V3 and subfamily B corresponding to V1 (which is the most abundant).

[0013] Recombinant subunit vaccines Bexsero® (GSK Vaccines) and Trumenba® (Wyeth Pharmaceuticals) contain the important meningococcal antigen, factor H binding protein (fHbp), which is a lipoprotein consisting of two beta barrels that tightly bind domains 6 and 7 of human complement factor H (CFH). fHbp has antigenic variability; the genomic sequence database contains over 900 different fHbp peptides, which fall into three variant groups or two subfamilies: VI (subfamily B), V2, and V3 (both subfamily A). Typically, immunization with a particular fHbp induces cross-protection against strains expressing fHbp belonging to the same but not different variant group, although there can be cross-protection between fHbp variant groups 2 and 3 (subfamily A). Bexsero® contains a single fHbp peptide (VI.1), along with two additional recombinant antigens and OMV, while Trumenba® consists of only two fHbp peptides (VI.55 and V3.45). To date, no vaccine studies have included V2 fHbp, although strains expressing this variant account for approximately 20-30% of all isolates (38% of UK cases). There is no vaccine with v2 fHbp because it is an inherently unstable antigen. fHbp on Neisseria meningitidis is a 27 KDa lipoprotein consisting of two beta barrels (N-terminal barrel and C-terminal barrel) connected by a short amino acid linker. The reason for the lack of V2 fHbp in currently licensed vaccines is the instability of its N-terminal beta barrel (Prymula, R. 2014; Johnson, S 2012). Furthermore, protein stability is important during the vaccine manufacturing process as it impacts yield and is an important issue for quality control. Also, the antigens in Bexsero® and Trumenba® have 36 and 4.8% exact sequence matches to the currently circulating serogroup B Neisseria meningitidis disease isolates in the UK, respectively, raising concerns about their ability to provide broad coverage against antigenically diverse pathogens. In addition, a modified V2 fHbp with increased stability compared to wild-type V2 fHbp has been disclosed, wherein the modified V2 fHbp comprises at least six mutations at Ser35, Asp107, Val112, Leu114, Ser137, and Gly138. Since a single fHbp cannot provide universal protection against meningococcal disease, immunization with a vaccine containing representatives from each of the three variants VI, V2, and V3 is necessary for broad Neisseria meningitidis serogroup B protection.

[0014] Both Bexsero® and Trumenba® vaccines were developed before it was recognized that complement factor H (CFH) binds meningococcus with high affinity via fHbp and that this impairs the immune response (Schneider, M 2009). Although fHbp has been shown to be an important protective antigen, it is currently unclear to what extent fHbp interacts with fH after immunization and whether any fHbp-fH interaction influences the overall immunogenicity of fHbp in humans. Studies in hfH transgenic mice and infant rhesus monkeys, the latter of which have a polymorphism in the fH gene that allows for high or low binding to fHbp (Konar M 2015), have shown that binding of fH to fHbp reduces the immunogenicity of fHbp (Beemink PT 2011) (Costa 2014) (Giuntini S 2015) (Granoff DM 2015) (Rossi R 2013).

[0015] Furthermore, upstream, downstream and formulation development are often rate-limiting steps for biopharmaceuticals to enter the market early and meet population demand.

[0016] Upstream process development includes scale-up of the fermentation process to ensure similar product yield and quality in large scale production as in small scale production. Various culture parameters, such as media composition, pH, agitation, aeration, temperature, cell density, inducer concentration, induction time and feeding strategy, influence protein expression levels depending on the expression system. Therefore, it is essential to evaluate each culture condition for the expression of each recombinant protein and the development of an efficient bioprocess.

[0017] Escherichia coli is the most widely used bacterial host for production of recombinant proteins due to: (1) its fast growth rate with a generation time span of 20 minutes under optimized conditions (Clark and Maaloe, 1967), (2) well developed tools for molecular manipulation and in-depth knowledge of its biology, and (3) the ability to achieve high cell densities using inexpensive culture reagents. In practice, however, many hurdles encountered along the production pipeline must be overcome. These include poor growth of the host strain, protein instability or toxicity, aggregation and inclusion body formation, incompatibility of environmental conditions (temperature, pH, salt concentration, etc.), and even no amplification of expression at all. This can be due to slower growth rates, lower final cell densities, and death when the protein of interest cannot be detected or is detected at very low levels (less than micrograms per liter of culture). E. coli cannot perform post-translational modifications, limiting the range of products that can be produced in this host organism in soluble and active form. In addition, E. coli cannot secrete recombinant proteins. Therefore, recombinant E. coli cells need to be disrupted to access the intracellular products, which are then purified, usually through several filtration steps.

[0018] Furthermore, high level expression of recombinant proteins in E. coli results in aggregation of the expressed proteins into inclusion bodies (IBs). This poses a serious challenge for production of soluble recombinant proteins with proper biological function at an industrial scale, as it requires extensive processing involving isolation from cells, solubilization, refolding, and purification to produce biologically active proteins. Altering culture conditions often presents the simplest solution to reduce IB formation in E. coli. However, culture conditions that favor production of soluble proteins can vary depending on the protein of interest involved and the E. coli host strain used, and therefore require experimental optimization. Factors such as the expression strain, fermentation medium, and operating conditions all play an important role in process scale-up to maintain or improve yields at larger scales, even at industrial scale, to provide large amounts of protein through a cost-effective, commercially viable manufacturing process. Therefore, it is important to identify the appropriate parameter or set of parameters that are critical for a specific process.

[0019] High level production of recombinant proteins will subsequently require efficient purification processes on an industrial scale as this contributes to the approval of therapeutic products for human use. Cell disruption is necessary to recover desired proteins expressed as intracellular inclusion bodies (IB). In small scale work, cell disruption can be very efficient; however, it is very poor when scaled up. Sonication has high energy requirements and has serious health and safety issues due to the noise. Also it is not continuous. Chemical cell lysis poses significant health and safety risks to the user and the cost of using large volumes of reagents required for large scale production can be prohibitively high. Furthermore, the presence of salt and detergents can be incompatible with protein assays. It can also affect the results of downstream applications such as mass spectrometry. Therefore, a suitable cell lysis method needs to be considered in the scaling up of the production process to obtain optimal results.

[0020] It is well known that increased product concentration in the upstream process leads to higher chromatography resin volumes and higher buffer requirements. Host cell proteins (HCPs) and DNA are the main sources of impurities and the HCPs of each process differ significantly from each other in their molecular weight, charge, hydrophobicity and structure. Therefore, they pose a challenge to chromatographic purification.

[0021] In the case of soluble active recombinant proteins that need to be purified, it is invaluable to have (i) means to detect it along the expression and purification protocol, (ii) to reach maximum solubility, and (iii) to easily purify it from the E. coli cell environment. Expressing amino acid fragments (peptide tags) or large polypeptides (fusion partners) in tandem with the desired protein to form a chimeric protein can allow these three objectives to be directly achieved. Peptide tags are less likely to cause interference when fused to the protein due to their small size. However, in some cases, they can have a negative impact on the tertiary structure or biological activity of the fused chimeric protein. Therefore, the peptide tag should also be removed as it can affect the protein conformation, hinder the interaction with the partner molecule or reduce the biological activity. In fact, the final solubility of the desired product is unpredictable when these tags are removed. In the case of tag removal by enzymatic digestion, the expression vector has a sequence encoding a protease cleavage site located downstream of the gene encoding the tag. The choice between different proteases is based on specificity, cost, number of amino acids left in the protein after cleavage and ease of removal after digestion. The Cysteine protease from Tobacco Etch Virus (TEV) is one of the most widely used proteases. However, the expression of TEV protease in E. coli has encountered difficulties regarding protein yield (reduced product yield) or low solubility of the protein at an industrial scale, which means that large volumes are required and often long incubation times are needed to achieve efficient cleavage.

[0022] Recombinant protein antigen-based vaccines often require adjuvants to achieve protection against the relevant disease. Aluminum salts are the most prevalent adjuvant in human vaccines approved by the U.S. Food and Drug Administration. The point of zero charge (PZC) of an adjuvant is the point at which the net surface charge is zero; the PZC of aluminum hydroxide is approximately 11, while the PZC of aluminum phosphate is approximately 4-5.5. Adsorption of proteins onto adjuvant surfaces is typically maximized when the net charge sign of the protein is opposite to that of the adjuvant surface, allowing for electrostatic attraction. Therefore, protein vaccine formulations are prepared with adsorption buffers to improve adsorption to the surface. However, protein conformation can change when the protein binds to the liquid-solid interface. Furthermore, the conformational changes induced by binding to the adjuvant can alter protein stability during long-term storage. For example, if adsorption is substantially complete, aggregation by pathways that occur in bulk solution is less likely to occur. Conversely, unfolding upon binding can expose residues that are normally buried to the solvent, facilitating degradation processes such as oxidation. (J Pharm Sci. 2009 September; 98(9): 2970-2993).

[0023] Because of the potential impact of degradation on the immunogenicity, toxicity, and efficacy of recombinant protein antigens, both physical and covalent stability are of primary concern after long-term storage of recombinant protein antigens adsorbed onto adjuvant surfaces.

[0024] There are various factors that affect the stability of recombinant protein antigens, including the percentage of antigen adsorbed onto the adjuvant surface, zeta potential, viscosity, solute concentration, pH changes, and temperature, which contribute to storage stability.

[0025] Zeta potential is one such physical property that is essential for the optimization of formulations of suspensions, emulsions, and protein solutions, as well as for predicting interactions with surfaces. Any suspended particle, macromolecule, or material surface displays a zeta potential. Knowledge of the zeta potential can reduce the time required to produce a trial formulation. It can also be used as an auxiliary tool for predicting long-term stability. In certain cases, particles in a dispersion can adhere to one another and form aggregates of increasing size, which can settle under the influence of gravity. Furthermore, it can be seen that the zeta potential depends on the nature of the buffer used.

[0026] There are various factors that affect the zeta potential, including the excipients used in the formulation, excipient compatibility, the concentration of the formulation components, pH, and conductivity.

[0027] Changes in pH affect the electrostatic forces, with the van der Waals forces remaining constant for a given system. At pHs close to the isoelectric point, the charge-charge repulsion between neutral molecules is minimal and attractive forces dominate, leading to flocculated systems, high viscosity, and most likely aggregation. This pH effect was observed by Liu et al. and Chari et al. in antibody solutions.

[0028] The stability and activity of recombinant protein antigens are affected by pH and temperature due to possible changes in the characteristics of the recombinant protein antigens, including gradual aggregation, degradation processes (such as oxidation) that affect physical and chemical stability.

[0029] Therefore, in summary, formulation development becomes more important for long-term storage of recombinant protein antigens. It is known that the effective concentration of available immunogens decreases with increasing aggregation. Therefore, there is a need for formulations and methods that overcome the problem of aggregation by stabilizing recombinant proteins against aggregation.

[0030] Therefore, there is a need for a vaccine against Neisseria meningitidis with maximum adsorption of recombinant protein antigens to the surface of adjuvants, showing low viscosity, no aggregation, long-term stability over a wide temperature range, improved immunogenicity, and at the same time affordability and safety.

[0031] Furthermore, there is a need for an efficient platform process for manufacturing an effective vaccine against Neisseria meningitidis that meets multiple criteria, including improved immunogenicity, safety, and affordability, in particular improved formulations that show low viscosity, no aggregation, and show long-term stability over a wide temperature range.

[0032] Furthermore, there is a need to prepare soluble, high-yield, and stable chimeric proteins, in which all the necessary protein antigens are immunogenic.

[0033] To overcome the above limitations of the prior art, the applicant proposes an improved upstream, downstream process and formulation development based on chimeric protein antigens of Neisseria meningitidis (meningococcus) serogroup B, and a method of preparing formulations based on such chimeric protein antigens. SUMMARY

[0034] Applicants provide 1) a chimeric antigen (ChA) against serogroup B N. meningitidis (MenB). The ChA utilizes fHbp (non-lipidated) as a molecular scaffold to present a surface exposed PorA VR2 loop, by inserting the VR2 loop ("10-20 amino acid" PorA VR2 loop rather than "entire PorA protein") into the beta turn region in fHbp. The ChA retains epitopes from both fHbp and PorA, and is found to elicit a functional immune response against both antigens. The integration of the VR2 loop does not alter the overall structure of fHbp, and the VR2 loop folds into a conformation recognized by bactericidal mAbs. 2) a soluble and high yield, stable chimeric protein, where both fHbp and PorA VR2 loop are immunogenic. 3) to create chimeras composed of the most prevalent fHbp and PorA antigens to maximize vaccine coverage, as the chimeras composition reflects the prevalent fHbp and PorA antigens circulating within a given geographic region. 4) to insert the PorA loop at a specific location in fHbp to obtain the desired reduction in Factor H binding (at least 10%, at least 50% reduction compared to wild type; preferably >70%) and have a molecular weight in the range of 20-40 kDa, while retaining immunogenic epitopes of both fHbp and PorA.

[0035]

[0036] 5) Insertion of one PorA loop into one fHbp (instead of 2 PorA loops on one fHbp) and insertion of maltose binding protein (MBP) tag along with His tag to ensure optimal expression, solubility and stability. 6) Insertion of TEV cleavage site between His-MBP tag and chimera to easily remove the tag during downstream processing and optimization of substrate (labeled recombinant protein / modified fHbp fusion protein) to enzyme ratio (20: 1 compared to 5: 1, 10: 1), temperature / incubation (30 °C, 15-18 hours). 7) Retention of at least 50 generations of plasmid, even without any antibiotic. 8) Use of combination of chemical lysis and homogenizer (instead of sonication or chemical lysis alone) for large scale lysis of cells (pressure 1000-1500 bar; 3-8 cycles), considering advantages of using homogenizer include disruption of cell wall at 4-15 °C, effective for neutral lipid extraction, easy to use in small volumes, faster processing, reliable operation, constant shear rate, and completely scalable. 9) Due to use of optimal concentration of inducer (IPTG (1 mM to 10 mM) or lactose (1 g / L to 50 g / L)); use of modified M9 salt medium (chemically defined medium) / fed batch mode supplemented with L-methionine (maintaining L-methionine concentration between 1-10 mM, preferably 2-5 mM compared to Luria Broth (LB) medium (complex medium) / batch mode); stopping glucose feed when OD at 590 / 600 nm is about 20-100 and starting glycerol feed and inducing culture by adding lactose and / or maintaining lactose at 1-50 g / L in fed batch mode, hence yield of labeled fHbp protein can be at least 700 mg of purified labeled protein (harvested) from 100 grams of wet cell mass. 10) Use of two step chromatography (ion exchange chromatography followed by affinity chromatography) instead of previously known 3 step chromatography (multi-step affinity chromatography, ion exchange chromatography followed by size exclusion chromatography). 11) Use of stabilized V2 fHbp by substituting amino acids in its N-terminal beta barrel (i.e. changing <5% of residues of the barrel). 12) Formulation comprising adjuvant (aluminum hydroxide), phosphate buffer, mannitol and polysorbate (polysorbate 20) conferring optimal pH (7-8), osmolality (200-500, preferably 200-400 mOsmol / kg) and zeta potential (-16 to -30 mV) resulting in maximum adsorption and retaining integrity / stability of the chimeric protein. 13) For TEV cleavage, 1:20 enzyme: substrate ratio incubation for 18 hours was found to be optimal. DETAILED DESCRIPTION OF DRAWINGS

[0037] The present application will now be described by way of example with reference to the accompanying drawings in which:

[0038] Figures 1-5Vector map showing recombinant protein / modified fHbp fusion protein according to an embodiment of the application;

[0039] Figure 6 Vector map showing TEV protease according to an embodiment of the application;

[0040] Figure 7 Flow chart showing seed development for 10 L scale fermentation batch for production of tagged fHbp protein;

[0041] Figure 8 Flow chart showing production of tagged fHbp protein in 10 L scale fermenter;

[0042] Figures 9a-9h Growth curve of tagged fHbp protein in 10 L scale fermentation batch along with SDS-PAGE gel image;

[0043] Figure 10 Flow chart showing seed development for 10 L scale fermentation batch for production of TEV protease;

[0044] Figure 11 Production of TEV protease in 10 L scale fermenter;

[0045] Figures 12a-12b Growth curve of TEV protease in 10 L scale fermentation batch along with SDS-PAGE gel image;

[0046] Figure 13 Percentage cleavage using homogenization;

[0047] Figures 14a-14b SDS-PAGE image of TEV protease cleavage reaction using different substrate: enzyme ratios;

[0048] Figure 15 Spectrophotometric analysis of TEV protease cleavage reaction using different substrate: enzyme ratios;

[0049] Figure 16 SDS-PAGE image of TEV protease cleavage reaction using different temperatures;

[0050] Figure 17 Spectrophotometric analysis of TEV protease cleavage reaction using different temperatures;

[0051] Figure 18 Flow chart showing purification of TEV protease by Ni-Sepharose Resin (pH 7.4);

[0052] Figure 19A flow diagram showing the purification of TEV protease through Ni-Sepharose Resin (pH 8.5) is shown;

[0053] Figures 20a-20b Purification profiles of TEV protease at pH 8.5 and pH 7.4 are shown, respectively;

[0054] Figure 21 A flow diagram showing the purification of tagged proteins through Ni-Sepharose Resin is shown;

[0055] Figure 22 A flow diagram showing the purification of tagged proteins expressed as inclusion bodies is shown;

[0056] Figure 23 A flow diagram showing the removal of tags from purified proteins is shown;

[0057] Figure 24 A flow diagram showing the ion exchange chromatography step for purification of recombinant protein / modified fHbp fusion proteins is shown;

[0058] Figure 25 A flow diagram showing the affinity chromatography step for purification of recombinant protein / modified fHbp fusion proteins is shown;

[0059] Figure 26 A graphical representation of the GMTs with individual data points and highlighted for each bleed day is shown;

[0060] Figure 27 Representative data showing the binding of human complement factor H to wild-type fHbp compared to Men B chimeric proteins is shown;

[0061] Figure 28 A graphical representation of the GMTs at each time point (serum bleed day) for the MenFive serogroup is shown;

[0062] Figure 29 A graphical representation of the GMTs at each time point (serum bleed day) for the MenB serogroup is shown; and

[0063] Figure 30 A standard curve for protein estimation using the micro bicinchoninic acid (BCA) method is shown.

[0064] OBJECT

[0065] Some of the objects of the present application, which are satisfied by at least one embodiment herein, are as follows:

[0066] The present disclosure aims to provide a highly efficient platform process for manufacturing an effective vaccine formulation against N. meningitidis which meets multiple criteria including improved immunogenicity, safety and affordability.

[0067] Another object of the present disclosure is to provide a highly efficient platform process for manufacturing an effective vaccine formulation comprising one or more recombinant protein / modified fHbp fusion proteins derived from N. meningitidis serogroup B and a pharmaceutically acceptable carrier or excipient.

[0068] Another object of the present disclosure is to develop and optimize the upstream bioprocess to increase cell density of the lead cell line and productivity of the recombinant protein / modified fHbp fusion protein.

[0069] Another object of the present disclosure is to develop and optimize the downstream bioprocess for production of recombinant protein / modified fHbp fusion protein at high yield and high purity.

[0070] Another object of the present disclosure is to develop and optimize the formulation comprising recombinant protein / modified fHbp fusion protein which shows improved immunogenicity, low viscosity, no aggregation; shows long term stability over a wide temperature range.

[0071] Another object of the present disclosure is to develop and optimize the formulation comprising chimeric antigen (ChA) against serogroup B N. meningitidis. ChA utilizes fHbp (non-lipidated) as a molecular scaffold to present the surface exposed PorA VR2 loop, which is achieved by inserting the VR2 loop (not the "entire PorA protein") into the beta-turn region in fHbp. ChA retains epitopes from both fHbp and PorA and was found to elicit functional immune responses against both antigens, where integration of the VR2 loop did not alter the overall structure of fHbp and the VR2 loop folded into a conformation recognized by bactericidal mAbs.

[0072] Another object of the present disclosure is to develop and optimize the formulation comprising soluble and high yield, stable chimeric protein, where both fHbp and PorA VR2 loop are immunogenic.

[0073] Another object of the present disclosure is to develop and optimize the formulation comprising chimera composed of the most popular fHbp and PorA antigens to maximize vaccine coverage.

[0074] Another object of the present disclosure is to develop and optimize the formulation comprising chimeric protein having a molecular weight in the range of 20 kDa to 40 kDa while retaining immunogenic epitopes of both fHbp and PorA:

[0075]

[0076] Another object of the present disclosure is to develop and optimize downstream including insertion of TEV cleavage site between His-MBP tag and chimera for easy removal of tag during downstream processing and optimization of substrate to enzyme ratio / temperature / incubation, retaining plasmid for maximum number of generations even without any antibiotic.

[0077] Another object of the present disclosure is to use a combination of chemical lysis and homogenizer (with optimized conditions instead of sonication or chemical method alone) for large scale lysis of cells.

[0078] Another object of the present disclosure is to develop high yield of chimeric fHbp-PorA protein due to use of optimal concentration of inducer; use of modified M9 salt media (chemically defined media) / fed batch mode supplemented with L-methionine; stopping glucose feed and starting glycerol feed when specific OD at 590 / 600 nm is reached and induction of culture by addition of lactose or IPTG.

[0079] Another object of the present disclosure is to develop two step chromatography instead of previously known method using at least 3 steps of chromatography.

[0080] Another object of the present disclosure is to develop and optimize formulation comprising chimeric protein, adjuvant (aluminum hydroxide), phosphate buffer, mannitol and polysorbate (polysorbate 20) thereby imparting optimal pH, osmolality and zeta potential leading to maximum adsorption and retaining integrity / stability of chimeric protein.

[0081] Yet another object of the present disclosure is to provide a method of vaccinating a host.

[0082] Other objects and advantages of the present disclosure will be more apparent from the following description, which is not intended to limit the scope of the present disclosure.

[0083] DETAILED DESCRIPTION

[0084] Before describing the compositions and formulations of the present application, it is to be understood that this application is not limited to the particular compositions and formulations described since such can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting since the scope of the present application will be limited only by the appended claims.

[0085] While the present disclosure can be susceptible to various modifications and alternative forms, some embodiments are shown by way of example in the drawings and are discussed in detail below. It should be understood that the disclosure can be practiced in a variety of embodiments and that the disclosure is not limited to any particular embodiment described and / or illustrated herein.

[0086] Aspects and embodiments are provided to give a full and enabling disclosure of the range of the disclosure. Numerous details are set forth to provide an understanding of embodiments of the disclosure. Those skilled in the art will understand, however, that the embodiments provided are not limited to the specifics set forth herein. In some embodiments, well-known compositions, well-known processes and well-known techniques have not been described in detail in order to not unnecessarily obscure the disclosure.

[0087] The terminology used in the present disclosure is for the purpose of describing particular embodiments only and is not intended to be limiting of the present disclosure. As used in the present disclosure, the forms "a", "an" and "the" can be intended to include one or more than one, unless the context clearly dictates otherwise.

[0088] The terms "comprises", "comprising", "including", and "having", are intended to be open-ended transitional phrases, thus specifying the presence of stated features, integers, steps, operations, elements, modules, units and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or groups thereof. The specific order of steps disclosed in the processes of the present disclosure should not be construed as necessarily requiring their performance in the order described or illustrated. It is also understood that additional or alternative steps can be employed.

[0089] The terms "comprising", "comprises" and "comprised of" as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, unrecited members, elements or method steps. It will be appreciated that the terms "comprising", "comprises" and "comprised of" as used herein include the terms "consisting of", "consists" and "consists of". More specifically, the term "comprising", as used herein, means that the claim encompasses all elements or method steps listed, but also includes additional unrecited elements or method steps. For example, a method comprising steps a), b) and c) encompasses, in its broadest form, a method consisting of steps a), b) and c). The phrase "consisting of means that the composition (or device or method) has only the recited elements (or steps). In contrast, the term "comprising" can also encompass a method that includes further steps in addition to steps a), b) and c) (such as steps d) and e)).

[0090] The terms first, second, third, and the like do not expressly limit the scope of the disclosure, as such terms can be used merely to distinguish one element, component, region, layer or section from another element, component, region, layer or section. Unless the disclosure clearly indicates otherwise, the use of terms such as first, second, third, and the like are not to imply a particular sequence or order unless clearly indicated by the disclosure.

[0091] Furthermore, the terms "first", "second", "third", or "(a)", "(b)", "(c)", "(d)", etc., and similar terms, are used herein to distinguish between similar elements and not necessarily to indicate a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the application described herein are capable of operating in other sequences than the one described or illustrated herein. Unless specifically stated otherwise as apparent from the prior description or claims themselves herein, the terms "first", "second", "third" or "(A)", "(B)", and "(C)" or "(a)", "(b)", "(c)", "(d)", "i", "ii", etc., where used in the description or the claims, refer to the steps of a method or use or assay in any order, i.e., the steps can be performed simultaneously or there can be time intervals of seconds, minutes, hours, days, weeks, months or even years between such steps.

[0092] In the following passages, different aspects of the application are defined in more detail. Each aspect so defined can be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous can be combined with any other feature or features indicated as being preferred or advantageous.

[0093] 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 of the application. Thus, 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, but can refer to different embodiments. Furthermore, the described features, structures, or characteristics can be combined in any suitable manner in one or more embodiments. In addition, the disclosure can include various alternative or additional steps, features, structures, or characteristics in addition to those specifically recited in this specification, as will occur to those skilled in the art. For example, although some embodiments described herein include certain features, not all embodiments need necessarily include those features, and different embodiments can include different features. The scope of the application is not limited to the embodiments specifically recited in this specification, but rather includes any and all embodiments that would normally occur to one skilled in the art upon reading this disclosure.

[0094] It should be understood that every feature or embodiment or combination of features or embodiments described herein is a non-limiting, illustrative example of any aspect of the application, and thus is intended to be combinable with any other feature or embodiment or combination of features or embodiments described herein. For example, where a feature is described as being in any one of a number of embodiments, such as “one embodiment,” “some embodiments,” “certain embodiments,” “further embodiments,” “a specific exemplary embodiment,” and / or “another embodiment,” each of these embodiments is intended to be a non-limiting example of a feature or combination of features described herein that can be combinable with any other feature or combination of features described herein without requiring recitation of every possible combination. Such features or combinations of features are applicable to any aspect of the application.

[0095] Furthermore, ranges defined throughout this specification include the end values, i.e. a range of 1 to 10, between 1 and 10 means that both 1 and 10 are included in the range. For the avoidance of doubt, the Applicant shall be entitled to any equivalents under the applicable law.

[0096] The term “about” as used herein when defining a value of a stated item, number, percentage, or term means a range of plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the value of the stated item, number, percentage, or term. Preferably, a range of plus or minus 10% is intended.

[0097] Where numerical ranges are used herein for describing concentrations, amounts, and / or other parameters, such ranges are understood to include both endpoints and any number sub-ranges therebetween. For example, a range of 1 to 5 microM is understood to include 1 microM and 5 microM, as well as any number sub-ranges therebetween, such as, for example, 1 microM to 3 microM. As used herein, the term “in vitro” means outside or external to an animal or human body. As used herein, the term “in vitro” is understood to include “ex vivo.” The term “ex vivo” generally refers to tissue or cells removed from an animal or human body and maintained or propagated outside the body, e.g., in a culture vessel. As used herein, the term “in vivo” means within or internal to an animal or human body.

[0098] Definitions:

[0099] To make the disclosure more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms can be found throughout the specification.

[0100] The terms "protein" or "polypeptide" or "(poly)peptide" or "peptide" as used herein, all of which are used interchangeably if not otherwise specified, include an isolated and / or purified and / or recombinant (poly)peptide which is essentially free of other host cell polypeptides. The term "peptide" as referred to herein comprises at least two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300 or even more amino acid residues, of which the alpha carboxyl of one is bound to the alpha amino of the other. Post-translational modifications of proteins or peptides used and considered herein are modifications of the newly formed protein or peptide and can involve the deletion, substitution or addition of amino acids, chemical modifications of certain amino acids, such as amidation, acetylation, phosphorylation, glycosylation, formation of pyroglutamic acid, oxidation / reduction of the sulfa group on methionine, or the addition of similar small molecules to certain amino acids.

[0101] The term "homolog" as used herein refers to a bacterial, fungal, plant or animal homolog of the oxidase or rubredoxin or rubredoxin reductase useful in the present application, preferably a plant homolog, but also includes truncated sequences, single-stranded DNA or RNA of coding and non-coding DNA sequences.

[0102] Sequence identity, homology or similarity is defined herein as the relationship between two or more amino acid sequences or two or more nucleic acid sequences, as determined by comparing the sequences. Typically, sequence identity or similarity is compared over the entire length of the sequences, but can also be compared only for a portion of the sequences aligned with each other. Preferably, sequence identity or similarity is compared herein over the entire length of the sequences. In the art, "identity" or "similarity" also means the degree of sequence relatedness between polypeptide sequences or nucleic acid sequences, as the case can be, as determined by the match between these sequences. Sequence alignments can be generated by many software tools, for example:

[0103] Needleman and Wunsch algorithm - Needleman, Saul B. & Wunsch, Christian D. (1970). "A general method applicable to the search for similarities in the amino acid sequence of two proteins". Journal of Molecular Biology 48 (3): 443-453.

[0104] For example, the algorithm is implemented as the "NEEDLE" program which performs a global alignment of two sequences. The NEEDLE program is for example included in the European Molecular Biology Open Software Suite (EMBOSS).

[0105] EMBOSS - a collection of various programs: The European Molecular Biology Open Software Suite (EMBOSS), Trends in Genetics 16 (6), 276 (2000).

[0106] BLOSUM (BLOcks Substitution Matrix) - often generated based on alignments of conserved regions (e.g. protein domains) (Henikoff S, Henikoff JG: Amino acid substitution matrices from protein blocks. Proceedings of the National Academy of Sciences of the USA. 1992 Nov 15; 89(22): 10915-9). One of many BLOSUMs is "BLOSUM62", which is often the "default" setting for many programs when aligning protein sequences.

[0107] BLAST (Basic Local Alignment Search Tool) - consists of several individual programs (BlastP, BlastN) which are used primarily to search for similar sequences in large sequence databases. The BLAST programs also produce local alignments. Usually used is the "BLAST" interface provided by NCBI (National Center for Biotechnology Information) which is an improved version ("BLAST2"). "Original" BLAST: Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) "Basic local alignment search tool." J. Mol. Biol. 215:403-410; BLAST2: Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402.

[0108] Sequence identity as used herein is preferably the value determined by the EMBOSS pairwise alignment algorithm "Needle". In particular, the "Longest Identity" can be calculated using the NEEDLE program (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite - Rice, P., et al. Trends in Genetics (2000) 16: 276-277; http: / / emboss.bioinformatics.nl) in the EMBOSS package using the NOBRIEF option ('Brief identity and similarity' to NO). Identity, homology or similarity between two aligned sequences is calculated as follows: the number of positions at which the two sequences show the same amino acid divided by the total length of the alignment minus the total number of gaps in the alignment. For alignment of amino acid sequences, the default parameters are: Matrix = Blosum62; Open Gap Penalty = 10.0; Gap Extension Penalty = 0.5. For alignment of nucleic acid sequences, the default parameters are: Matrix = DNAfull; Open Gap Penalty = 10.0; Gap Extension Penalty = 0.5.

[0109] Sequence identity is usually provided as "% sequence identity" or "% identity". To determine the percent identity between two amino acid sequences, in a first step a pairwise sequence alignment is generated between the two sequences, wherein the two sequences are aligned over their entire, whole or full length (i.e. a pairwise global alignment). The alignment is generated using the programs or software described herein. The preferred alignment for the purposes of the present application is the one that enables the highest sequence identity.

[0110] The term sequence "identity" as used herein refers to the percent identity between two aligned sequences using the standard NCBI BLAST parameters (http: / / blast.ncbi.nlm.nih.gov).

[0111] The nucleic acid (or polynucleotide) of the present application comprises a nucleic acid sequence encoding the fusion protein of the present application. The nucleic acid sequence encoding the fusion protein of the present application is preferably a recombinant and / or isolated and / or purified nucleic acid sequence. The nucleic acid sequence encoding the fusion protein of the present application can be generated and isolated using known molecular biology standard techniques, the sequence information provided herein and the organism.

[0112] The term "nucleic acid" as used herein includes reference to a deoxyribonucleotide or ribonucleotide polymer (i.e., a polynucleotide) in either single- or double-stranded form, and unless otherwise indicated, includes known analogues of natural nucleotides that have similar binding properties, i.e., they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids). A polynucleotide can be a full-length or subsequence of a natural or heterologous structural gene or regulatory gene. Unless otherwise indicated, the term also includes reference to the specified sequence as well as the complementary sequence. Thus, DNA or RNA with a backbone that is modified is a "polynucleotide" as the term is used herein, as are DNA or RNA comprising rare bases (e.g., inosine) or modified bases (e.g., tritylated bases, to name just two examples). It is understood that a wide variety of modifications to DNA and RNA have been made to serve many useful purposes known to those skilled in the art. The term "polynucleotide" as used herein includes such chemically-modified, enzymatically-modified, or metabolically-modified forms of polynucleotides, as well as the characteristic chemical forms of DNA and RNA of viruses and cells, including simple and complex cells, among other things. Each nucleic acid sequence encoding a polypeptide (such as an oxidase or heme dioxygenase or heme dioxygenase reductase) herein also describes all possible silent variations of a nucleic acid by virtue of the genetic code. The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, if a conservative modification is used, then it can be one that encodes the same or a conservatively modified variant of an amino acid sequence due to the degeneracy of the genetic code. The term "degeneracy of the genetic code" refers to the fact that numerous functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons and the encoded polypeptide will be unchanged. Such nucleic acid variations are "silent variations," and represent a class of conservatively modified variations. The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues.

[0113] The terms "polypeptide," "peptide," and "protein" also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. Such naturally occurring amino acid analogues are those that have essentially the same properties as the corresponding naturally occurring amino acid, i.e., they are specifically reactive with an antibody induced in response to the protein consisting entirely of the naturally occurring amino acid. The terms "polypeptide," "peptide," and "protein" also encompass modifications, including but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation. In the context of the present application, oligomers (e.g., oligonucleotides, oligopeptides) are considered to be members of the group of polymers. Oligomers have relatively few monomeric units, typically 2-100, especially 6-100, including, for example, the primer sequences used in the Examples to clone oxidases or rubredoxin or rubredoxin reductase useful in the present application.

[0114] The term "heterologous," when used in reference to a nucleic acid (DNA or RNA) or protein of the present application, refers to a nucleic acid or protein that does not naturally occur as part of the organism, cell, genome, or DNA or RNA sequence in which it is present, or that is present in a cell or at a position or positions in the genome or DNA or RNA sequence that differ from that in which it is found in nature. A heterologous nucleic acid or protein of the present application is not endogenous to the cell, but is obtained from another cell, or is synthetically or recombinantly produced. Typically, although not necessarily, such a nucleic acid encodes a protein that is not normally produced by the cell in which the DNA is expressed. A gene that is endogenous to a particular host cell but is modified from its natural form, e.g., by use of DNA shuffling, is also referred to as heterologous. The term "heterologous" also includes non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the term "heterologous" can refer to a DNA segment that is foreign or heterologous to the cell, or that is homologous to the cell but is in a position and / or number of copies not normally found in the host cell nucleic acid. The foreign DNA segment is expressed to produce a foreign polypeptide.

[0115] A "homologous" DNA sequence of the present application is a DNA sequence that is naturally associated with the host cell into which it is introduced. One of skill in the art will recognize that any nucleic acid or protein that is heterologous or foreign in the cell in which it is expressed is encompassed by the term heterologous nucleic acid or protein herein.

[0116] The term "modified", "modification", "mutated" or "mutation" used herein with respect to a protein or polypeptide in comparison to another protein or polypeptide equally applies to a nucleotide or nucleic acid sequence. The term is used to indicate that the modified nucleotide or nucleic acid sequence encoding the protein or polypeptide has at least one difference in the nucleotide or nucleic acid sequence compared to the nucleotide or nucleic acid sequence of the protein or polypeptide with which it is compared. The term is used whether the modified or mutated protein has actually been obtained by mutagenesis of the nucleic acid encoding the amino acids or modification or otherwise of the polypeptide or protein, e.g. using artificial gene synthesis methods. Mutagenesis is a well known method in the art and includes, for example, site-directed mutagenesis, e.g. by PCR or by oligonucleotide-mediated mutagenesis, as described in Sambrook, J., and Russell, D.W. Molecular Cloning: A Laboratory Manual. 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (2001). The term "modified", "modification", "mutated" or "mutation" used herein with respect to a gene is used to indicate that the nucleotide sequence of the gene or its regulatory sequences differs in at least one nucleotide from the nucleotide sequence with which it is compared. The modification or mutation can in particular be a substitution of a nucleotide with a different nucleotide, a deletion of a nucleotide or an insertion of a nucleotide.

[0117] The terms "modified fHbp / modified fHbp fusion protein / recombinant protein / chimera / chimeric protein / chimeric molecule / chimeric antigen / fusion protein / clones" are used interchangeably throughout this specification and refer to a protein produced by joining two or more genes that originally encoded separate or identical proteins, resulting in a polypeptide containing a combination of sequences from different gene products or sources. Translation of such recombinant / chimeric / fusion genes results in a single polypeptide with functional properties derived from each of the original proteins and some additional features.

[0118] The term "tagged protein" as used herein refers to a protein with a specific peptide sequence (also called a tag) grafted to it. Tags serve various purposes and can be added to either end of the target protein, be C-terminal specific or N-terminal specific, or be specific for both the C- and N-termini. Some tags are also inserted at a site within the protein of interest; they are called internal tags.

[0119] Affinity tags are added to proteins so that they can be purified from their crude biological source using affinity techniques. Affinity tags include chitin binding protein (CBP), maltose binding protein (MBP), Strep tag and glutathione-S-transferase (GST). The poly(His) tag is a widely used protein tag that binds to matrices loaded with immobilized metal ions.

[0120] The term "peptide loop" as used herein is intended to mean a single chain polypeptide sequence anchored at both ends, for example to a scaffold such as fHbp. The term "loop" does not imply or require that the polypeptide adopts any particular secondary structure.

[0121] The term "foreign" as used herein in the context of "foreign peptide loop" is understood to mean that the peptide loop is derived from a different source relative to the fHbp protein (i.e. it is not fHbp or a fragment thereof). However, it can be from the same organism as fHbp. For example, a modified fHbp can comprise a fHbp from Neisseria meningitidis modified with a (foreign) peptide loop derived from Neisseria meningitidis PorA.

[0122] The term "isolated", when applied to a modified fHbp of the application, means that the protein: (i) is encoded by a nucleic acid using recombinant DNA methods or viral vectors; or (ii) is synthesized, for example by chemical synthesis methods; or (iii) is separated from biological material and then purified. An isolated polypeptide of the application includes a protein expressed from a nucleotide sequence encoding the protein or from a recombinant vector containing a nucleotide sequence encoding the protein.

[0123] The term "immunogenic" or "antigen" as used herein refers to a molecule capable of eliciting an immune response in a human or animal body. The immune response can be protective.

[0124] The term "protective" as used herein refers to prevention of disease, reduction in the risk of infection, transmission and / or progression of disease, reduction in the severity of disease, cure of a condition or disease, alleviation of symptoms or reduction in the severity of a disease or disease symptoms.

[0125] The term "prevention" as used herein refers to prevention of disease or protective treatment. Prevention can include reduction in the risk of infection, transmission and / or progression, or reduction in the severity of disease.

[0126] The term "treatment" as used herein refers to cure of a condition or disease, alleviation of symptoms or reduction in the severity of a disease or disease symptoms.

[0127] The term "freeze-drying / lyophilize / lyophilization" refers to the process of freezing a suspension / solution and then removing water by sublimation under low pressure.

[0128] The term "sublimation" refers to a change in the physical properties of a composition in which the composition changes directly from a solid state to a gaseous state without becoming a liquid.

[0129] The present disclosure contemplates a highly efficient platform process for manufacturing an effective vaccine against N. meningitidis that meets multiple criteria, including improved immunogenicity, safety, and affordability, in particular, improved formulation that shows low viscosity, no aggregation; shows long-term stability over a wide temperature range.

[0130] According to one aspect of the application, there is provided a modified Factor H binding protein (fHbp). In embodiments, the modified Factor H binding protein (fHbp) is a fusion protein and comprises a wild-type fHbp variant and at least one exogenous peptide loop.

[0131] In one embodiment, the fHbp is a meningococcal fHbp. In another embodiment, the fHbp is a gonococcal fHbp.

[0132] In embodiments, the modified Factor H binding protein (fHbp) has an amino acid sequence that is at least 75% identical to any one of SEQ ID NOs 6 to 10.

[0133] In embodiments, the modified Factor H binding protein (fHbp) comprises an amino acid sequence that is at least 75% identical to any one of SEQ ID NOs 6 to 10.

[0134] The skilled person will appreciate that one, two, three or four or more amino acid substitutions, deletions or additions can be made to the modified fHbp of the application herein without significantly removing its immunogenic function or affecting stability. Substitutions can be to similar amino acid residues, for example with similar MW, charge, hydrophobicity or moiety, or synthetic analogues. Such modifications are contemplated as part of the application.

[0135] In one embodiment, the modified fHbp can have at least 75.0%, or 80.0%, or 85.0%, 90.0%, 95.0%, 95.0%, 98.0%, 99.0%, or 99.5% identity to any of the modified fHbp sequences described herein.

[0136] In one embodiment, the modified Factor H binding protein (fHbp) is selected from an amino acid sequence that is at least 75.0%, or 80.0%, or 85.0%, 90.0%, 95.0%, 95.0%, 98.0%, 99.0%, or 99.5% identical to any one of SEQ ID NOs 6 to 10.

[0137] In one embodiment, the modified Factor H binding protein (fHbp) is selected from an amino acid sequence having at least 75.0% identity to the sequence of SEQ ID No 6. In a preferred embodiment, the modified Factor H binding protein (fHbp) is selected from an amino acid sequence having at least 75.0%, or 80.0%, or 85.0%, 90.0%, 95.0%, 95.0%, 98.0%, 99.0%, or 99.5% identity to the sequence of SEQ ID No 6.

[0138] In one embodiment, the modified Factor H binding protein (fHbp) is selected from an amino acid sequence having at least 75.0% identity to the sequence of SEQ ID No 7. In a preferred embodiment, the modified Factor H binding protein (fHbp) is selected from an amino acid sequence having at least 75.0%, or 80.0%, or 85.0%, 90.0%, 95.0%, 95.0%, 98.0%, 99.0%, or 99.5% identity to the sequence of SEQ ID No 7.

[0139] In one embodiment, the modified Factor H binding protein (fHbp) is selected from an amino acid sequence having at least 75.0% identity to the sequence of SEQ ID No 8. In a preferred embodiment, the modified Factor H binding protein (fHbp) is selected from an amino acid sequence having at least 75.0%, or 80.0%, or 85.0%, 90.0%, 95.0%, 95.0%, 98.0%, 99.0%, or 99.5% identity to the sequence of SEQ ID No 8.

[0140] In one embodiment, the modified Factor H binding protein (fHbp) is selected from an amino acid sequence having at least 75.0% identity to the sequence of SEQ ID No 9. In a preferred embodiment, the modified Factor H binding protein (fHbp) is selected from an amino acid sequence having at least 75.0%, or 80.0%, or 85.0%, 90.0%, 95.0%, 95.0%, 98.0%, 99.0%, or 99.5% identity to the sequence of SEQ ID No 9.

[0141] In one embodiment, the modified Factor H binding protein (fHbp) is selected from an amino acid sequence having at least 75.0% identity to the sequence of SEQ ID No 10. In a preferred embodiment, the modified Factor H binding protein (fHbp) is selected from an amino acid sequence having at least 75.0%, or 80.0%, or 85.0%, 90.0%, 95.0%, 95.0%, 98.0%, 99.0%, or 99.5% identity to the sequence of SEQ ID No 10.

[0142] According to embodiments of the application, the at least one exogenous peptide loop is immunogenic and is derived from a bacterial membrane protein.

[0143] In embodiments, the fHbp variant is selected from v1, v2 and v3 and is modified with at least one PorA loop inserted into a beta-turn region of the fHbp. In one embodiment, the fHbp can comprise fHbp v1. In another embodiment, the fHbp can comprise fHbp v2. In another embodiment, the fHbp can comprise fHbp v3.

[0144] In embodiments, the variant of wild-type fHbp comprises a wild-type meningococcal orthologue of fHbp. For example, the variant of fHbp can comprise Ghfp, the gonococcal homologue of fHbp. Ghfp is non-functional and is closely related to v3 fHbp (>95% aa identity, dissociation constant KD>100 mM with factor H).

[0145] In another embodiment, the PorA loop is selected from VR1 and VR2. In one embodiment, the PorA loop is VR1. In another embodiment, the PorA loop is VR2.

[0146] In embodiments, the modified factor H binding protein is modified to reduce factor H binding activity. In a preferred embodiment, the modified fHbp has >80% reduced binding to human factor H (fH) compared to wild-type fHbp.

[0147] According to another aspect of the application, there is provided a nucleic acid which essentially or at least encodes a modified fHbp according to the application herein.

[0148] In embodiments, the application relates to a nucleic acid sequence encoding a modified fHbp, wherein the nucleic acid sequence has at least 75.0%, or 80.0%, or 85.0%, 90.0%, 95.0%, 95.0%, 98.0%, 99.0%, or 99.5% identity to the sequence of any one of SEQ ID NOs. 1 to 5.

[0149] In one embodiment, the application relates to a nucleic acid sequence encoding a modified fHbp, wherein the nucleic acid sequence has at least 75.0% identity to the sequence of SEQ ID No 1. In a preferred embodiment, the application relates to a nucleic acid sequence encoding a modified fHbp, wherein the nucleic acid sequence has at least 75.0%, or 80.0%, or 85.0%, 90.0%, 95.0%, 95.0%, 98.0%, 99.0%, or 99.5% identity to the sequence of SEQ ID No 1.

[0150] In one embodiment, the present application relates to a nucleic acid sequence encoding a modified fHbp, wherein the nucleic acid sequence has at least 75.0% identity to the sequence of SEQ ID No 2. In a preferred embodiment, the present application relates to a nucleic acid sequence encoding a modified fHbp, wherein the nucleic acid sequence has at least 75.0%, or 80.0%, or 85.0%, 90.0%, 95.0%, 95.0%, 98.0%, 99.0%, or 99.5% identity to the sequence of SEQ ID No 2.

[0151] In one embodiment, the present application relates to a nucleic acid sequence encoding a modified fHbp, wherein the nucleic acid sequence has at least 75.0% identity to the sequence of SEQ ID No 3. In a preferred embodiment, the present application relates to a nucleic acid sequence encoding a modified fHbp, wherein the nucleic acid sequence has at least 75.0%, or 80.0%, or 85.0%, 90.0%, 95.0%, 95.0%, 98.0%, 99.0%, or 99.5% identity to the sequence of SEQ ID No 3.

[0152] In one embodiment, the present application relates to a nucleic acid sequence encoding a modified fHbp, wherein the nucleic acid sequence has at least 75.0% identity to the sequence of SEQ ID No 4. In a preferred embodiment, the present application relates to a nucleic acid sequence encoding a modified fHbp, wherein the nucleic acid sequence has at least 75.0%, or 80.0%, or 85.0%, 90.0%, 95.0%, 95.0%, 98.0%, 99.0%, or 99.5% identity to the sequence of SEQ ID No 4.

[0153] In one embodiment, the present application relates to a nucleic acid sequence encoding a modified fHbp, wherein the nucleic acid sequence has at least 75.0% identity to the sequence of SEQ ID No 5. In a preferred embodiment, the present application relates to a nucleic acid sequence encoding a modified fHbp, wherein the nucleic acid sequence has at least 75.0%, or 80.0%, or 85.0%, 90.0%, 95.0%, 95.0%, 98.0%, 99.0%, or 99.5% identity to the sequence of SEQ ID No 5.

[0154] In embodiments, a nucleic acid sequence encoding a modified fHbp of the present application is provided. In embodiments, the nucleic acid is a vector, such as a viral vector.

[0155] According to another aspect, the present application relates to an immunogenic composition comprising at least one modified fHbp as described herein.

[0156] In embodiments, the present application relates to an immunogenic composition comprising at least one modified fHbp as disclosed herein or at least one modified fHbp encoded by a nucleic acid sequence encoding a modified fHbp as disclosed herein.

[0157] In embodiments, an immunogenic composition comprising at least one modified fHbp of the present application is provided.

[0158] In embodiments, the present application relates to an immunogenic composition comprising at least one modified fHbp of the present application, wherein the modified fHbp comprises

[0159] at least one modified fHbp of an amino acid sequence that is at least 75% identical to any one of the amino acid sequences of SEQ ID NO. 6 to 10, or combinations thereof, or

[0160] at least one modified fHbp encoded by a nucleic acid sequence that is at least 75% identical to any one of the nucleic acid sequences of SEQ ID NO. 1 to 5, or combinations thereof.

[0161] In embodiments, the present application relates to an immunogenic composition comprising at least one modified fHbp of the present application, wherein the modified fHbp comprises

[0162] - fHbp V3.45 M5: PorA316-320 / exP1.14 (of SEQ ID NO. 6 or encoded by SEQ ID NO. 1), or

[0163] - fHbp V2.19 M6: PorA316-320 / exP1.4 (of SEQ ID NO. 7 or encoded by SEQ ID NO. 2), or

[0164] - fHbp V1.14: PorA307-311 / exP1.9 (of SEQ ID NO. 8 or encoded by SEQ ID NO. 3), or

[0165] - fHbp V1.1: PorA307-311 / exP1.4 (of SEQ ID NO. 9 or encoded by SEQ ID NO. 4), or

[0166] - fHbp V1.1: PorA307-311 / exP1.9 (of SEQ ID NO. 10 or encoded by SEQ ID NO. 5), or combinations thereof.

[0167] In another embodiment, the immunogenic composition comprises two or more different modified fHbpps.

[0168] In yet another embodiment, the immunogenic composition comprises three or more different modified fHbps.

[0169] In yet another embodiment, the immunogenic composition comprises four different modified fHbps.

[0170] In embodiments, the immunogenic composition comprises a pharmaceutically acceptable carrier. In further embodiments, the immunogenic composition comprises an adjuvant. In another embodiment, the immunogenic composition further comprises at least one other prophylactically or therapeutically active molecule.

[0171] The at least one other prophylactically or therapeutically active molecule comprises:

[0172] - a protein: capsular polysaccharide vaccine; or

[0173] - a conjugate vaccine, wherein an antigen comprising a fHbp scaffold loaded with an exogenous peptide loop is introduced as a protein carrier molecule in the conjugate vaccine.

[0174] The modified fHbp, nucleic acid or composition of the application can be used as a medicament, or for the treatment or prevention of a pathogen infection or colonization in a subject.

[0175] The present application also contemplates an immunogenic composition comprising a combination of a modified fHbp, a nucleic acid and at least one other prophylactically or therapeutically active molecule.

[0176] In embodiments, the at least one other prophylactically or therapeutically active molecule comprises a protein: capsular polysaccharide conjugate vaccine.

[0177] In embodiments, the protein: capsular polysaccharide vaccine comprises any one of a serogroup C or A capsular with a bacterial toxoid, a bivalent vaccine (conjugated with serogroup C and A capsular polysaccharides with a bacterial toxoid), a tetravalent- (serogroup A, C, Y, W polysaccharides conjugated with a bacterial toxoid) or a pentavalent- (serogroup A, C, Y, W, X polysaccharides conjugated with a bacterial toxoid) conjugate vaccine.

[0178] In another embodiment, the at least one other prophylactically or therapeutically active molecule comprises a conjugate vaccine, wherein an antigen comprising a fHbp scaffold loaded with an exogenous peptide loop is introduced as a protein carrier molecule in the conjugate vaccine, optionally wherein the conjugate vaccine comprises any serogroup capsular polysaccharide selected from A, C, Y, W or X strains, or a combination thereof.

[0179] In embodiments, a Factor H binding protein (fHbp) is used as an epitope display scaffold.

[0180] Fhbp found on the surface of N. meningitidis is a 27 kDa lipoprotein consisting of two beta barrels (N-terminal barrel and C-terminal barrel) connected by a short amino acid linker. While the charged carbohydrates on the surface of vascular endothelium engage fH, the charged amino acids in fHbp bind to the same site on fH with nanomolar affinity. Based on differences in nucleotide and predicted amino acid sequences, fHbp from different N. meningitidis strains are classified using several schemes. Factor H binding protein (fHbp, also known in the art as lipoprotein 2086 (Fletcher et al (2004) Infect Immun 72:2088-2100), genome-derived Neisserial antigen (GNA) 1870 (Masignani et al. (2003) J Exp Med 197:789-99) or "741") is a surface-exposed lipoprotein expressed in N. meningitidis bacteria. Based on differences in nucleotide and predicted amino acid sequences, fHbp from different N. meningitidis strains are classified using several schemes. These include two subfamilies (A and B) (Murphy E, et al. (2009) The Journal of Infectious Diseases 200: 379- 389) or three variant groups (V1, V2 and V3) (Masignani V, et al. (2003) The Journal of Experimental Medicine 197: 789-799), where subfamily A corresponds to V2 and V3, and subfamily B corresponds to V1 (which is the most abundant).

[0181] fHbp belonging to the same variant group have more than 85% amino acid similarity between them, while there is only 60-70% similarity between the three variant groups. fHbp is also an antigen that elicits serum bactericidal antibody responses in immunized individuals, and is a key component of investigational vaccines currently being evaluated in clinical trials for the prevention of meningococcal disease (particularly serogroup B). However, immunization with proteins belonging to one variant fHbp family generates variant-specific responses, with no cross-reactivity with other variant groups. Thus, a single fHbp cannot provide universal protection against meningococcal disease, and thus immunization with representatives from each of the three variants V1, V2 and V3 is necessary for a broad-based vaccine.

[0182] Meningococcal outer membrane vesicles (OMVs) are PorA, an integral outer membrane protein (OMP) in Neisseria meningitidis. However, the sequence of this protein is diverse and the prevalence of specific variants varies by geographic region. Variants of PorA are identified by sequence in the variable region (VR) of the protein, which is located in a surface-exposed peptide loop and is the target of the immune response. PorA has seven extracellular peptide loops; the fourth loop is variable region 2 (VR2) and is the target of most serum bactericidal activity (SBA) generated by PorA following natural infection and immunization with OMVs. SBA is a correlate of known protection against meningococcal disease. Despite the sequence diversity, approximately 70% of UK isolates are covered by a vaccine comprising six PorA proteins (http: / / pubmlst.org / neisseria / PorA / ).

[0183] It has been shown herein that immunogenic peptides, such as those from PorA, can be introduced into Factor H binding protein (fHbp) that acts as a molecular scaffold. The peptides introduced into fHbp are presented to the immune system and are capable of eliciting a protective response, such as SBA. Advantageously, the fHbp molecule provides an ideal molecular scaffold for stabilizing peptide loops for epitope display, particularly for epitopes that are difficult to stabilize and display in their native conformation, such as loops from integral OMPs, such as PorA. In particular, many OMPs, such as PorA, are difficult to express due to insolubility of their transmembrane domains. PorA has a 16-β-strand barrel structure, with surface-exposed loops between strands 1 and 2 (loop 1), strands 7 and 8 (loop 4), strands 9 and 10 (loop 5), and strands 11 and 12 (loop 7) proving to be the most effective antigens. fHbp contains two β barrels, thus peptide loop sequences from OMPs can be inserted into the tips of the loops between the β-strands of fHbp to present extracellular loop fragments from integral OMPs in their native conformation for immunization. Thus, the modified fHbp scaffold molecules of the present invention can be used as prophylactic or therapeutic vaccines against Nm or gonococci, with a single protein presenting key epitopes from two different antigens.

[0184] In one embodiment, the modified fHbp is a fusion protein, such as a recombinant fusion protein. In another embodiment, the modified fHbp is an isolated modified fHbp molecule. In yet another embodiment, the modified fHbp molecule of the present invention is included in a multivalent vaccine as a single protein. In yet another embodiment, the modified fHbp is included in an OMV vaccine.

[0185] In embodiments where more than one exogenous peptide loop is inserted into fHbp or a variant thereof, the exogenous peptide loops are the same, e.g., the same sequence, or substantially similar. For example, some epitopes (e.g., PorA epitopes) can not elicit a sufficient functional response when presented alone on fHbp. In such cases, the present application can be used to provide the same epitope at multiple sites on the same modified fHbp molecule, thereby enhancing the immunological recognition of the epitope. Alternatively, the exogenous peptide loops are different from one another. For example, where the exogenous peptide loops are derived from a single protein (e.g., PorA), the different exogenous peptide loops are from different regions of the protein (e.g., PorA). In one embodiment, the different exogenous peptide loops are derived from overlapping and different regions of the protein (e.g., PorA). In embodiments where more than one exogenous peptide loop is inserted into fHbp or a variant thereof, the exogenous peptide loops are derived from different species or strains. For example, when a multivalent vaccine against multiple different antigens, including different organisms, is desired.

[0186] In another aspect of the present application, an effective vaccine formulation is developed comprising at least one recombinant protein / modified fHbp fusion protein, an adjuvant and one or more pharmaceutically acceptable excipients.

[0187] In embodiments, the vaccine formulation comprising at least one recombinant protein / modified fHbp fusion protein additionally comprises an antigen selected from the group consisting of antigenic peptide subvariants fHbp 3.45, fHbp 1.55, fHbp-fHbp-fHbp or any other fHbp fusion protein, fHbp-cholera toxin, multiple PorA with single fHbp fusion, recombinant Neisseria meningitidis group B NHBA fusion protein, recombinant Neisseria meningitidis group B NadA protein, recombinant Neisseria meningitidis group B fHbp fusion protein or outer membrane vesicles (OMV) from Neisseria meningitidis group B strain NZ98 / 254.

[0188] In one embodiment, the recombinant protein can comprise transferrin binding protein, Neisserial heparin binding protein, Neisserial surface protein A, PorA, meningococcal enterobactin receptor FetA, Neisserial adhesin A or Factor H binding protein (fHbp).

[0189] More preferably, the recombinant protein / modified fHbp fusion protein can comprise a modified Factor H binding protein (fHbp) comprising fHbp or a variant thereof modified by the addition of at least one exogenous peptide loop from a different antigen to act as a molecular scaffold.

[0190] More preferably, the recombinant protein / modified fHbp fusion protein can comprise a modified Factor H binding protein (fHbp) comprising fHbp or a variant thereof modified by the addition of at least two exogenous peptide loops from different antigens to act as a molecular scaffold.

[0191] For example, the vaccine formulation can comprise a fHbp variant selected from v1 or v2 or v3. The formulation can comprise a fHbp variant selected from v1 and v2. The formulation can comprise a fHbp variant selected from v2 and v3. The formulation can comprise a fHbp variant selected from v1, v2 and v3.

[0192] The recombinant protein / modified fHbp fusion protein is a fHbp variant selected from v1, v2 and v3 modified with at least one PorA loop comprising at least 10 amino acids inserted into a beta-turn region in fHbp. In embodiments, the PorA loop is selected from VR1 and VR2 and has a reduction in binding to human factor H (fH) of >80% compared to wild-type fHbp.

[0193] All activities relating to the generation of recombinant modified factor H binding proteins (fHbp) were carried out at Oxford University Innovation Limited. The contents of PCT / GB2013 / 052215 and PCT / GB2017 / 052535 are incorporated herein in their entirety.

[0194] In embodiments, a vaccine formulation is developed against N. meningitidis wherein the vaccine formulation can comprise one or more recombinant protein / modified fHbp fusion proteins derived from N. meningitidis and a pharmaceutically acceptable carrier or excipient.

[0195] In one embodiment, the recombinant protein / modified fHbp fusion protein is derived from N. meningitidis serogroups A, B, C, H, I, K, L, 29E, W135, X, Y and Z.

[0196] In a preferred embodiment, the recombinant protein / modified fHbp fusion protein is derived from N. meningitidis serogroup B.

[0197] In one embodiment, the vaccine formulation can comprise one or more different modified factor H binding protein (fHbp) variants as antigens selected from the group consisting of:

[0198]

[0199]

[0200] In embodiments, the recombinant protein / modified fHbp fusion protein has a molecular weight in the range of 10 kDa to 200 kDa, preferably up to 50 kDa.

[0201] In an embodiment, the recombinant protein / modified fHbp fusion protein has a molecular weight in the range of 20 kDa to 40 kDa. In one embodiment, the formulation can comprise four different modified factor H binding protein (fHbp) variants selected from the group summarized in Table 4.

[0202]

[0203] In one embodiment, the formulation can comprise four different modified factor H binding protein (fHbp) variants selected from Table 1 as antigens.

[0204] In one embodiment, the recombinant protein / modified fHbp fusion protein is adsorbed on adjuvants to improve the immunogenicity of the antigen and maximize protection against the related disease.

[0205] In one embodiment, the adsorption of the recombinant protein / modified fHbp fusion protein on adjuvants is evaluated and optimized for the percentage of adsorption on adjuvants. According to the embodiments of the present application, adsorption buffers are used to improve surface adsorption, reduce aggregation and unfolding upon binding, and reduced potency of the recombinant protein / modified fHbp fusion protein as an antigen after adsorption.

[0206] In one embodiment, the recombinant protein / modified fHbp fusion protein is adsorbed on an adjuvant selected from an aluminum adjuvant base salt such as aluminum hydroxide, aluminum phosphate, aluminum hydroxyl phosphate, and potassium aluminum sulfate, or an immunostimulatory component-based adjuvant selected from oil and water emulsions (MF-59, liposomes, lipopolysaccharides, saponins, lipid A, lipid A derivatives, monophosphoryl lipid A, GLA, 3-deacylated monophosphoryl lipid A, AS01, AS03, AF3) (including all 3 MPLA suppliers we are exploring, including synthetic etc.; MPL from Salmonella enterica serovar Minnesota Re 595) (e.g., Sigma Aldrich Catalog # L6895)), IL-2, RANTES, GM-CSF, TNF-a, IFN-g, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L, and 41BBL, 3-deacylated monophosphoryl lipid A, AS01, AS03, AF3), oligonucleotides, oligonucleotides containing at least one unmethylated CpG, and / or liposomes, Freund's adjuvant, Freund's complete adjuvant, Freund's incomplete adjuvant, polymers, copolymers such as polyoxyethylene-polyoxypropylene copolymers, including block copolymers, polymeric p 1005, CRL-8300 adjuvant, muramyl dipeptide, agonists of the following, for example: TLR1 / 2 (which can be a synthetic ligand) (e.g., Pam3Cys), TLR2 (e.g., CFA, Pam2Cys), TLR3 (e.g., polyFC, poly A:U), TLR-4 agonists (e.g., MPLA, lipid A, and LPS), TLR5 (e.g., flagellin), TLR7 (e.g., gardiquimod, imiquimod, loxoribine, resiquimod®), TLR7 / 8 (e.g., R0848), TLR8 (e.g., imidazoquinoline, ssPolyU, 3M-012), TLR9 (e.g., ODN 1826 (Type B), ODN 2216 (Type A), and / or TLR11 / 12 (e.g., inhibitory proteins), TLR-4 agonists, flagellin, flagellin derived from Gram-negative bacteria, TLR-5 agonists, fragments of flagellin capable of binding to TLR-5 receptors, Alpha-C-galactosylceramide, chitosan, interleukin-2, QS-21, ISCOMS, squalene mixtures (SAF-1), Quil A, cholera toxin B subunit, polyphosphazene and its derivatives, mycobacterial cell wall preparations, mycolic acid derivatives, non-ionic block copolymer surfactants, OMVs, fHbp, combinations of saponins with sterols and lipids, TLR-agonists (MPL, CpG, poly-IC, imiquimod), dmLT, 1,25-dihydroxyvitamin D3, CAF01, poly[di(carboxylatophenoxy)-phosphazene] (PCPP), and Venezuelan equine encephalitis (VEE) replicon particles, or combinations thereof.In an embodiment, the amount of adjuvant is in the range of 0.5 mg / ml to 4.5 mg / ml.

[0207] In an embodiment, the adjuvant is aluminium hydroxide having a particle size of greater than 500 nm.

[0208] In an embodiment, the percentage of adsorption of the recombinant protein / modified fHbp fusion protein adsorbed on the adjuvant is in the range of 70% to 100%.

[0209] In another embodiment, fHbp V3.45 M5 PorA 316-320 exP1.14 The percentage of adsorption adsorbed on the adjuvant is in the range of 80% to 100%.

[0210] In another embodiment, fHbp V1.14 PorA 307-311 exP1.9 The percentage of adsorption adsorbed on the adjuvant is in the range of 80% to 90%.

[0211] In another embodiment, fHbp V2.19 PorA 316-320 exP1.4 The percentage of adsorption adsorbed on the adjuvant is in the range of 80% to 90%.

[0212] In another embodiment, fHbp V1.1 PorA 307-311 exP1.4 The percentage of adsorption adsorbed on the adjuvant is in the range of 80% to 90%.

[0213] In another embodiment, fHbp V1.1 PorA 307-311 exP1.9 The percentage of adsorption adsorbed on the adjuvant is in the range of 70% to 80%.

[0214] In an embodiment, the pharmaceutically acceptable carrier or excipient can be selected from a buffer, a sugar, a sugar alcohol or polyol, a surfactant, a polymer, a salt, an amino acid or a pH adjusting agent, a hydrolyzed protein, a preservative, and a liquid carrier.

[0215] Examples of a buffering agent are selected from the group consisting of carbonates, phosphates, acetates, HEPES, succinates, TRIS, borates, citrates, lactates, gluconates, and tartrates, and more complex organic buffers including phosphate buffers containing sodium and / or potassium phosphates in proportions selected to achieve a desired pH. In another example, the buffering agent contains tris(hydroxymethyl)aminomethane, or "Tris", which is formulated to achieve a desired pH. In yet another example, the buffering agent can be minimal essential medium containing Hanks salts. Other buffering agents are contemplated by the present disclosure, such as HEPES, piperazine-N,N'-bis(PIPES), and 2-ethanesulfonic acid (MES). The buffering agent helps to stabilize the recombinant protein / modified fHbp fusion protein of the present disclosure. In embodiments, the amount of buffering agent is in the range of 0.1 mM to 300 mM.

[0216] Examples of a sugar as an excipient are selected from the group consisting of trehalose, mannose, raffinose, lactobionic acid, glucose, maltitol, isomaltitol, maltose, lactose, dextrose, fructose, or combinations thereof. In embodiments, the amount of sugar is in the range of 5 mg / ml to 100 mg / ml.

[0217] Examples of a sugar alcohol or polyol as an excipient are selected from the group consisting of mannitol, lactitol, sorbitol, glycerol, xylitol, maltitol, lactitol, erythritol, isomaltitol, and hydrogenated starch hydrolysate, or combinations thereof. In embodiments, the amount of sugar alcohol or polyol is in the range of 5 mg / ml to 100 mg / ml.

[0218] Surfactants can be classified according to their 'HLB' (hydrophilic / lipophilic balance). Preferred surfactants of the present invention have an HLB of at least 10, preferably at least 15, and more preferably at least 16. Examples of surfactants as an excipient can include non-ionic surfactants such as polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 85, nonoxynol 9, octoxynol 40, octoxynol 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyl-660 hydroxystearate, polyoxyl-35 castor oil, and poloxamer - 0.001% - 0.05%, in DOWFAX 2B® (Dow Chemical Company), and the like. In embodiments, the amount of surfactant is in the range of 0.001% to 0.05%. TMcopolymer of ethylene oxide (EO), propylene oxide (PO) and / or butylene oxide (BO) sold under the trade name Pluronic®, such as linear EO / PO block copolymers; octoxynol, which can vary in the number of repeating ethoxy (oxy-1,2-ethanediyl) groups, with octoxynol-9 (Triton X-100, or t-octylphenoxy polyethoxyethanol) of particular interest; (octylphenoxy)polyethoxyethanol (IGEPAL CA-630 / NP-40); phospholipids, such as phosphatidylcholine (lecithin); nonylphenol ethoxylates, such as Tergitol NP series; polyoxyethylene fatty ether derived from lauryl alcohol, cetyl alcohol, stearyl alcohol, and oleyl alcohol (known as Brij surfactants), such as triethylene glycol monolauryl ether (Brij 30); and sorbitan esters (commonly known as SPANs), such as sorbitan trioleate (Span 85) and sorbitan monolaurate. Non-ionic surfactants are preferred. A preferred surfactant for inclusion in the emulsion is Tween 20. In embodiments, the amount of surfactant is in the range of 0.01 mg / ml to 2 mg / ml. Examples of polymers can include dextran, carboxymethylcellulose, hyaluronic acid, cyclodextrin, and the like. TM NP series; polyoxyethylene fatty ether derived from lauryl alcohol, cetyl alcohol, stearyl alcohol, and oleyl alcohol (known as Brij surfactants), such as triethylene glycol monolauryl ether (Brij 30); and sorbitan esters (commonly known as SPANs), such as sorbitan trioleate (Span 85) and sorbitan monolaurate. Non-ionic surfactants are preferred. A preferred surfactant for inclusion in the emulsion is Tween 20. In embodiments, the amount of surfactant is in the range of 0.01 mg / ml to 2 mg / ml. Examples of polymers can include dextran, carboxymethylcellulose, hyaluronic acid, cyclodextrin, and the like.

[0219] Examples of salts can include NaCl, KCl, KH2PO4, Na2HPO4.2H2O, CaCl2, MgCl2, and the like.

[0220] Examples of amino acids as excipients can include tricine, leucine, isoleucine, glycine, glutamine, L-arginine, L-arginine hydrochloride, lysine, L-alanine, tryptophan, phenylalanine, tyrosine, valine, cysteine, glycine, methionine, proline, serine, threonine.

[0221] Examples of hydrolyzed proteins can include gelatin, lactalbumin hydrolysate, monosodium glutamate, collagen hydrolysate, keratin hydrolysate, peptides, casein hydrolysate, and whey protein hydrolysate, serum albumin.

[0222] Examples of preservatives can include 2-phenoxyethanol, benzethonium chloride (Phemerol), phenol, m-cresol, thiomersal, formaldehyde, parabens (e.g. methyl-, ethyl-, propyl- or butyl-parabens), benzalkonium chloride, benzyl alcohol, chlorobutanol, p-chloro-m-cresol, or benzyl alcohol or combinations thereof. The vaccine composition can include a preservative for a single immunisation or can include material for multiple immunisations (i.e. a "multi-dose" kit). It is preferred to include a preservative in a multi-dose arrangement. Alternatively (or additionally) to including a preservative in a multi-dose composition, the composition can be contained in a container with a sterile adaptor for removal of the material. In embodiments, the amount of preservative is in the range of 1 mg / mL to 10 mg / ml.

[0223] Examples of suitable liquid carriers can include WFI (water for injection) and saline.

[0224] In embodiments, the vaccine formulation comprises:

[0225] - at least one recombinant protein / at least one modified fHbp;

[0226] - aluminium hydroxide;

[0227] - mannitol;

[0228] - phosphate; and

[0229] - polysorbate.

[0230] In another embodiment, the vaccine formulation comprises:

[0231] - at least one modified fHbp having an amino acid sequence that is at least 75% identical to any one of the amino acid sequences of SEQ ID NO. 6 to 10; or

[0232] - at least one modified fHbp encoded by a nucleic acid sequence that is at least 75% identical to any one of the nucleic acid sequences of SEQ ID NO. 1 to 5; and

[0233] - aluminium hydroxide;

[0234] - mannitol;

[0235] - phosphate; and

[0236] - polysorbate.

[0237] According to embodiments of the application, the vaccine formulation is formulated in a dose in the range of 0.1 mL to 1 mL, for example from 0.2 mL to 0.8 mL, 0.4 mL to 0.6 mL. In one embodiment, the vaccine formulation is formulated in a dose of 0.5 mL.

[0238] According to embodiments of the application, the recombinant protein / modified fHbp fusion protein is present in the vaccine formulation in an amount ranging from 15 μg / ml to 200 μg / ml, e.g., from 20 μg / ml to 200 μg / ml, 25 μg / ml to 200 μg / ml, 25 μg / ml to 150 μg / ml, 30 μg / ml to 200 μg / ml, 30 μg / ml to 150 μg / ml, 35 μg / ml to 200 μg / ml, 35 μg / ml to 150 μg / ml, 40 μg / ml to 200 μg / ml, 40 μg / ml to 150 μg / ml, 45 μg / ml to 200 μg / ml, 45 μg / ml to 150 μg / ml, 50 μg / ml to 200 μg / ml, 50 to 150 μg / ml, 75 μg / ml to 200 μg / ml, 75 to 150 μg / ml, 120 μg / ml to 200 μg / ml, 120 μg / ml to 150 μg / ml.

[0239] According to embodiments of the application, the recombinant protein / modified fHbp fusion protein is present in the vaccine formulation in an amount of 15 μg / ml, 20 μg / ml, 25 μg / ml, 30 μg / ml, 35 μg / ml, 40 μg / ml, 45 μg / ml, 50 μg / ml, 55 μg / ml, 60 μg / ml, 65 μg / ml, 70 μg / ml, 75 μg / ml, 80 μg / ml, 85 μg / ml, 90 μg / ml, 95 μg / ml, 100 μg / ml, 105 μg / ml, 110 μg / ml, 115 μg / ml, 120 μg / ml, 125 μg / ml, 130 μg / ml, 135 μg / ml, 140 μg / ml, 145 μg / ml, 150 μg / ml, 200 μg / ml.

[0240] In embodiments, the vaccine formulation comprises (i) at least one recombinant protein / modified fHbp fusion protein; (ii) aluminum hydroxide in an amount ranging from 0.5 mg / ml to 4.5 mg / ml; (iii) mannitol in an amount ranging from 5 mg / ml to 100 mg / ml; (iv) phosphate buffer in an amount ranging from 1 mM to 10 mM; and (v) polysorbate 20 in an amount ranging from 0.01 mg / ml to 2 mg / ml; wherein each recombinant protein / modified fHbp fusion protein is present in an amount ranging from 15 μg / ml to 150 μg / ml.

[0241] In an embodiment, the vaccine formulation comprises (i) at least two recombinant protein / modified fHbp fusion proteins; (ii) aluminum hydroxide in an amount ranging from 0.5 mg / ml to 4.5 mg / ml; (iii) mannitol in an amount ranging from 5 mg / ml to 100 mg / ml; (iv) phosphate buffer in an amount ranging from 1 mM to 10 mM; and (v) polysorbate 20 in an amount ranging from 0.01 mg / ml to 2 mg / ml; wherein each recombinant protein / modified fHbp fusion protein is present in an amount ranging from 15 pg / ml to 150 pg / ml.

[0242] In an embodiment, the vaccine formulation comprises (i) at least three recombinant protein / modified fHbp fusion proteins; (ii) aluminum hydroxide in an amount ranging from 0.5 mg / ml to 4.5 mg / ml; (iii) mannitol in an amount ranging from 5 mg / ml to 100 mg / ml; (iv) phosphate buffer in an amount ranging from 1 mM to 10 mM; and (v) polysorbate 20 in an amount ranging from 0.01 mg / ml to 2 mg / ml; wherein each recombinant protein / modified fHbp fusion protein is present in an amount ranging from 15 pg / ml to 150 pg / ml.

[0243] In an embodiment, the vaccine formulation comprises (i) at least four recombinant protein / modified fHbp fusion proteins; (ii) aluminum hydroxide in an amount ranging from 0.5 mg / ml to 4.5 mg / ml; (iii) mannitol in an amount ranging from 5 mg / ml to 100 mg / ml; (iv) phosphate buffer in an amount ranging from 1 mM to 10 mM; and (v) polysorbate 20 in an amount ranging from 0.01 mg / ml to 2 mg / ml; wherein each recombinant protein / modified fHbp fusion protein is present in an amount ranging from 15 pg / ml to 150 pg / ml.

[0244] In an embodiment, the vaccine formulation comprises:

[0245] at least one modified fHbp having an amino acid sequence which is at least 75% identical to any one of the amino acid sequences SEQ ID NO. 6 to 10; or

[0246] at least one modified fHbp encoded by a nucleic acid sequence which is at least 75% identical to any one of the nucleic acid sequences SEQ ID NO. 1 to 5;

[0247] and

[0248] o aluminum hydroxide;

[0249] o mannitol;

[0250] o phosphate; and

[0251] o polysorbate.

[0252] In embodiments, the vaccine formulation comprises:

[0253] - at least one modified fHbp of an amino acid sequence that is at least 75% identical to any one of the amino acid sequences of SEQ ID NO. 6 to 10; or

[0254] - at least one modified fHbp encoded by a nucleic acid sequence that is at least 75% identical to any one of the nucleic acid sequences of SEQ ID NO. 1 to 5; and

[0255] - aluminium hydroxide in an amount ranging from 0.5 mg / ml to 4.5 mg / ml;

[0256] - mannitol in an amount ranging from 5 mg / ml to 100 mg / ml;

[0257] - phosphate buffer in an amount ranging from 1 mM to 10 mM; and

[0258] - polysorbate 20 in an amount ranging from 0.01 mg / ml to 2 mg / ml.

[0259] In embodiments, the vaccine formulation comprises:

[0260] - (fHbp V3.45 M5:PorA316-320 / exP1.14 of SEQ ID NO. 6 or encoded by SEQ ID NO. 1) in an amount ranging from 15 pg / ml to 150 pg / ml; or

[0261] - (fHbp V2.19 M6:PorA316-320 / exP1.4 of SEQ ID NO. 7 or encoded by SEQ ID NO. 2) in an amount ranging from 15 pg / ml to 150 pg / ml; or

[0262] - (fHbp V1.14:PorA307-311 / exP1.9 of SEQ ID NO. 8 or encoded by SEQ ID NO. 3) in an amount ranging from 15 pg / ml to 150 pg / ml; or

[0263] - (fHbp V1.1:PorA307-311 / exP1.4 of SEQ ID NO. 9 or encoded by SEQ ID NO. 4) in an amount ranging from 15 pg / ml to 150 pg / ml; and

[0264] - aluminium hydroxide in an amount ranging from 0.5 mg / ml to 4.5 mg / ml;

[0265] - mannitol in an amount ranging from 5 mg / ml to 100 mg / ml;

[0266] - phosphate buffer in an amount ranging from 1 mM to 10 mM; and

[0267] - polysorbate 20 in an amount ranging from 0.01 mg / ml to 2 mg / ml.

[0268] In embodiments, the vaccine formulation comprises:

[0269] - (fHbp V3.45 M5:PorA316-320 / exP1.14 of SEQ ID NO. 6 or encoded by SEQ ID NO. 1) in an amount ranging from 15 pg / ml to 150 pg / ml;

[0270] - (fHbp V2.19 M6:PorA316-320 / exP1.4 of SEQ ID NO. 7 or encoded by SEQ ID NO. 2) in an amount ranging from 15 pg / ml to 150 pg / ml;

[0271] - (fHbp V1.14:PorA307-311 / exP1.9 of SEQ ID NO. 8 or encoded by SEQ ID NO. 3) in an amount ranging from 15 pg / ml to 150 pg / ml;

[0272] - (fHbp V1.1 :PorA307-311 / exP1.4 of SEQ ID NO. 9 or encoded by SEQ ID NO. 4) in an amount ranging from 15 pg / ml to 150 pg / ml;

[0273] - aluminium hydroxide in an amount ranging from 0.5 mg / ml to 4.5 mg / ml;

[0274] - mannitol in an amount ranging from 5 mg / ml to 100 mg / ml;

[0275] - phosphate buffer in an amount ranging from 1 mM to 10 mM; and

[0276] - polysorbate 20 in an amount ranging from 0.01 mg / ml to 2 mg / ml.

[0277] In embodiments, the vaccine formulation comprises:

[0278] - (SEQ ID NO. 6 or encoded by SEQ ID NO. 1 ) fHbp V3.45 M5:PorA316-320 / exP1.14 in an amount ranging from 15 pg / ml to 150 pg / ml; or

[0279] - (SEQ ID NO. 7 or encoded by SEQ ID NO. 2) fHbp V2.19 M6:PorA316-320 / exP1.4 in an amount ranging from 15 pg / ml to 150 pg / ml; or

[0280] - (SEQ ID NO. 10 or encoded by SEQ ID NO. 5) fHbp V1.1 :PorA307-311 / exP1.9 in an amount ranging from 15 pg / ml to 150 pg / ml; or

[0281] - (SEQ ID NO. 8 or encoded by SEQ ID NO. 3) fHbp V1.14:PorA307-311 / exP1.9 in an amount ranging from 15 pg / ml to 150 pg / ml; and

[0282] - aluminium hydroxide in an amount ranging from 0.5 mg / ml to 4.5 mg / ml;

[0283] - mannitol in an amount ranging from 5 mg / ml to 100 mg / ml;

[0284] - phosphate buffer in an amount ranging from 1 mM to 10 mM; and

[0285] - polysorbate 20 in an amount ranging from 0.01 mg / ml to 2 mg / ml.

[0286] In embodiments, the vaccine formulation comprises:

[0287] - (SEQ ID NO. 6 or encoded by SEQ ID NO. 1 ) fHbp V3.45 M5:PorA316-320 / exP1.14 in an amount ranging from 15 pg / ml to 150 pg / ml; or

[0288] - (SEQ ID NO. 7 or encoded by SEQ ID NO. 2) fHbp V2.19 M6:PorA316-320 / exP1.4 in an amount ranging from 15 pg / ml to 150 pg / ml; or

[0289] - (SEQ ID NO. 10 or encoded by SEQ ID NO. 5) fHbp V1.1 :PorA307-311 / exP1.9 in an amount ranging from 15 pg / ml to 150 pg / ml;

[0290] - (SEQ ID NO. 8 or encoded by SEQ ID NO. 3) fHbp V1.14:PorA307-311 / exP1.9 in an amount ranging from 15 pg / ml to 150 pg / ml;

[0291] - Aluminium hydroxide in an amount ranging from 0.5 mg / ml to 4.5 mg / ml;

[0292] - Mannitol in an amount ranging from 5 mg / ml to 100 mg / ml;

[0293] - Phosphate buffer in an amount ranging from 1 mM to 10 mM; and

[0294] - Polysorbate 20 in an amount ranging from 0.01 mg / ml to 2 mg / ml.

[0295] In embodiments, the vaccine formulation comprises 2-phenoxyethanol in an amount ranging from 1 mg / mL to 10 mg / ml.

[0296] According to embodiments of the application, the vaccine formulation comprises (i) at least one fusion protein comprising a stabilized non-functional / non-lipidated fHbp and a PorA VR2 loop, and (ii) at least one polysaccharide-protein conjugate.

[0297] In embodiments, the vaccine formulation comprises (i) at least one fusion protein comprising a stabilized non-functional / non-lipidated fHbp and a PorA VR2 loop, and at least one conjugate selected from the group consisting of: (a) a conjugate of (i) a capsular polysaccharide of serogroup A N. meningitidis and (ii) tetanus toxoid; (b) a conjugate of (i) a capsular polysaccharide of serogroup C N. meningitidis and (ii) CRM197; (c) a conjugate of (i) a capsular polysaccharide of serogroup Y N. meningitidis and (ii) CRM197; (d) a conjugate of (i) a capsular polysaccharide of serogroup W135 N. meningitidis and (ii) CRM197; and (d) a conjugate of (i) a capsular polysaccharide of serogroup X N. meningitidis and (ii) tetanus toxoid.

[0298] In embodiments, the vaccine formulation comprises (i) at least two fusion proteins, each fusion protein consisting of one fHbp variant type coupled to one PorA VR2 loop, and at least one conjugate selected from the group consisting of: (a) a conjugate of (i) a capsular polysaccharide of serogroup A N. meningitidis and (ii) tetanus toxoid; (b) a conjugate of (i) a capsular polysaccharide of serogroup C N. meningitidis and (ii) CRM197; (c) a conjugate of (i) a capsular polysaccharide of serogroup Y N. meningitidis and (ii) CRM197; (d) a conjugate of (i) a capsular polysaccharide of serogroup W135 N. meningitidis and (ii) CRM197; and (d) a conjugate of (i) a capsular polysaccharide of serogroup X N. meningitidis and (ii) tetanus toxoid.

[0299] In embodiments, the vaccine formulation comprises (i) at least three fusion proteins, each fusion protein consisting of one fHbp variant type coupled to two PorA VR2 loops, and at least one conjugate selected from the group consisting of: (a) a conjugate of (i) a capsular polysaccharide of serogroup A N. meningitidis and (ii) tetanus toxoid; (b) a conjugate of (i) a capsular polysaccharide of serogroup C N. meningitidis and (ii) CRM197; (c) a conjugate of (i) a capsular polysaccharide of serogroup Y N. meningitidis and (ii) CRM197; (d) a conjugate of (i) a capsular polysaccharide of serogroup W135 N. meningitidis and (ii) CRM197; and (d) a conjugate of (i) a capsular polysaccharide of serogroup X N. meningitidis and (ii) tetanus toxoid.

[0300] In embodiments, the vaccine formulation comprises (i) at least four fusion proteins, each fusion protein consisting of one fHbp variant type coupled to three PorA VR2 loops, and at least one conjugate selected from the group consisting of: (a) a conjugate of (i) a capsular polysaccharide of serogroup A N. meningitidis and (ii) tetanus toxoid; (b) a conjugate of (i) a capsular polysaccharide of serogroup C N. meningitidis and (ii) CRM197; (c) a conjugate of (i) a capsular polysaccharide of serogroup Y N. meningitidis and (ii) CRM197; (d) a conjugate of (i) a capsular polysaccharide of serogroup W135 N. meningitidis and (ii) CRM197; and (d) a conjugate of (i) a capsular polysaccharide of serogroup X N. meningitidis and (ii) tetanus toxoid.

[0301] In embodiments, the recombinant protein / modified fHbp fusion protein of the present application is co-administered with one or more vaccines selected from the group consisting of BEXSERO, MENVEO, MENACTRA, NIMENRIX, MenQuadFi, MENFIVE, MenAfriVac, Men AC, and Men ACHib.

[0302] In a preferred embodiment, the recombinant protein / modified fHbp fusion protein of the present application is co-administered with MENFIVE.

[0303] In an embodiment, the vaccine formulation comprises 2-phenoxyethanol in an amount ranging from 1 mg / mL to 10 mg / ml.

[0304] In an embodiment, the vaccine formulation has a zeta potential ranging from -16 mV to -30 mV.

[0305] In an embodiment, the vaccine formulation has an osmolality ranging from 200 mOsmol / kg to 500 mOsmol / kg.

[0306] The above vaccine formulation is manufactured by the following broad steps, which are further explained in detail in subsequent paragraphs:

[0307] - culturing host cells comprising expression vectors in a nutrient medium;

[0308] - inducing the host cells to express the protein;

[0309] - harvesting and isolating the host cells;

[0310] - lysing the harvested cells and isolating host cell debris to obtain the tagged protein;

[0311] - purifying the tagged protein;

[0312] - removing the tag from the tagged protein to obtain the recombinant protein / modified fHbp fusion protein;

[0313] - purifying the recombinant protein / modified fHbp fusion protein; and

[0314] - preparing the vaccine formulation comprising the purified recombinant protein / modified fHbp fusion protein.

[0315] According to another aspect of the present application, the upstream bioprocess is developed and optimized to increase cell density of the lead cell line and productivity of the recombinant protein / modified fHbp fusion protein.

[0316] In an embodiment, the upstream bioprocess can include batch, fed-batch, continuous or perfusion culture mode for production of the recombinant protein / modified fHbp fusion protein.

[0317] In an embodiment, the upstream bioprocess can include a fed-batch method, which includes the following steps:

[0318] (a) preparing an aqueous fermenter nutrient medium and a feed solution;

[0319] (b) inoculating the fermenter nutrient medium with a host cell line;

[0320] (c) continuously feeding with a feed solution;

[0321] (d) inducing protein expression;

[0322] (e) harvesting and cell separation.

[0323] In one embodiment, multiple experiments were performed during upstream bioprocess development in order to achieve high productivity required for the intended commercial process. The cultivation process development work was focused on four main areas: (1) confirming high performance of each cell expressing strain identified during the screening process and selecting clones with improved basal cultivation process; (2) developing enhanced media composition capable of supporting high productivity cultivation performance; (3) adjusting fermentation process parameters and process controls to achieve high cell density and recombinant protein / modified fHbp fusion protein expression; and (4) feeding strategy and optimization of amino acid addition to prevent stalling during transcription and translation.

[0324] In one embodiment, the aqueous fermenter nutrient medium can include undefined medium, Terrific Broth (TB) medium, Lysogenia Broth, Luria Broth or Luria-Bertani medium, chemically defined medium, M9 minimal medium, chemically defined M9 minimal salts medium, 2xYT medium, or Super Optimal broth (SOC) medium with catabolite repression or combinations thereof.

[0325] However, preferably, the composition of Luria Broth (LB medium) is summarized in Table 5.

[0326]

[0327] NaCl - Sodium Chloride

[0328] However, preferably, the composition of chemically defined M9 minimal salts medium is summarized in Table 6.

[0329]

[0330] K2HPO4 - Potassium Dihydrogen Phosphate

[0331] KH2PO4 - Potassium Dihydrogen Phosphate

[0332] (NH4)2SO4 - Ammonium Sulfate

[0333] MgSO4, 7H2O - Magnesium Sulfate Heptahydrate

[0334] TES - trace elements solution

[0335] A 14% liquid ammonia solution and a 6% orthophosphoric acid were used to maintain the pH during the fermentation batch in a pH range of 6.8-8.2.

[0336] However, preferably the composition of the trace elements solution is summarized in Table 7.

[0337]

[0338] However, preferably the composition of the Terrific Broth (TB) medium is summarized in Table 8.

[0339]

[0340] In one embodiment, the aqueous fermenter nutrient medium can additionally comprise an antibiotic selected from kanamycin, neomycin, streptomycin, tobramycin, paromomycin, amphotericin B, ampicillin, erythromycin, gentamicin, nystatin, penicillin-streptomycin, polymyxin B, tetracycline, thiabenzazole or tylosin or a combination thereof.

[0341] In one embodiment, the host cell line can comprise a bacterial expression host system.

[0342] More preferably, the bacterial expression host system is E. coli and the E. coli strain is selected from the group comprising BL21 (DE3), BL21 (DE3) pLysS*, BL21 (DE3) pLysE*, BL21 star (DE3), BL21-A1, BLR (DE3), HMS174 (DE3)**, Tuner (DE3), Origami2 (DE3)**, Rosetta2 (DE3)*, Rosetta gami (DE3), Lemo21 (DE3)*, T7 Express, m15 pREP4*, C41 (DE3), C43 (DE3) or B834 (DE3).

[0343] * indicates presence of additional plasmid

[0344] Most preferably, the bacterial expression host system is the E. coli strain B834 (DE3).

[0345] In an embodiment, the fermentation conditions following inoculation of the host cell line can include a temperature in the range of 35°C to 39°C; a pH in the range of 5.0 to 9.0; dissolved oxygen in the range of 10% to 100%; agitation in the range of 100-1800 rpm; gas flow rate 0-2 VVM (volume of gas per minute per unit volume of liquid).

[0346] In an embodiment, the inoculated fermenter nutrient medium can include continuous feeding with a feed solution after depletion of the carbon source.

[0347] Preferably, the composition of the feed solution comprising glucose is summarized in Table 9.

[0348]

[0349] (Glucose / Dextrose / Dextrose monohydrate are used interchangeably in this application)

[0350] Preferably, the composition of the feed solution comprising glycerol is summarized in Table 10.

[0351]

[0352] In an embodiment, the glycerol concentration in the glycerol feed composition is in the range of 30% to 80%. In an embodiment, the glycerol concentration in the glycerol feed composition is 50%.

[0353] In an embodiment, the feed rate of the glucose feed is in the range of 0.3 to 3.0 mL / min / L of culture.

[0354] In an embodiment, the glucose feed can be replaced with a glycerol feed solution, and subsequently the host cell line can be induced to express the recombinant protein / modified fHbp fusion protein.

[0355] In an embodiment, the feed rate of the glycerol feed is in the range of 0.3 to 3.0 mL / min / L of culture.

[0356] In an embodiment, an inducer selected from lactose or its non-hydrolyzed analogue isopropyl β-D-l-thiogalactopyranoside (IPTG) is used to induce the host cell line to express the recombinant protein / modified fHbp fusion protein.

[0357] In an embodiment, the inducing parameters can include inducing at an OD measured at 590 / 600 nm of 20-100. In an embodiment, the inducing agent is IPTG at a concentration ranging from 1 mM to 10 mM. In another embodiment, the inducing agent is IPTG at a concentration ranging from 5 mM to 10 mM. In an embodiment, the inducing agent is lactose at a concentration ranging from 1 g / L to 50 g / L. In another embodiment, the inducing agent is lactose at a concentration ranging from 5 g / L to 50 g / L.

[0358] In an embodiment, the inducing / induction temperature is in the range of 17°C to 37°C and the inducing hours is in the range of 3 to 24 hours.

[0359] In an embodiment, multiple experiments are performed during the inducing process to optimize the inducing density and length, culture temperature, culture pH, temperature variation, and glucose / glycerol feed rate.

[0360] In an embodiment, three different fed-batch methods / regimens for chimeric protein production in a fermenter are summarized in Table 11.

[0361]

[0362] In an embodiment, the culture is harvested 3-7 hours after induction.

[0363] In an embodiment, the cells are separated from the fermenter nutrient medium by centrifugation after harvesting.

[0364] Preferably, the centrifugation parameters can include 6000-8000 relative centrifugal force (rcf) for 30-60 minutes at 1 °C to 5 °C.

[0365] In an embodiment, the wet cell mass (g / L) obtained after harvesting can include 150-350 g / L of the harvested fermentation broth.

[0366] According to yet another aspect of the present application, a downstream bioprocess is developed and optimized to produce the recombinant protein / modified fHbp fusion protein at high yield and high purity.

[0367] In an embodiment, the downstream bioprocess can include any of the following steps:

[0368] a) cell lysis / cell disruption;

[0369] b) cell separation and clarification;

[0370] c) purification of the tagged protein;

[0371] d) affinity tag removal;

[0372] e) Protein purification;

[0373] f) Concentration / diafiltration / buffer exchange.

[0374] In an embodiment, a number of experiments are performed during the downstream bioprocess development to achieve high yield and high purity required for the intended commercial process.

[0375] In an embodiment, the cell culture is lysed or disrupted after harvest and cell separation to make the intracellular product accessible.

[0376] In an embodiment, the cell lysis / cell disruption is performed by a method selected from the group comprising chemical, biological, physical or mechanical mode or a combination thereof. Amongst which, the chemical method of cell lysis can comprise a detergent, a solvent, an acid, a base or a combination thereof. Amongst which, the biological method of cell lysis can comprise lysozyme. Amongst which, the physical method of cell lysis can comprise freeze-thaw, acoustic cavitation, hydrodynamic cavitation or osmotic shock or a combination thereof. Amongst which, the mechanical mode of cell lysis can comprise grinding (e.g. bead beater) or high pressure homogenization or a combination thereof.

[0377] In an embodiment, the cell lysis / cell disruption is performed by a mechanical mode comprising high pressure homogenization.

[0378] In another embodiment, the cell lysis / cell disruption is performed by chemical lysis using a lysis buffer.

[0379] In yet another embodiment, the cell lysis / cell disruption is performed by a combination of chemical lysis followed by a mechanical mode comprising high pressure homogenization. In an embodiment, the high pressure homogenization comprises a pressure in the range of 1000-1500 bar, a cycle in the range of 3-8, a flow rate of 9 L / hour ± 5%, a temperature in the range of 4°C to 15°C.

[0380] In an embodiment, the flow rate is increased as the process is scaled up.

[0381] In an embodiment, a homogenizer is used for mechanical lysis.

[0382] In an embodiment, the lysis buffer comprises sodium phosphate in the range of 10-100 mM, pH 6.0-8.0, imidazole in the range of 10-50 mM, sodium chloride (NaCl) in the range of 100-500 mM.

[0383] Lysis buffer: A lysis buffer is a buffered solution used for lysing cells for use.

[0384] In an embodiment, the efficiency of disruption and potential product loss of the intracellular product after cell lysis / cell disruption is evaluated by measuring any one or all of the following parameters including total protein release, cell viability and particle size distribution.

[0385] In an embodiment, the recombinant protein / modified fHbp fusion protein expression level of the intracellular product after cell lysis / cell disruption is assessed by a method selected from the group comprising SDS-PAGE, Western Blot, ELISA, enzymatic assay.

[0386] Preferably, the recombinant protein / modified fHbp fusion protein can comprise a fHbp-PorA chimeric protein.

[0387] In an embodiment, the fHbp-PorA chimeric protein can comprise an affinity tag selected from the group comprising a small peptide tag and / or a large polypeptide.

[0388] Affinity tag: When designing a project that requires a soluble active recombinant protein / modified fHbp fusion protein to be purified (as is often the case), it is invaluable to have the means to (i) detect it along the expression and purification protocol, (ii) achieve maximum solubility, and (iii) purify it easily from the E. coli cell environment. Expressing an amino acid fragment (peptide tag) or a large polypeptide (fusion partner) in tandem with the desired protein to form a tagged fusion protein can allow these three goals to be achieved directly.

[0389] Preferably, the fHbp-PorA chimeric protein can comprise a small peptide tag selected from the group comprising poly-Arg-, FLAG-, poly-His-, c-Myc-, S-, and Strep II-tags and a large polypeptide (fusion partner) selected from the group comprising maltose binding protein (MBP), N-utility protein A (NusA), thioredoxin (Trx), glutathione S-transferase (GST), ubiquitin, and SUMO.

[0390] Most preferably, the fHbp-PorA chimeric protein can comprise poly-His and MBP tags; wherein the expression vector can comprise pET28a-His-MBP-TEV-fHbp-PorA.

[0391] In an embodiment, the cell mass after cell lysis / cell disruption is subjected to cell separation and clarification.

[0392] Preferably, the cell separation is performed by centrifugation, wherein the intracellular product comprising the recombinant protein / modified fHbp fusion protein is separated in the supernatant.

[0393] In an embodiment, the centrifugation is performed at 4000-10000 RPM for 30-60 minutes at 1 °C to 5 °C.

[0394] In an embodiment, the supernatant after cell separation is subjected to clarification to remove lysate proteins.

[0395] In an embodiment, the clarification is performed by using filters selected from the group comprising reduced pore size (e.g., 6μ, 5μ, 0.8μ, 0.65μ, 0.45μ, 0.2μ). Suitable commercially available filters and filtration devices are well known to the person skilled in the art and can be selected by the person skilled in the art. Exemplary filtration devices can be made of polypropylene or cellulose acetate or polyethersulfone and commercially available filters can be Millipak (Millipore), Kleenpak (Pall) and Sartobran TM P filtration device.

[0396] In an embodiment, the supernatant comprising the labeled recombinant protein / modified fHbp fusion protein after cell separation and clarification is subjected to purification comprising a chromatography based purification method, ultrafiltration, diafiltration or a combination thereof.

[0397] In an embodiment, the chromatography based purification method comprises ion exchange or affinity chromatography or a combination of both.

[0398] In another embodiment, the chromatography based purification method comprises affinity chromatography based purification wherein the labeled recombinant protein / modified fHbp fusion protein binds to the affinity column and is further washed with increasing concentration of buffers comprising sodium phosphate + NaCl + imidazole. The labeled protein is eluted with elution buffer.

[0399] Preferably, the affinity column can comprise a immobilized metal affinity column resin.

[0400] Immobilized metal affinity chromatography (IMAC) resin is a high binding capacity resin used for purification of His-tagged proteins, wherein it relies on the affinity of His for immobilized transition metals.

[0401] According to an embodiment, the immobilized metal affinity column resin comprises metal ions selected from copper, zinc, nickel and the like.

[0402] Preferably, the immobilized metal affinity column resin comprises uncharged or pre-charged Ni 2+ resin.

[0403] In an embodiment, the uncharged form is charged with selected metal ions to achieve greater purification flexibility. In another embodiment, the immobilized metal affinity column resin can comprise Ni Sepharose 6 Fast Flow Column.

[0404] In an embodiment, the wash buffer comprises 20-50mM sodium phosphate buffer, 50-300mM sodium chloride (NaCl) pH 7.4 and increasing concentration of imidazole comprising 20-80mM (5-6 CV each).

[0405] In an embodiment, elution of the target protein is performed using an elution buffer comprising 20-50 mM sodium phosphate buffer, 50-120 mM sodium chloride (NaCl) pH 7.4 and imidazole comprising 100-300 mM.

[0406] In an embodiment, the eluate comprising the labeled recombinant protein / modified fHbp fusion protein after elution is further subjected to concentration and diafiltration.

[0407] In an embodiment, concentration and diafiltration is performed using tangential flow filtration (TFF), typically through a filter having a molecular weight cut-off (MWCO) in the range of 5-50 kDa and 10-50 mM sodium phosphate buffer at pH 7.4.

[0408] In an embodiment, concentration and diafiltration is performed using tangential flow filtration (TFF), typically through a filter having a molecular weight cut-off (MWCO) in the range of 5-50 kDa and 10-50 mM sodium phosphate buffer at pH 7.4 or Tris-HCl buffer at pH 8.5.

[0409] In an embodiment, the labeled recombinant protein / modified fHbp fusion protein is subjected to TEV protease digestion to remove the affinity tag.

[0410] In an embodiment, the recombinant protein / modified fHbp fusion protein comprising poly-His and MBP tag is subjected to TEV protease digestion to remove the His-MBP tag, wherein the TEV protease digestion comprises incubating the recombinant protein comprising poly-His and MBP tag with TEV protease.

[0411] Tobacco etch virus (TEV) protease is the 27-kDa catalytic domain of the polyprotein

[0412] In an embodiment, the His-GST-TEV protease has a molecular size of about 50 kDa.

[0413] In a preferred embodiment, the tagged recombinant protein / modified fHbp fusion protein is subjected to TEV protease digestion to remove the affinity tag, wherein the HIS-GST-TEV protease has at least 75% or 80.0%, or 85.0%, or 90.0% or 95.0% or 98.0%, or 99.0%, or 99.5% identity to the amino acid SEQ ID NO. 12 or is encoded by a nucleic acid sequence having at least 75.0%, or 80.0%, or 85.0%, or 90.0% or 95.0% or 98.0%, or 99.0%, or 99.5% identity to the sequence of SEQ ID NO. 11.

[0414] In an embodiment, the ratio of tagged recombinant protein / modified fHbp fusion protein: TEV protease is in the range of 5: 1 to 30: 1 or higher based on the activity and purity of the TEV protease. In an embodiment, the ratio of tagged recombinant protein / modified fHbp fusion protein (substrate): TEV protease is 20: 1.

[0415] In an embodiment, the incubation temperature is in the range of 4°C to 35°C and the incubation time is in the range of 15 to 20 hours.

[0416] In an embodiment, the TEV protease digestion is carried out in the presence of 10-50 mM sodium phosphate buffer pH 7.4 followed by addition of dithiothreitol (DTT) at a final concentration of 0.5 mM to 5 mM.

[0417] In an embodiment, the TEV protease used for tag removal / digestion is prepared by a method comprising the steps of:

[0418] - culturing the host cell comprising the expression vector in a nutrient medium;

[0419] - inducing the host cell to express the TEV protease;

[0420] - harvesting and isolating the host cell;

[0421] - lysing the harvested cell and isolating the host cell to obtain the TEV protease; and

[0422] - purifying the TEV protease.

[0423] In embodiments, the host cell can comprise E. coli, and the E. coli strain can be selected from the group comprising BL21 (DE3), BL21 (DE3) pLysS*, BL21 (DE3) pLysE*, BL21 star (DE3), BL21-A1, BLR (DE3), HMS174 (DE3)**, Tuner (DE3), Origami2 (DE3)**, Rosetta2 (DE3)*, Rosetta gami (DE3), Lemo21 (DE3)*, T7 Express, m15 pREP4*, C41 (DE3), C43 (DE3), Rosetta™ (DE3) pLysS, or B834 (DE3).

[0424] In embodiments, the host cell for TEV protease expression is E. coli Rosetta™ (DE3) pLysS.

[0425] In embodiments, the TEV protease prepared by the above method comprises a TEV protease having SEQ ID NO. 12 or encoded by SEQ ID NO. 11.

[0426] In embodiments, the TEV protease is purified using the following non-limiting steps:

[0427] - lysing the harvested cells;

[0428] - separating the host cell debris and collecting the supernatant;

[0429] - washing the supernatant at least once using a wash buffer;

[0430] - eluting the purified TEV protease with an elution buffer;

[0431] - concentrating the purified TEV protease.

[0432] In embodiments, the wash buffer comprises sodium phosphate, sodium chloride, and imidazole.

[0433] In embodiments, the supernatant is subjected to at least 4 wash steps with a wash buffer having a pH of 7.4.

[0434] In another embodiment, the supernatant is subjected to at least 2 wash steps with a wash buffer having a pH of 8.5.

[0435] The methods and parameters for preparation and purification of TEV protease are similar to those described above for the tagged recombinant protein / modified fHbp fusion protein, and are detailed in subsequent examples.

[0436] In one embodiment, after tag removal, the recombinant protein / modified fHbp fusion protein is subjected to purification comprising a chromatography based purification method, ultrafiltration, diafiltration, or a combination thereof.

[0437] According to embodiments, the chromatography is selected from column chromatography, ion exchange chromatography, anion exchange chromatography, cation exchange chromatography, column chromatography, flash chromatography, gel filtration / size exclusion / gel permeation (molecular sieve) chromatography, affinity chromatography, paper chromatography, thin layer chromatography, gas chromatography, dye ligand chromatography, hydrophobic interaction chromatography, pseudo-affinity chromatography, liquid chromatography, high pressure liquid chromatography (HPLC), immobilized metal affinity chromatography, anion exchange chromatography, cation exchange chromatography, multimodal chromatography, multimodal anion exchange chromatography, electrostatic interaction chromatography, hydrogen bond chromatography, reverse phase chromatography, and combinations thereof.

[0438] In embodiments, the chromatography based purification method comprises ion exchange or affinity chromatography or a combination of both.

[0439] In another embodiment, the chromatography based purification method comprises ion exchange followed by affinity based chromatography purification.

[0440] In yet another embodiment, the ion exchange chromatography comprises anion exchange chromatography.

[0441] In yet another embodiment, the anion exchange chromatography comprises strong anion exchange chromatography.

[0442] In embodiments, the affinity column can comprise an immobilized metal affinity column resin.

[0443] Immobilized metal affinity chromatography (IMAC) resin is a high binding capacity resin used for purification of His-tagged proteins, where it relies on the affinity of His for immobilized transition metals.

[0444] In embodiments, the immobilized metal affinity column resin comprises an uncharged or pre-charged Ni 2+ resin.

[0445] In embodiments, the uncharged form is charged with a selected metal ion to achieve greater purification flexibility.

[0446] In embodiments, the immobilized metal affinity column resin comprises a Ni Sepharose 6 Fast Flow Column.

[0447] The chromatography can be multimodal anion exchange resin Capto™ Adhere, Capto adhere ImpRes, Capto MMC ImpRes, or any other chromatography comprising a mixed mode combination of ion exchange, electrostatic interaction, hydrogen bonding, and hydrophobic interaction.

[0448] In an embodiment, concentration and diafiltration is carried out using tangential flow filtration (TFF), typically through filters having a molecular weight cut-off (MWCO) in the range of 5 KDa - 50 Kda.

[0449] In an embodiment, the eluate comprising the recombinant protein / modified fHbp fusion protein is sterilized by direct flow filtration (DFF) through at least one sterilizing grade filter to obtain a filtrate comprising the sterilized recombinant protein / modified fHbp fusion protein. Wherein, the sterilizing grade filter can be selected from a group comprising 0.8μ, 0.45μ, 0.2μ. Further, commercially available filters and filtration devices are well known in the art and can be selected by a person skilled in the art. Exemplary filtration devices can be made of polypropylene or cellulose acetate or polyethersulfone or polyvinylidene fluoride and commercially available filters can be Millipak (Millipore), Kleenpak (Pall) and Sartobran TM P filtration device.

[0450] In an embodiment, the recombinant protein / modified fHbp fusion protein further comprises inclusion bodies (IB).

[0451] Inclusion bodies (IB): Accumulation of protein aggregates / insoluble proteins is referred to as IB. IB formation is caused by an imbalance equilibrium between protein aggregation and solubilization.

[0452] In an embodiment, the recombinant protein / modified fHbp fusion protein comprising inclusion bodies (IB) is subjected to:

[0453] a) inclusion body washing and recovery;

[0454] b) inclusion body (IB) solubilization and refolding;

[0455] In an embodiment, the recombinant protein / modified fHbp fusion protein comprising inclusion bodies (IB) is subjected to urea denaturation and inclusion body binding affinity column solubilized in urea and further washed with decreasing concentration of urea and refolding buffer that facilitates correct refolding. The target protein is eluted with elution buffer.

[0456] In an embodiment, denaturation is carried out using a denaturation buffer composition comprising 20-50 mM sodium phosphate buffer, 50-120 mM sodium chloride (NaCl) pH 7.4 and 5-10 M urea.

[0457] In another embodiment, the washing with reduced concentration of urea includes washing with 20-50 mM sodium phosphate buffer, 50-120 mM sodium chloride (NaCl) pH 7.4. In another embodiment, the reduced concentration of urea includes 8M, 6M, 4M, 2M, 1M (each for 5-6 CV).

[0458] In an embodiment, the elution of the target protein is carried out using an elution buffer comprising 40mM-400mM imidazole.

[0459] In an embodiment, the affinity column includes a immobilized metal affinity column resin.

[0460] Immobilized metal affinity chromatography (IMAC) resin is a high binding capacity resin used for purification of His-tagged proteins, wherein it relies on the affinity of His for immobilized transition metals.

[0461] In an embodiment, the immobilized metal affinity column resin includes uncharged or pre-charged Ni 2+ resin.

[0462] In another embodiment, the uncharged form is charged with a selected metal ion to achieve greater flexibility in purification.

[0463] In yet another embodiment, the immobilized metal affinity column resin includes Ni Sepharose 6 Fast Flow Column.

[0464] In an embodiment, the supernatant comprising inclusion bodies is subjected to protein precipitation prior to urea denaturation.

[0465] In an embodiment, the protein precipitation is carried out using ammonium sulfate.

[0466] In an embodiment, the supernatant is subjected to filtration using 0.22 µM filter after urea denaturation.

[0467] In an embodiment, the eluate comprising the target recombinant protein / modified fHbp fusion protein is further subjected to concentration and diafiltration after elution.

[0468] In an embodiment, the concentration and diafiltration is carried out using tangential flow filtration (TFF), typically through a filter having a molecular weight cut-off (MWCO) in the range of 5kDa-50kDa.

[0469] In an embodiment, the concentration of the recombinant protein / modified fHbp fusion protein is greater than 0.95 mg / ml.

[0470] In another embodiment, the glucose feed is stopped and the glycerol feed is started when the OD at 590 / 600 nm is 20-100, and the culture is induced by adding lactose and / or maintaining lactose at 1-50 g / L in fed-batch mode.

[0471] In an embodiment, the purified chimeric / recombinant protein / modified fHbp fusion protein is stored at 2°C to 8°C until further use. In another embodiment, the purified chimeric / recombinant protein / modified fHbp fusion protein is stored at 2°C to 8°C in the presence of a stabilizer. In an embodiment, the stabilizer is selected from TRIS, Tween / polysorbate, non-ionic detergents such as polyethylene glycol lauryl ether (BRIJ 35), sucrose (up to 5%), and the like.

[0472] According to another aspect of the present application, the recombinant protein / modified fHbp fusion protein formulation is optimized to improve immunogenicity, improve stability and maintain long term storage stability of the recombinant protein / modified fHbp fusion protein antigen.

[0473] In an embodiment, the optimized vaccine formulation has low viscosity, is free from aggregation, and has long term stability over a wide temperature range. In an embodiment, the optimized vaccine formulation includes a solid or liquid carrier.

[0474] In an embodiment, the vaccine formulation is entirely liquid. Suitable forms of liquid formulations include solutions, suspensions, emulsions, syrups, isotonic aqueous solutions, viscous compositions buffered to a selected pH, and elixirs.

[0475] In an embodiment, the vaccine formulation includes a polymer or other agent to control the consistency of the composition, and / or to control the release of the antigen / secreted protein from the composition.

[0476] In an embodiment, the vaccine formulation is in the form of a transdermal formulation, including a lotion, gel, spray, ointment, or other suitable dosage form. If nasal or respiratory (mucosal) administration (e.g., aerosol inhalation or insufflation) is desired, the composition can be in such a form and dispensed from a squeeze spray dispenser, pump dispenser, or aerosol dispenser. Aerosols are typically pressurized by hydrocarbons. Pump dispensers can preferably dispense metered doses or doses of a particular particle size. When in the form of a solution, suspension, and gel, in some embodiments, the immunogenic composition contains a substantial amount of water (preferably purified water) in addition to the active ingredients.

[0477] In an embodiment, the vaccine formulation is stable at 2-8°C for 12 to 36 months; at 25°C for 2 to 6 months; at 37°C for 1 week to 4 weeks, at 42°C for 2-7 days, and at 55°C for 2-7 days.

[0478] In one embodiment, the vaccine formulation is a lyophilized / freeze-dried formulation.

[0479] In one embodiment, the final pH of the formulation can be in the range of pH 6.0 to pH 8.0.

[0480] According to a further aspect of the application, there is provided a modified fHbp, nucleic acid or formulation according to the application for use in the treatment or prevention of a pathogen infection or colonisation in a subject. According to a yet further aspect of the application, there is provided a method of treating or preventing a pathogen infection or colonisation in a subject comprising administering to the subject a modified fHbp, nucleic acid or composition according to the application. According to a further aspect of the application, there is provided a method of vaccination comprising administering to the subject a modified fHbp, nucleic acid or composition according to the application.

[0481] In one embodiment, the modified fHbp is immunogenic, involves administration or injection of an immunologically effective amount of the immunogenic formulation to a human subject by parenteral or subcutaneous or intradermal, intramuscular or intraperitoneal or intravenous administration or sustained release from an implant or by eye drops or nasal or rectal or buccal or vaginal, peroral or gastric or mucosal or sublingual, alveolar or gingival or olfactory or respiratory mucosal administration or any other immunological route.

[0482] As used herein, "co-administration" means that different immunogenic compositions / vaccines can be administered separately or as a combination.

[0483] Where the vaccines are administered separately, they are typically administered at different sites, for example one vaccine is administered to the left upper arm and the second vaccine is administered to the right upper arm. Thus, the two vaccines can be administered contralaterally (e.g. arms or legs or arms and legs on opposite sides of the body) or ipsilaterally (e.g. arms and legs on the same side of the body). Although the vaccines are administered separately, they are administered at substantially the same time (e.g. during the same medical consultation or visit to a healthcare professional or vaccination centre), for example within 1 hour of each other.

[0484] However, rather than separate co-immunisation, administration as a combination can be performed. Thus, co-immunisation can use a combination vaccine, i.e. a single composition in which different immunogens are mixed. Combination vaccines offer the advantage to the subject of receiving fewer injection times, which can lead to a clinical advantage of increased compliance.

[0485] The compositions of the application are typically administered directly to the patient. Direct delivery can be accomplished by parenteral injection (e.g., subcutaneous, intraperitoneal, intravenous, intramuscular, or interstitially into a tissue) or by rectal, oral, vaginal, topical, transdermal, intranasal, ocular, aural, pulmonary, or other mucosal administration. Intramuscular administration into the thigh or upper arm is preferred. Injection can be via a needle (e.g., a hypodermic needle), but alternatively needle-free injection can be used. A typical intramuscular dose is about 0.5 ml.

[0486] The compositions can also be provided in the form of a "multiple dose" kit, i.e., a single container containing sufficient composition for multiple immunizations. Multiple doses can include a preservative, or the multiple dose container can have a sterile fitting for removal of individual doses of the composition.

[0487] The subject to be immunized is a human, who can be of any age, e.g., 0-12 months old, 1-5 years old, 5-18 years old, 18-55 years old, or greater than 55 years old. Preferably, the subject to be immunized is an adolescent (e.g., 12-18 years old) or an adult (18 years old or greater).

[0488] Alternatively, the subject is an adolescent or adult who has been immunized against N. meningitidis during childhood (e.g., before age 12) and receives a booster dose of the immunogenic composition according to the application.

[0489] In embodiments, the vaccine formulation of the application demonstrates cross-protection against N. gonorrhoeae strains and N. meningitidis serogroups ACWYX.

[0490] The administration can be provided in a therapeutically effective amount. The skilled person will be able to determine the appropriate dose and repetition for administration.

[0491] The vaccine formulation can be formulated into a single dose vial or a multiple dose vial (2 dose or 5 dose or 10 dose vials) or a multiple dose kit or a pre-filled syringe, wherein the vaccine formulation can be given in a single dose regimen or, preferably, in a multiple dose regimen, wherein 1-3 separate doses are given at subsequent time intervals after the primary vaccination process if needed after 1-3 years. The dose regimen will also depend at least in part on the booster doses needed to confer protective immunity.

[0492] In embodiments, the vaccine formulation is formulated for administration to a human subject, an elderly, an adolescent, an adult or a child less than 2 years old or a child greater than 2 years old, according to a one dose or two dose regimen or three dose regimen, the regimen consisting of a first dose and / or a second dose and / or a third dose, the second dose being administered between 3 months and 2 years after the first dose, the third dose being administered between 3 months and 2 years after the second dose.

[0493] In embodiments, the subject is a mammal, e.g., a human.

[0494] In embodiments, the infection is a bacterial infection. In another embodiment, the infection is meningitis, for example, Neisseria meningitidis or Neisseria gonorrhoeae.

[0495] According to another aspect of the application, there is provided a combination of a modified fHbp according to the application and at least one other prophylactically or therapeutically active molecule.

[0496] In embodiments, the at least one other prophylactically or therapeutically active molecule comprises a vaccine or antigen different from the modified fHbp according to the application herein. According to embodiments of the application, the antigen is selected from, but not limited to, diphtheria toxoid (D), tetanus toxoid (T), whole cell pertussis (wP), hepatitis B virus surface antigen (HBsAg), Haemophilus influenzae b PRP-carrier protein conjugate (Hib), Haemophilus influenzae (a, c, d, e, f serotypes and strains not encapsulated by a capsule), Neisseria meningitidis A antigen, Neisseria meningitidis C antigen, Neisseria meningitidis W-135 antigen, Neisseria meningitidis Y antigen, Neisseria meningitidis X antigen, Streptococcus pneumoniae antigen, Neisseria meningitidis B bleb or purified antigen, Staphylococcus aureus antigen, anthrax, BCG, hepatitis (A, C, D, E, F and G strains) antigen, human papillomavirus, HIV, Salmonella typhi antigen, acellular pertussis, modified adenylate cyclase, malaria antigen (RTS, S), measles, mumps, rubella, dengue, zika, ebola, Japanese encephalitis, rotavirus, diarrhoeal antigen, flavivirus, smallpox, yellow fever, shingles and varicella virus antigen.

[0497] In another embodiment, the application contemplates compositions comprising, in addition to the fHbp-PorA chimeric antigen, antigens for immunization against other diseases or infections. According to embodiments, the composition comprises the following additional antigens:

[0498] - protein antigens from PorB, Fet A, OmpC, NHBA, NadA, meningococcal antigen 287, NspA, HmbR, NhhA, App, 936,

[0499] - saccharide antigens from Neisseria meningitidis serogroups A, C, W, Y and / or X,

[0500] - saccharide antigens from Streptococcus pneumoniae,

[0501] - diphtheria antigens, such as diphtheria toxoid, such as CRM197 mutant,

[0502] - tetanus antigens, such as tetanus toxoid,

[0503] - an antigen from Bordetella pertussis, acellular or whole cell pertussis antigens,

[0504] - a saccharide antigen from Haemophilus influenzae B,

[0505] - a polio antigen, e.g. IPV,

[0506] - a measles, mumps and / or rubella antigen,

[0507] - an influenza antigen, e.g. hemagglutinin and / or neuraminidase surface proteins,

[0508] - an antigen (protein or saccharide) from Streptococcus agalactiae (group B streptococcus),

[0509] - an antigen (protein or saccharide) from Streptococcus pyogenes (group A streptococcus),

[0510] - an antigen (protein or saccharide) from Staphylococcus aureus,

[0511] - an antigen (protein or saccharide) from certain species of Salmonella Spp.

[0512] In one embodiment, the at least one other prophylactically or therapeutically active molecule comprises a monovalent capsular polysaccharide-protein conjugate vaccine. The monovalent protein capsular polysaccharide vaccine can comprise any of a serogroup C or A capsular polysaccharide from Neisseria meningitidis conjugated to a bacterial toxoid, a bivalent vaccine (with serogroup C and A capsular polysaccharides conjugated to a bacterial toxoid), a tetravalent (serogroups A, C, Y, W) or pentavalent (A, C, Y, W, X) conjugate vaccine. Alternatively, the at least one other prophylactically or therapeutically active molecule can comprise a conjugate vaccine wherein an antigen comprising a fHbp scaffold loaded with an exogenous peptide loop (such as a PorA loop) can be introduced as a protein carrier molecule in the conjugate vaccine. The conjugate vaccine can comprise any of the serogroup capsular polysaccharides from A, C, Y, W or X strains, alone or in combination.

[0513] The combination vaccine can be selected from a hexavalent (ACWYX-B), tetravalent (AC-Hib-B), trivalent (AC-B), bivalent (A-B, X-B, C-B).

[0514] In embodiments, the liquid Neisseria meningitidis serogroup B vaccine is reconstituted with a lyophilized ACWYX conjugate vaccine for bedside administration.

[0515] According to embodiments, the present application relates to a method of inducing an immune response against N. meningitidis in a mammal. The method comprises administering to the mammal an effective amount of an immunogenic composition comprising i) at least one fusion protein comprising a stabilized non-functionalized / non-lipidated fHbp and a PorA VR2 loop of PorA and at least one conjugate selected from the group consisting of: (a) a conjugate of (i) a capsular polysaccharide of serogroup A N. meningitidis and (ii) tetanus toxoid; (b) a conjugate of (i) a capsular polysaccharide of serogroup C N. meningitidis and (ii) CRM197; (c) a conjugate of (i) a capsular polysaccharide of serogroup Y N. meningitidis and (ii) CRM197; (d) a conjugate of (i) a capsular polysaccharide of serogroup W135 N. meningitidis and (ii) CRM197; and (d) a conjugate of (i) a capsular polysaccharide of serogroup X N. meningitidis and (ii) tetanus toxoid.

[0516] One aspect of the present application relates to a method of inducing an immune response against N. meningitidis in a mammal. The method comprises administering to the mammal an effective amount of an immunogenic composition comprising i) at least two fusion proteins, each fusion protein consisting of one fHbp variant type coupled to one PorA VR2 loop and at least one conjugate selected from the group consisting of: (a) a conjugate of (i) a capsular polysaccharide of serogroup A N. meningitidis and (ii) tetanus toxoid; (b) a conjugate of (i) a capsular polysaccharide of serogroup C N. meningitidis and (ii) CRM197; (c) a conjugate of (i) a capsular polysaccharide of serogroup Y N. meningitidis and (ii) CRM197; (d) a conjugate of (i) a capsular polysaccharide of serogroup W135 N. meningitidis and (ii) CRM197; and (d) a conjugate of (i) a capsular polysaccharide of serogroup X N. meningitidis and (ii) tetanus toxoid.

[0517] Another aspect of the present application relates to a method of inducing an immune response against N. meningitidis in a mammal. The method comprises administering to the mammal an effective amount of an immunogenic composition comprising i) at least three fusion proteins, each fusion protein consisting of one fHbp variant type coupled to two PorA VR2 loops and at least one conjugate selected from the group consisting of: (a) a conjugate of (i) a capsular polysaccharide of serogroup A N. meningitidis and (ii) tetanus toxoid; (b) a conjugate of (i) a capsular polysaccharide of serogroup C N. meningitidis and (ii) CRM197; (c) a conjugate of (i) a capsular polysaccharide of serogroup Y N. meningitidis and (ii) CRM197; (d) a conjugate of (i) a capsular polysaccharide of serogroup W135 N. meningitidis and (ii) CRM197; and (d) a conjugate of (i) a capsular polysaccharide of serogroup X N. meningitidis and (ii) tetanus toxoid.

[0518] Yet another aspect of the application relates to a method of inducing an immune response against N. meningitidis in a mammal. The method comprises administering to the mammal an effective amount of an immunogenic composition comprising i) at least four fusion proteins, each fusion protein consisting of one fHbp variant type coupled to three PorA VR2 loops, and at least one conjugate selected from the following: (a) a conjugate of (i) a capsular polysaccharide of serogroup A N. meningitidis and (ii) tetanus toxoid; (b) a conjugate of (i) a capsular polysaccharide of serogroup C N. meningitidis and (ii) CRM197; (c) a conjugate of (i) a capsular polysaccharide of serogroup Y N. meningitidis and (ii) CRM197; (d) a conjugate of (i) a capsular polysaccharide of serogroup W135 N. meningitidis and (ii) CRM197; and (d) a conjugate of (i) a capsular polysaccharide of serogroup X N. meningitidis and (ii) tetanus toxoid.

[0519] The fHbp-PorA chimeric antigens for use in the application comprise an amino acid sequence that is 50% or more identical (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to a sequence disclosed in the Sequence Listing of SEQ ID 1-10. The Sequence Listing is included in standard ST.26 format and is incorporated herein by reference.

[0520] List of Men B strains used to test fHbp-PorA chimeric vaccines

[0521] fHbp isogroup strains expressing different fHbp (V1.1, V1.14, V2.19 and V3.45) in the H44 / 76 AfHbp AporA construct were used to test fHbp-PorA chimeric vaccines:

[0522] A. fHbp expressing strains:

[0523] 1. H44 / 76 AfHbp AporA: fHbp V1.1

[0524] 2. H44 / 76 AfHbp AporA: fHbp V1.14

[0525] 3. H44 / 76 AfHbp AporA: fHbp V2.19

[0526] 4. H44 / 76 AfHbp AporA: fHbp V3.45

[0527] B. PorA expressing strains:

[0528] 1. H44 / 76 AfHbp APorA:PorA 1.4

[0529] 2. H44 / 76 AfHbp APorA:PorA 1.9

[0530] C. Clinical isolates:

[0531] M11.240413 (expressing fHbp V1.13 and PorA 1.9)

[0532] The following clinical strains of N. meningitidis will be used in the SBA at Oxford / UKHSA to test the fHbp-PorA chimeric vaccine:

[0533]

[0534] In addition, N. meningitidis strains M15 240912, M16 240272, M15 240460 or any other suitable strain can be used in the SBA.

[0535] According to a further aspect of the application, there is provided the use of a Factor H binding protein (fHbp) as an epitope display scaffold. The use as an epitope display scaffold can include the use of any of the modified Factor H binding proteins (fHbp) described herein. In addition to potential use as a vaccine, the compositions or modified fHbp according to the application can be used as a diagnostic reagent and can be used as a measure of the immunological competence of a vaccine.

[0536] The immune response elicited by the modified fHbp of the application can affect the ability of a subject immunised against N. meningitidis (Nm) infection with the modified fHbp of the application. Preferably, the ability of a subject immunised against Nm infection with the modified fHbp of the application is hindered or prevented. The immune response elicited can recognise and destroy Nm.

[0537] Alternatively or in addition, the immune response elicited can hinder or prevent replication of Nm. Alternatively or in addition, the immune response elicited can hinder or prevent Nm from causing disease in a human or non-human animal.

[0538] The foregoing description of specific implementations will so fully reveal the general nature of the methods and procedures herein that others can adapt and / or merely apply such methods and procedures as the specific circumstances indicate without undue experimentation. Consequently, the particular implementation as set forth is not to be understood as limiting in nature. Rather, it is to be understood that the methods and procedures as broadly described herein are amenable to modification and / or adaptation by those of ordinary skill in the art having the benefit of this disclosure without undue experimentation and, accordingly, such modifications and / or adaptations are to be considered within the scope of the disclosed implementations as set forth herein.

[0539] Throughout this specification, the term "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0540] The use of the phrases "one or more" or "at least one" implies the use of one or more elements or ingredients or quantities, as in the embodiments of the application, can achieve one or more desired purposes or results.

[0541] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present disclosure. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure

[0542] Unless otherwise stated in the present specification, numerical values given for various physical parameters, dimensions and quantities are only approximations and are considered to be within the scope of the present application.

[0543] While considerable emphasis has been placed herein on the particular features of the preferred embodiments, it will be appreciated that various modifications can be made by those skilled in the art and that many changes can be made in the preferred embodiments without departing from the principles of the present disclosure. These and other variations and modifications of the preferred embodiments will be apparent to those skilled in the art from the disclosure, and it is the intent that all such modifications and changes be considered as being within the scope of the present disclosure. Accordingly, the description is to be construed as limited in scope only as far as recited in the appended claims.

[0544] Technical advantages:

[0545] 1. The present invention provides a highly efficient platform process for manufacturing an effective vaccine against N. meningitidis, which meets multiple criteria, including improved immunogenicity, safety and affordability.

[0546] 2. The present invention provides a method of developing and optimizing upstream bioprocess to increase cell density of the lead cell line and productivity of recombinant protein / modified fHbp fusion protein.

[0547] 3. The present invention provides a method of developing and optimizing downstream bioprocess to produce recombinant protein / modified fHbp fusion protein at high yield and high purity.

[0548] 4. The improved formulation overcomes the limitations of the prior art and shows low viscosity, no aggregation; shows long term stability over a wide temperature range, indirectly maintaining the desired characteristics of recombinant protein / modified fHbp fusion protein, including high stability and immunogenicity.

[0549] 5. The new formulation / composition i) no aggregates and particle formation, higher osmolality, optimized zeta potential, low viscosity iii) stability of desired physicochemical and immunogenic characteristics of recombinant protein / modified fHbp fusion protein and immunogenicity on storage for 12 months at 2-8°C, 6 months at 25°C and 30 days at 40°C.

[0550] 6. Chimeric antigen (ChA) against N. meningitidis serogroup B. ChA utilizes fHbp (non-lipidated) as a molecular scaffold to present a surface-exposed PorA VR2 loop, by inserting the VR2 loop ("10-20 amino acid" PorA VR2 loop rather than "entire PorA protein") into the beta-turn region in fHbp. ChA retains epitopes from both fHbp and PorA, and was found to elicit functional immune responses against both antigens. Integration of the VR2 loop does not alter the overall structure of fHbp, and the VR2 loop folds into a conformation recognized by bactericidal mAbs.

[0551] 7. The soluble and high yield chimeric protein is stable, with both fHbp and PorA VR2 loop being immunogenic.

[0552] 8. The chimer produced consists of the most prevalent fHbp and PorA antigens to maximize vaccine coverage, as the chimer composition reflects the prevalent fHbp and PorA antigens circulating within a given geographic region.

[0553] 9. Insertion of a PorA loop into specific positions in fHbp results in a desired reduction of Factor H binding (at least 10%, at least 50% reduction compared to wild type; preferably >70%) and has a molecular weight in the range of 20 to 40 kDa while retaining immunogenic epitopes of both fHbp and PorA (Table 12).

[0554]

[0555] The present application is explained in more detail by the following embodiments and combinations of embodiments, which are derived from the respective dependencies and linkages:

[0556] I. A modified Factor H binding protein (fHbp) comprising a wild type fHbp variant and at least one exogenous peptide loop, wherein

[0557] a. the modified Factor H binding protein (fHbp) is selected from an amino acid sequence having at least 75% identity to any one of the sequences of SEQ ID No 6 to 10,

[0558] b. wherein the at least one exogenous peptide loop is immunogenic,

[0559] c. wherein the at least one exogenous peptide loop is derived from a bacterial membrane protein,

[0560] d. wherein the modified fHbp is a fusion protein,

[0561] e. wherein the fHbp variant is selected from v1, v2 and v3, which is modified with the insertion of at least one PorA loop into the beta-turn region in fHbp, the PorA loop comprising at least 10 amino acids; and

[0562] f. wherein the PorA loop is selected from VR1 and VR2.

[0563] II. The modified Factor H binding protein as described in embodiment I, wherein the modified fHbp comprises a wild type fHbp variant and at least one exogenous bacterial membrane protein peptide loop, or wherein the modified fHbp is a fusion protein.

[0564] III. The modified Factor H binding protein as described in any of the preceding embodiments, wherein the modified fHbp is modified to reduce Factor H binding activity.

[0565] IV. A nucleic acid sequence encoding a modified fHbp, wherein the nucleic acid sequence has at least 75% identity to any one of the sequences of SEQ ID NO. 1 to 5.

[0566] V. An immunogenic composition comprising at least one modified fHbp as described in any of the preceding embodiments or a nucleic acid sequence encoding a modified fHbp as described in embodiment IV.

[0567] VI. The immunogenic composition of embodiment V, wherein the composition comprises two or more different modified fHbps.

[0568] VII. The immunogenic composition of any of the preceding embodiments, wherein the composition comprises a pharmaceutically acceptable carrier.

[0569] VIII. The immunogenic composition of any of the preceding embodiments, wherein the composition further comprises an adjuvant.

[0570] IX. The immunogenic composition of any of the preceding embodiments, wherein the composition further comprises at least one other prophylactically or therapeutically active molecule comprising a monovalent protein: capsular polysaccharide vaccine.

[0571] X. The immunogenic composition of any of the preceding embodiments, wherein the fHbp scaffold bearing the exogenous peptide loop is incorporated as a protein carrier molecule in a conjugate vaccine.

[0572] XI. The immunogenic composition of any of the preceding embodiments, comprising a recombinant protein / modified fHbp fusion protein and in combination with at least one additional antigen selected from:

[0573] - a protein antigen from PorB, Fet A, OmpC, NHBA, NadA, meningococcal antigen 287, NspA, HmbR, NhhA, App, 936,

[0574] - a saccharide antigen from N. meningitidis serogroups A, C, W, Y and / or X,

[0575] - a saccharide antigen from S. pneumoniae,

[0576] - a diphtheria antigen, e.g. diphtheria toxoid, e.g. CRM197 mutant,

[0577] - a tetanus antigen, e.g. tetanus toxoid,

[0578] - an antigen from B. pertussis, acellular or whole cell pertussis antigen,

[0579] - a saccharide antigen from H. influenzae B,

[0580] - a polio antigen, e.g. IPV,

[0581] - a measles, mumps and / or rubella antigen,

[0582] - an influenza antigen, such as hemagglutinin and / or neuraminidase surface proteins,

[0583] - an antigen (protein or sugar) from Streptococcus agalactiae (group B streptococcus),

[0584] - an antigen (protein or sugar) from Streptococcus pyogenes (group A streptococcus),

[0585] - an antigen (protein or sugar) from Staphylococcus aureus,

[0586] - an antigen (protein or sugar) from certain species of Salmonella.

[0587] XII. The immunogenic composition as described in any of the preceding embodiments, wherein the fHbp scaffold bearing an exogenous peptide loop is incorporated as a protein carrier molecule in a polysaccharide conjugate vaccine selected from (A, X, C, W, Y), bivalent (A-B, X-B, C-B), trivalent (AC-B, AC-Hib), tetravalent (AC-Hib-B), pentavalent (ACWYX) or hexavalent (ACWYX-B).

[0588] XIII. The modified fHbp as described in any of embodiments I to III, the nucleic acid as described in embodiment IV, or the composition as described in any of embodiments V to IX, for use as a medicament, or for use in the treatment or prevention of a pathogen infection or colonization in a subject.

[0589] XIV. The modified fHbp as described in any of embodiments I to III, the nucleic acid as described in embodiment IV, or the composition as described in any of embodiments V to IX, in combination with at least one other prophylactically or therapeutically active molecule.

[0590] XV. The combination as described in embodiment XIII or IX, wherein the at least one other prophylactically or therapeutically active molecule comprises a conjugate vaccine comprising any serogroup capsular polysaccharide selected from A, C, Y, W or X strains or combinations thereof.

[0591] XVI. The combination as described in embodiment XV or the composition as described in embodiment IX, wherein the protein: capsular polysaccharide vaccine comprises any serogroup C or A capsular with a bacterial toxoid, bivalent vaccine (with serogroup C and A capsular polysaccharides conjugated to a bacterial toxoid), tetravalent (serogroup A, C, Y, W polysaccharides conjugated to a bacterial toxoid) or pentavalent (serogroup A, C, Y, W, X polysaccharides conjugated to a bacterial toxoid) conjugate vaccine.

[0592] XVII. The factor H binding protein (fHbp) as described in embodiments I to III as an epitope display scaffold and when used as an epitope display scaffold.

[0593] XVIII. A vaccine formulation comprising at least one recombinant protein / modified fHbp fusion protein, an adjuvant and one or more pharmaceutically acceptable excipients, wherein the recombinant protein / modified fHbp fusion protein is at least one selected from transferrin binding protein, Neisserial heparin binding protein, Neisserial surface protein A, PorA, meningococcal enterobactin receptor FetA, Neisserial adhesin A, factor H binding protein (fHbp) as described in any one of the preceding embodiments or a combination thereof.

[0594] XIX. The vaccine formulation as described in embodiment XVIII, wherein the recombinant protein is fHbp as described in any one of the preceding embodiments and wherein the fHbp is derived from Neisseria meningitidis serogroups A, B, C, H, I, K, L, 29E, W135, X, Y and Z.

[0595] XX. The vaccine formulation as described in any one of the preceding embodiments, wherein the recombinant protein is fHbp as described in any one of the preceding embodiments and wherein the fHbp is derived from Neisseria meningitidis serogroup B.

[0596] XXI. The vaccine formulation as described in any one of the preceding embodiments, wherein the recombinant protein is fHbp as described in any one of the preceding embodiments and wherein the fHbp has a molecular weight in the range of 10 kDa to 200 kDa, preferably up to 50 kDa.

[0597] XXII. The vaccine formulation as described in any of the preceding embodiments, wherein the adjuvant is selected from the group consisting of aluminum hydroxide, aluminum phosphate, aluminum hydroxyphosphate, and potassium aluminum sulfate, MF-59, liposomes, lipopolysaccharide, saponin, lipid A, lipid A derivative, monophosphoryl lipid A, GLA, 3-deacylated monophosphoryl lipid A, AS01, AS03, AF3, IL-2, RANTES, GM-CSF, TNF-a, IFN-g, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L and 41BBL, oligonucleotides, oligonucleotides comprising at least one unmethylated CpG and / or liposomes, Freund's adjuvant, Freund's complete adjuvant, Freund's incomplete adjuvant, polymers, copolymers such as polyoxyethylene-polyoxypropylene copolymers, including block copolymers, polymer p1005, CRL-8300 adjuvant, muramyl dipeptide, agonists of TLR1 / 2, TLR2, TLR3, TLR-4 agonists, TLR5, TLR7, TLR7 / 8, TLR8, TLR9, ODN 2216 (type A), TLR11 / 12, TLR-4 agonists, flagellin, flagellin derived from gram-negative bacteria, TLR-5 agonists, fragments of flagellin capable of binding to TLR-5 receptors, Alpha-C-galactosylceramide, chitosan, interleukin-2, QS-21, squalene, Quil A, cholera toxin B subunit, polyphosphazene and derivatives thereof, mycobacterial cell wall preparations, mycolic acid derivatives, non-ionic block copolymer surfactants, OMV, fHbp, a combination of saponin with sterol and lipid, dmLT, 1,25-dihydroxyvitamin D3, CAF01, poly[di(carboxylatophenoxy)-phosphazene] (PCPP), and Venezuelan equine encephalitis (VEE) replicon particles or combinations thereof.

[0598] XXIII. The vaccine formulation as described in any of the preceding embodiments, wherein the adjuvant is aluminum hydroxide having a particle size > 500 nm.

[0599] XXIV. The vaccine formulation as described in any of the preceding embodiments, wherein the one or more pharmaceutically acceptable excipients are

[0600] a. a buffer selected from the group consisting of a carbonate, a phosphate, an acetate, HEPES, a succinate, TRIS, a borate, a citrate, a lactate, a gluconate, a tartrate, or a combination thereof;

[0601] b. a sugar selected from the group consisting of trehalose, mannose, raffinose, lactobionic acid, glucose, maltitol, isomaltitol, maltose, lactose, dextrose, fructose, or a combination thereof;

[0602] c. a sugar alcohol or polyol selected from mannitol, lactitol, sorbitol, glycerol, xylitol, maltitol, lactitol, erythritol, isomalt, and hydrogenated starch hydrolysate, or a combination thereof;

[0603] d. a surfactant selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 85, nonoxynol-9, octoxynol-9, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyl-660 hydroxystearate, polyoxyl-35 castor oil, soybean lecithin, poloxamer, copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO), octoxynol, phospholipids, nonoxynol ethoxylate, polyoxyethylene fatty ether derived from lauryl alcohol, cetyl alcohol, stearyl alcohol, and oleyl alcohol, sorbitan esters, or a combination thereof;

[0604] e. a polymer selected from dextran, carboxymethylcellulose, hyaluronic acid, cyclodextrin, or a combination thereof;

[0605] f. a salt selected from NaCl, KCl, KH2PO4, Na2HPO4.2H2O, CaCl2, MgCl2, or a combination thereof;

[0606] g. an amino acid selected from tris(hydroxymethyl)methylaminomethane, leucine, isoleucine, glycine, glutamine, L-arginine, L-arginine hydrochloride, lysine, L-alanine, tryptophan, phenylalanine, tyrosine, valine, cysteine, glycine, methionine, proline, serine, threonine, or a combination thereof;

[0607] h. a hydrolyzed protein selected from gelatin, lactalbumin hydrolysate, monosodium glutamate, collagen hydrolysate, keratin hydrolysate, peptides, casein hydrolysate, whey protein hydrolysate, serum albumin, or a combination thereof;

[0608] i. a preservative selected from phenoxyethanol, benzalkonium chloride (Phemerol), phenol, m-cresol, thiomersal, formaldehyde, parahydroxybenzoic acid ester, benzalkonium bromide, benzyl alcohol, chlorobutanol, p-chloro-m-cresol, benzyl alcohol, or a combination thereof; and

[0609] j. a liquid carrier selected from water for injection (WFI) or saline.

[0610] XXV. The vaccine formulation as described in any of the preceding embodiments, wherein the vaccine formulation comprises:

[0611] - at least one recombinant protein / at least one modified fHbp as described in any of the preceding embodiments;

[0612] - aluminum hydroxide;

[0613] - mannitol;

[0614] - phosphate; and

[0615] - polysorbate.

[0616] XXVI. The vaccine formulation as described in any one of the preceding embodiments, wherein the vaccine formulation comprises:

[0617] - at least one modified fHbp of an amino acid sequence having at least 75% identity to any one of the amino acid sequences of SEQ ID NO. 6 to 10; or

[0618] - at least one modified fHbp encoded by a nucleic acid sequence having at least 75% identity to any one of the nucleic acid sequences of SEQ ID NO. 1 to 5; and

[0619] - aluminium hydroxide;

[0620] - mannitol;

[0621] - phosphate; and

[0622] - polysorbate.

[0623] XXVII. The vaccine formulation as described in any one of the preceding embodiments, comprising (i) at least one recombinant protein / modified fHbp fusion protein; (ii) aluminium hydroxide in an amount ranging from 0.5 mg / ml to 4.5 mg / ml; (iii) mannitol in an amount ranging from 5 mg / ml to 100 mg / ml; (iv) phosphate buffer in an amount ranging from 1 mM to 10 mM; and (v) polysorbate 20 in an amount ranging from 0.01 mg / ml to 2 mg / ml; wherein each recombinant protein / modified fHbp fusion protein is present in an amount ranging from 15 pg / ml to 150 pg / ml.

[0624] XXVIII. The vaccine formulation as described in any one of the preceding embodiments, comprising (i) at least two recombinant protein / modified fHbp fusion proteins; (ii) aluminium hydroxide in an amount ranging from 0.5 mg / ml to 4.5 mg / ml; (iii) mannitol in an amount ranging from 5 mg / ml to 100 mg / ml; (iv) phosphate buffer in an amount ranging from 1 mM to 10 mM; and (v) polysorbate 20 in an amount ranging from 0.01 mg / ml to 2 mg / ml; wherein each recombinant protein / modified fHbp fusion protein is present in an amount ranging from 15 pg / ml to 150 pg / ml.

[0625] XXIX. The vaccine formulation as described in any one of the preceding embodiments, comprising (i) at least three recombinant protein / modified fHbp fusion proteins; (ii) aluminum hydroxide in an amount ranging from 0.5 mg / ml to 4.5 mg / ml; (iii) mannitol in an amount ranging from 5 mg / ml to 100 mg / ml; (iv) phosphate buffer in an amount ranging from 1 mM to 10 mM; and (v) polysorbate 20 in an amount ranging from 0.01 mg / ml to 2 mg / ml; wherein each recombinant protein / modified fHbp fusion protein is present in an amount ranging from 15 pg / ml to 150 pg / ml.

[0626] XXX. The vaccine formulation as described in any one of the preceding embodiments, comprising (i) at least four recombinant protein / modified fHbp fusion proteins; (ii) aluminum hydroxide in an amount ranging from 0.5 mg / ml to 4.5 mg / ml; (iii) mannitol in an amount ranging from 5 mg / ml to 100 mg / ml; (iv) phosphate buffer in an amount ranging from 1 mM to 10 mM; and (v) polysorbate 20 in an amount ranging from 0.01 mg / ml to 2 mg / ml; wherein each recombinant protein / modified fHbp fusion protein is present in an amount ranging from 15 pg / ml to 150 pg / ml.

[0627] XXXI. The vaccine formulation as described in any one of the preceding embodiments, wherein the vaccine formulation comprises:

[0628] - at least one modified fHbp having an amino acid sequence that is at least 75% identical to any one of the amino acid sequences of SEQ ID NO. 6 to 10; or

[0629] - at least one modified fHbp encoded by a nucleic acid sequence that is at least 75% identical to any one of the nucleic acid sequences of SEQ ID NO. 1 to 5;

[0630] and

[0631] - aluminum hydroxide in an amount ranging from 0.5 mg / ml to 4.5 mg / ml;

[0632] - mannitol in an amount ranging from 5 mg / ml to 100 mg / ml;

[0633] - phosphate buffer in an amount ranging from 1 mM to 10 mM; and

[0634] - polysorbate 20 in an amount ranging from 0.01 mg / ml to 2 mg / ml.

[0635] XXXII. The vaccine formulation as described in any one of the preceding embodiments, comprising:

[0636] a. (SEQ ID NO. 6 of or encoded by SEQ ID NO. 1 ) fHbp V3.45 M5:PorA316-320 / exP1.14 in an amount ranging from 15 pg / ml to 150 pg / ml; or

[0637] b. (SEQ ID NO. 7 of or encoded by SEQ ID NO. 2) fHbp V2.19 M6:PorA316-320 / exP1.4 in an amount ranging from 15 pg / ml to 150 pg / ml; or

[0638] c. (SEQ ID NO. 8 of or encoded by SEQ ID NO. 3) fHbp V1.14:PorA307-311 / exP1.9 in an amount ranging from 15 pg / ml to 150 pg / ml; or

[0639] d. (SEQ ID NO. 9 of or encoded by SEQ ID NO. 4) fHbp V1.1 :PorA307-311 / exP1.4 in an amount ranging from 15 pg / ml to 150 pg / ml; and

[0640] e. aluminium hydroxide in an amount ranging from 0.5 mg / ml to 4.5 mg / ml;

[0641] f. mannitol in an amount ranging from 5 mg / ml to 100 mg / ml;

[0642] g. phosphate buffer in an amount ranging from 1 mM to 10 mM; and

[0643] h. polysorbate 20 in an amount ranging from 0.01 mg / ml to 2 mg / ml.

[0644] XXXIII. The vaccine formulation as described in any of the preceding embodiments, comprising:

[0645] a. (SEQ ID NO. 6 of or encoded by SEQ ID NO. 1 ) fHbp V3.45 M5:PorA316-320 / exP1.14 in an amount ranging from 15 pg / ml to 150 pg / ml;

[0646] b. (SEQ ID NO. 7 of or encoded by SEQ ID NO. 2) fHbp V2.19 M6:PorA316-320 / exP1.4 in an amount ranging from 15 pg / ml to 150 pg / ml;

[0647] c. (SEQ ID NO. 8 or encoded by SEQ ID NO. 3) fHbp V1.14:PorA307-311 / exP1.9 in an amount ranging from 15 pg / ml to 150 pg / ml;

[0648] d. (SEQ ID NO. 9 or encoded by SEQ ID NO. 4) fHbp V1.1 :PorA307-311 / exP1.4 in an amount ranging from 15 pg / ml to 150 pg / ml;

[0649] e. Aluminum hydroxide in an amount ranging from 0.5 mg / ml to 4.5 mg / ml;

[0650] f. Mannitol in an amount ranging from 5 mg / ml to 100 mg / ml;

[0651] g. Phosphate buffer in an amount ranging from 1 mM to 10 mM; and

[0652] h. Polysorbate 20 in an amount ranging from 0.01 mg / ml to 2 mg / ml.

[0653] XXXIV. The vaccine formulation of any of the preceding embodiments, comprising:

[0654] a. (SEQ ID NO. 6 or encoded by SEQ ID NO. 1) fHbp V3.45 M5:PorA316-320 / exP1.14 in an amount ranging from 15 pg / ml to 150 pg / ml; or

[0655] b. (SEQ ID NO. 7 or encoded by SEQ ID NO. 2) fHbp V2.19 M6:PorA316-320 / exP1.4 in an amount ranging from 15 pg / ml to 150 pg / ml; or

[0656] c. (SEQ ID NO. 10 or encoded by SEQ ID NO. 5) fHbp V1.1 :PorA307-311 / exP1.9 in an amount ranging from 15 pg / ml to 150 pg / ml; or

[0657] d. (SEQ ID NO. 8 or encoded by SEQ ID NO. 3) fHbp V1.14:PorA307-311 / exP1.9 in an amount ranging from 15 pg / ml to 150 pg / ml; and

[0658] e. Aluminum hydroxide in an amount ranging from 0.5 mg / ml to 4.5 mg / ml;

[0659] f. mannitol in an amount ranging from 5 mg / ml to 100 mg / ml;

[0660] g. phosphate buffer in an amount ranging from 1 mM to 10 mM; and

[0661] h. polysorbate 20 in an amount ranging from 0.01 mg / ml to 2 mg / ml.

[0662] XXXV. The vaccine formulation as described in any one of the preceding embodiments, comprising:

[0663] a. (SEQ ID NO. 6 or encoded by SEQ ID NO. 1) fHbp V3.45 M5:PorA316-320 / exP1.14 in an amount ranging from 15 pg / ml to 150 pg / ml;

[0664] b. (SEQ ID NO. 7 or encoded by SEQ ID NO. 2) fHbp V2.19 M6:PorA316-320 / exP1.4 in an amount ranging from 15 pg / ml to 150 pg / ml;

[0665] c. (SEQ ID NO. 10 or encoded by SEQ ID NO. 5) fHbp V1.1 :PorA307-311 / exP1.9 in an amount ranging from 15 pg / ml to 150 pg / ml;

[0666] d. (SEQ ID NO. 8 or encoded by SEQ ID NO. 3) fHbp V1.14:PorA307-311 / exP1.9 in an amount ranging from 15 pg / ml to 150 pg / ml;

[0667] e. aluminum hydroxide in an amount ranging from 0.5 mg / ml to 4.5 mg / ml;

[0668] f. mannitol in an amount ranging from 5 mg / ml to 100 mg / ml;

[0669] g. phosphate buffer in an amount ranging from 1 mM to 10 mM; and

[0670] h. polysorbate 20 in an amount ranging from 0.01 mg / ml to 2 mg / ml.

[0671] XXXVI. The vaccine formulation as described in any one of the preceding embodiments, wherein the formulation comprises 2-phenoxyethanol in an amount ranging from 1 mg / mL to 10 mg / ml.

[0672] XXXVII. The vaccine formulation as described in any of the preceding embodiments, wherein the vaccine composition is stable for a period of six months at 2-8 °C, 25 °C and 40 °C.

[0673] XXXVIII. The vaccine formulation as described in any of the preceding embodiments, having a zeta potential in the range of -16 mV to -30 mV; an osmolality in the range of 200 mOsmol / kg to 500 mOsmol / kg.

[0674] XXXIX. The vaccine formulation as described in any of the preceding embodiments, further comprising one or more antigens selected from diphtheria toxoid (D), tetanus toxoid (T), whole cell pertussis (wP), hepatitis B virus surface antigen (HbsAg), Haemophilus influenzae b PRP-carrier protein conjugate (Hib), Haemophilus influenzae (serotypes a, c, d, e, f and non-capsulated strains), Neisseria meningitidis A antigen, Neisseria meningitidis C antigen, Neisseria meningitidis W-135 antigen, Neisseria meningitidis Y antigen, Neisseria meningitidis X antigen, Streptococcus pneumoniae antigen, Neisseria meningitidis B bleb or purified antigen, Staphylococcus aureus antigen, anthrax, BCG, hepatitis (A, C, D, E, F and G strains) antigen, human papillomavirus, HIV, Salmonella typhi antigen, acellular pertussis, modified adenylate cyclase, malaria antigen (RTS, S), measles, mumps, rubella, dengue, zika, ebola, chikungunya, Japanese encephalitis, rotavirus, diarrhea antigen, flavivirus, smallpox, yellow fever, shingles, varicella virus antigen and combinations thereof.

[0675] XL. The vaccine formulation as described in any of the preceding embodiments, comprising (i) at least one fusion protein comprising a stabilized non-functional / non-lipidated fHbp and a PorA VR2 loop, and (ii) at least one polysaccharide-protein conjugate.

[0676] XLI. The vaccine formulation as described in any of the preceding embodiments, wherein the recombinant protein / modified fHbp fusion protein is co-administered with one or more vaccines selected from BEXSERO, MENVEO, MENACTRA, NIMENRIX, MenQuadFi, MENFIVE, MenAfriVac, Men AC and Men ACHib.

[0677] XLII. The vaccine formulation as described in any of the preceding embodiments, wherein the recombinant protein / modified fHbp fusion protein is co-administered with MENFIVE.

[0678] XLIII. The vaccine formulation as described in any of the preceding embodiments, comprising i) at least one fusion protein comprising a stabilized non-functional / non-lipidated fHbp and a PorA VR2 loop, and at least one conjugate selected from the group consisting of: (a) a conjugate of (i) a capsular polysaccharide of serogroup A N. meningitidis and (ii) tetanus toxoid; (b) a conjugate of (i) a capsular polysaccharide of serogroup C N. meningitidis and (ii) CRM197; (c) a conjugate of (i) a capsular polysaccharide of serogroup Y N. meningitidis and (ii) CRM197; (d) a conjugate of (i) a capsular polysaccharide of serogroup W135 N. meningitidis and (ii) CRM197; and (d) a conjugate of (i) a capsular polysaccharide of serogroup X N. meningitidis and (ii) tetanus toxoid.

[0679] XLIV. The vaccine formulation as described in any of the preceding embodiments, comprising i) at least two fusion proteins, each fusion protein consisting of one fHbp variant type coupled to one PorA VR2 loop, and at least one conjugate selected from the group consisting of: (a) a conjugate of (i) a capsular polysaccharide of serogroup A N. meningitidis and (ii) tetanus toxoid; (b) a conjugate of (i) a capsular polysaccharide of serogroup C N. meningitidis and (ii) CRM197; (c) a conjugate of (i) a capsular polysaccharide of serogroup Y N. meningitidis and (ii) CRM197; (d) a conjugate of (i) a capsular polysaccharide of serogroup W135 N. meningitidis and (ii) CRM197; and (d) a conjugate of (i) a capsular polysaccharide of serogroup X N. meningitidis and (ii) tetanus toxoid.

[0680] XLV. The vaccine formulation as described in any of the preceding embodiments, comprising i) at least three fusion proteins, each fusion protein consisting of one fHbp variant type coupled to two PorA VR2 loops, and at least one conjugate selected from the group consisting of: (a) a conjugate of (i) a capsular polysaccharide of serogroup A N. meningitidis and (ii) tetanus toxoid; (b) a conjugate of (i) a capsular polysaccharide of serogroup C N. meningitidis and (ii) CRM197; (c) a conjugate of (i) a capsular polysaccharide of serogroup Y N. meningitidis and (ii) CRM197; (d) a conjugate of (i) a capsular polysaccharide of serogroup W135 N. meningitidis and (ii) CRM197; and (d) a conjugate of (i) a capsular polysaccharide of serogroup X N. meningitidis and (ii) tetanus toxoid.

[0681] XLVI. The vaccine formulation as described in any one of the preceding embodiments, comprising i) at least four fusion proteins, each fusion protein consisting of one fHbp variant type coupled to three PorA VR2 loops, and at least one conjugate selected from the group consisting of: (a) a conjugate of (i) a capsular polysaccharide of serogroup A N. meningitidis and (ii) tetanus toxoid; (b) a conjugate of (i) a capsular polysaccharide of serogroup C N. meningitidis and (ii) CRM197; (c) a conjugate of (i) a capsular polysaccharide of serogroup Y N. meningitidis and (ii) CRM197; (d) a conjugate of (i) a capsular polysaccharide of serogroup W135 N. meningitidis and (ii) CRM197; and (d) a conjugate of (i) a capsular polysaccharide of serogroup X N. meningitidis and (ii) tetanus toxoid.

[0682] XLVII. The vaccine formulation as described in any one of the preceding embodiments, for use in the treatment or prevention of infection and / or disease caused by N. meningitidis serogroup B.

[0683] XLVIII. The vaccine formulation as described in any one of the preceding embodiments, wherein the vaccine formulation demonstrates cross-protection against Neisseria gonorrhea strains and N. meningitidis serogroups ACWYX.

[0684] XLIX. The vaccine formulation as described in any one of the preceding embodiments, wherein the percentage of adsorption of the recombinant protein / modified fHbp fusion protein on the adjuvant is in the range of 70% to 100%.

[0685] L. The vaccine formulation as described in any one of the preceding embodiments, wherein the percentage of adsorption of the fHbp V3.45 M5 PorA 316-320 exP1.14 on the adjuvant is in the range of 80% to 100%.

[0686] LI. The vaccine formulation as described in any one of the preceding embodiments, wherein the percentage of adsorption of the fHbp V1.14 PorA 307-311 exP1.9 on the adjuvant is in the range of 80% to 90%.

[0687] LII. The vaccine formulation as described in any one of the preceding embodiments, wherein the percentage of adsorption of the fHbp V2.19 PorA 316-320 exP1.4 on the adjuvant is in the range of 80% to 90%.

[0688] LIII. The vaccine formulation as described in any one of the preceding embodiments, wherein the percentage of adsorption of the fHbp V1.1 PorA 307-311 exP1.4 on the adjuvant is in the range of 80% to 90%.

[0689] LIV. The vaccine formulation as described in any one of the preceding embodiments, wherein the percentage of adsorption of fHbp V1.1 PorA 307-311 ex P1.9 on the adjuvant is in the range of 70% to 80%.

[0690] LV. A method for manufacturing the vaccine formulation as described in any one of the preceding embodiments, the method comprising the steps of:

[0691] a. culturing a host cell comprising an expression vector in a nutrient medium;

[0692] b. inducing the host cell to express the protein;

[0693] c. harvesting and isolating the host cell;

[0694] d. lysing the harvested cell and isolating host cell debris to obtain the marker protein;

[0695] e. purifying the marker protein;

[0696] f. removing the tag from the marker protein to obtain the recombinant protein / modified fHbp fusion protein;

[0697] g. purifying the recombinant protein / modified fHbp fusion protein; and

[0698] h. preparing the vaccine formulation comprising the purified recombinant protein / modified fHbp fusion protein.

[0699] LVI. The method as described in any one of the preceding embodiments, wherein the host cell is a bacterial expression host system.

[0700] LVII. The method as described in any one of the preceding embodiments, wherein the bacterial expression host system is an E. coli strain selected from BL21(DE3), BL21(DE3)pLysS*, BL21(DE3)pLysE*, BL21 star (DE3), BL21-A1, BLR(DE3), HMS174(DE3)**, Tuner(DE3), Origami2(DE3)**, Rosetta2(DE3)*, Rosetta gami(DE3), Lemo21(DE3)*, T7 Express, m15 pREP4*, C41(DE3), C43(DE3), or B834(DE3).

[0701] LVIII. The method as described in any one of the preceding embodiments, wherein the nutrient medium is selected from the group consisting of undefined medium, Terrific Broth (TB) medium, Lysogenia Broth, Luria Broth or Luria-Bertani medium, chemically defined medium, M9 minimal medium, chemically defined M9 minimal salts medium, 2xYT medium, or Super Optimal broth (SOC) medium with catabolite repression, and combinations thereof.

[0702] LIX. The method as described in any one of the preceding embodiments, wherein the concentration of L-methionine is maintained in the range of 1 mM to 10 mM during the growth of the host cell in step (a), wherein the fermentation is in fed-batch mode.

[0703] LX. The method as described in any one of the preceding embodiments, wherein the host cell is grown at a temperature in the range of 35 °C to 39 °C; at a pH in the range of 5.0 to 9.0; at dissolved oxygen in the range of 10% to 100%; at agitation in the range of 100-1800 rpm; at a gas flow rate in the range of 0-2 volume of gas per minute per unit volume of liquid (VVM).

[0704] LXI. The method as described in any one of the preceding embodiments, wherein the host cell is induced using an inducer selected from the group consisting of lactose and its non-hydrolyzed analogue isopropyl beta-D-l-thiogalactopyranoside (IPTG).

[0705] LXII. The method as described in any one of the preceding embodiments, wherein the concentration of lactose is in the range of 1 g / L to 50 g / L and the concentration of IPTG is in the range of 1 mM to 10 mM.

[0706] LXIII. The method as described in any one of the preceding embodiments, wherein the host cell is lysed using a method selected from the group consisting of chemical mode, biological mode, physical mode, mechanical mode, and combinations thereof.

[0707] LXIV. The method as described in any one of the preceding embodiments, wherein the host cell is lysed using a combination of chemical mode and mechanical mode.

[0708] LXV. The method as described in any one of the preceding embodiments, wherein the host cell is lysed using a lysis buffer having a pH in the range of 7-9 followed by mechanical lysis of 3-8 cycles at a pressure in the range of 1000-1500 bar.

[0709] LXVI. The method as described in any one of the preceding embodiments, wherein the mechanical lysis is performed using a homogenizer.

[0710] LXVII. The method of any of the preceding embodiments, wherein the tagged protein in step (e) is purified using a chromatography step, followed by concentration and diafiltration.

[0711] LXVIII. The method of any of the preceding embodiments, wherein the tag is removed using TEV protease having a protein:TEV protease ratio in the range of 5: 1 to 30: 1.

[0712] LXIX. The method of any of the preceding embodiments, wherein the recombinant protein / modified fHbp fusion protein in step (g) is purified using a chromatography step, followed by concentration and diafiltration.

[0713] LXX. The method of any of the preceding embodiments, wherein the chromatography is selected from the group consisting of column chromatography, ion exchange chromatography, anion exchange chromatography, cation exchange chromatography, column chromatography, flash chromatography, gel filtration / size exclusion / gel permeation (molecular sieve) chromatography, affinity chromatography, paper chromatography, thin layer chromatography, gas chromatography, dye ligand chromatography, hydrophobic interaction chromatography, pseudo-affinity chromatography, liquid chromatography, high pressure liquid chromatography (HPLC), immobilized metal affinity chromatography, anion exchange chromatography, cation exchange chromatography, multimodal chromatography, multimodal anion exchange chromatography, electrostatic interaction chromatography, hydrogen bond chromatography, reverse phase chromatography, and combinations thereof.

[0714] LXXI. The method of any of the preceding embodiments, wherein the tagged protein is expressed as inclusion bodies (IBs) and purified by urea unfolding the protein and refolding the protein on column.

[0715] LXXII. The method of any of the preceding embodiments, wherein the vaccine formulation is prepared by adsorbing the individual recombinant protein / modified fHbp fusion proteins onto an adjuvant, followed by addition to an excipient mixture comprising a sugar alcohol, a buffer, a stabilizer, and a liquid carrier.

[0716] LXXIII. The method of any of the preceding embodiments, wherein the excipient mixture comprises a preservative.

[0717] LXXIV. The method of any of the preceding embodiments, wherein the TEV protease is produced by a method comprising:

[0718] a. culturing a host cell comprising an expression vector in a nutrient medium;

[0719] b. inducing the host cell to express the TEV protease;

[0720] c. harvesting and isolating the host cell;

[0721] d. lysing the harvested host cells and isolating the host cells to obtain the TEV protease; and

[0722] e. purifying the TEV protease; and

[0723] f. concentrating / diafiltering and storing the purified TEV protease.

[0724] LXXV. The method as described in any one of the preceding embodiments, wherein the host cell for expressing the TEV protease is an E. coli strain selected from BL21(DE3), BL21(DE3)pLysS*, BL21(DE3)pLysE*, BL21 star (DE3), BL21-A1, BLR(DE3), HMS174(DE3)**, Tuner(DE3), Origami2(DE3)**, Rosetta2(DE3)*, Rosettagami(DE3), Lemo21(DE3)*, T7Express, m15pREP4*, C41(DE3), C43(DE3), Rosetta(DE3)pLysS, or B834(DE3).

[0725] LXXVI. The method as described in any one of the preceding embodiments, wherein the glucose feed is stopped when the OD at 590 / 600 nm is 20-100, and the glycerol feed is started, and the culture is induced by adding lactose and / or maintaining lactose at 1-50 g / L in fed-batch mode.

[0726] LXXVII. A method of inducing an immune response against a N. meningitidis serogroup B strain by administering to an individual a vaccine formulation as described in any one of the preceding embodiments, wherein the administering step induces an immune response against a N. meningitidis serogroup B strain. Example

[0727] The foregoing description of the implementations has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the

[0728] The disclosure is further described in view of the following examples, which are presented for purposes of illustration only and are not to be construed as limiting the scope of the disclosure.

[0729] The method of manufacturing a vaccine formulation according to the disclosure comprises the following broad steps, which are explained in further detail in the subsequent paragraphs:

[0730] - culturing the host cell comprising the expression vector in a nutrient medium;

[0731] - inducing host cells to express proteins;

[0732] - harvesting and isolating host cells;

[0733] - lysing the harvested cells and isolating host cell debris to obtain tagged proteins;

[0734] - purifying tagged proteins;

[0735] - removing tags from tagged proteins to obtain recombinant proteins / modified fHbp fusion proteins;

[0736] - purifying recombinant proteins / modified fHbp fusion proteins;

[0737] - preparing vaccine formulations comprising purified recombinant proteins / modified fHbp fusion proteins.

[0738] Example-1: Expression of tagged fHbp protein and TEV protease

[0739] Source of biological resources used in the present disclosure:

[0740] 1) Host cells: E. coli B834 (DE3) for expression of recombinant proteins; Catalogue No. D48175, Sigma Aldrich (Transferred from Oxford University to Serum Institute of India Pvt. Ltd.)

[0741] 2) Plasmid*: pET-28a(+) DNA; Catalogue No. D48556, EMD Millipore (Transferred from Oxford University to Serum Institute of India Pvt. Ltd.)

[0742] 3) Host cells: Rosetta™ (DE3) pLysS competent cells for expression of TEV protease; Catalogue No. D49062, Sigma Aldrich

[0743] 4) Plasmid*: pET-28a(+) DNA; Catalogue No. D48556, EMD Millipore (Transferred from Oxford University to Serum Institute of India Pvt. Ltd.)

[0744] (*Gene of interest cloned / inserted into backbone of pET-28a(+) plasmid)

[0745] 1A: Expression of tagged fHbp protein

[0746] Thirty-three clone constructs expressing the fHbp-PorA chimeric protein were received from the University of Oxford. Of these 33 clones, 24 were screened for protein expression, details of which are provided in Table 3. From these 24 screened clones, five were ultimately selected for vaccine formulation based on molecular and biochemical characterization, as listed in Table 13. Sequence lists of the recombinant protein / modified fHbp fusion protein and the TEV protease are provided in Table 13a.

[0747]

[0748]

[0749] The expression vector used to express the fHbp protein marker is pET28a-His-MBP-TEV-fHbp-PorA. See Table 14 below and... Figures 1-5 The document provides details of the vector map and nucleotide sequence.

[0750]

[0751] The labeled fHbp protein was produced in a 10L fermentation batch. Details of the culture media used are provided in Tables 15 to 19.

[0752] Stock solutions for culture medium and feed preparation:

[0753] 1) Alkaline solution: 14% ammonia solution

[0754] 2) Acid solution: 6% phosphoric acid

[0755] 3) Defoamer solution: 10% defoamer (STRUKTOL® J 673 A)

[0756]

[0757]

[0758]

[0759] Details of the feed solution:

[0760] Methionine stock solution: 45 mg / mL L-methionine solution

[0761]

[0762]

[0763] Inducing agent: lactose monohydrate solution

[0764] exist Figure 7 The document provides a seed development process for a 10L scale fermentation batch for producing the labeled fHbp protein. Figure 8 The document provides a process for producing labeled fHbp proteins in a 0L scale fermenter.

[0765] according to Figures 7 to 8 The process provided in the paper is used to grow each labeled fHbp protein separately.

[0766] During the purification of the labeled protein from cell lysates, the clone fHbp V3.45 M5:PorA was encountered. 316 -320 / exP1.14 and fHbp V2.19 M6:PorA 316-320 / exP1.4 Protein precipitation occurred. To address this issue, during fermentation, the temperature setpoint was changed from 37°C to 33°C during induction.

[0767] The effects of temperature changes are shown below:

[0768] Cloning fHbp V2.19 M6: PorA 316-320 / exP1.4 :

[0769] - Low temperature induction (33°C) resulted in the expression of the major part of the labeled fHbp protein in a soluble form.

[0770] -Lowering the temperature by 4°C solved the protein precipitation problem during downstream processing.

[0771] Cloning fHbp V3.45 M5: PorA 316-320 / exP1.14 :

[0772] - Low temperature induction (33℃) resulted in the expression of the major portion of the labeled fHbp protein in a soluble form.

[0773] - Although the problem has been addressed to some extent, it persists during downstream processing.

[0774] exist Figures 9a to 9h The study provides growth curves and SDS-PAGE gel images of labeled fHbp protein from a 10L fermentation batch. Figure 9a and 9b The growth curves and SDS-PAGE gel images of fHbpV3.45 M5:PorA316-320 / exP1.14 (SEQ ID NO.1, 6) in a 10L fermentation batch are shown respectively. Figure 9c and 9dThe growth curves and SDS-PAGE gel images of fHbpV2.19 M6:PorA316-320 / exP1.4 (SEQ ID NO.2, 7) in a 10L fermentation batch are shown respectively. Figure 9e and 9f The growth curves and SDS-PAGE gel images of fHbpV1.14:PorA307-311 / exP1.9 (SEQ ID NO. 3, 8) in 10L fermentation batches were described respectively; and Figure 9g and 9h The growth curves and SDS-PAGE gel images of fHbpV1.1:PorA307-311 / exP1.4 (SEQ ID NO.4, 9) in a 10L fermentation batch are presented respectively.

[0775] Analysis from SDS-PAGE ( Figure 9b , 9d As can be seen from 9f and 9h, the major portions of all recombinantly labeled fHbp proteins (60kDa to 80kDa) are expressed in soluble form.

[0776] 1B: Expression of TEV protease

[0777] The expression vector used to express the TEV protease is His-Gst-TEV protease (pET28a) (kanamycin / chloramphenicol). Details of the vector map and nucleotide sequence are provided in Table 20 and the figure below.

[0778]

[0779] TEV protease was produced in a 10L fermentation batch. Details of the culture media used are provided in Tables 21 to 24.

[0780] Stock solutions for culture medium and feed preparation:

[0781] 1) Alkaline solution: 14% ammonia solution

[0782] 2) Acid solution: 6% phosphoric acid

[0783] 3) Defoamer solution: 10% defoamer (STRUKTOL® J 673 A)

[0784]

[0785]

[0786]

[0787] Details of the feed solution –

[0788] L-methionine stock: 45 mg / mL L-methionine solution

[0789]

[0790] Inducer - IPTG solution

[0791] A seed development process for 10 L scale fermentation batch for production of TEV protease is provided in Figure 10 A process for production of TEV protease in 10 L scale fermenter is provided in Figure 11

[0792] During purification of TEV protease from cell lysate, protein precipitation was encountered. To address this issue, during fermentation, at the time of induction, temperature set point was changed from 37 °C to 20 °C, i.e., culture was induced with IPTG at 20 °C for 12-16 hours.

[0793] The impact of temperature change is given below:

[0794] - Low temperature induction (20 °C) resulted in major portion of TEV protein being expressed in soluble form.

[0795] - Low temperature induction (20 °C) addressed the issue of protein precipitation during downstream processing.

[0796] - As the culture was induced at 20 °C, the induction time was increased to 12-16 hours.

[0797] Growth curves for TEV protease for 10 L scale fermentation batch are provided in Figure 12a and Figure 12b SDS-PAGE gel images, respectively. From SDS-PAGE analysis Figure 12b ) it can be seen that major portion of TEV protease (50 kDa) was expressed in soluble form.

[0798] Example-2: Purification of labeled fHbp protein and TEV protease

[0799] Harvested cells obtained in Example-1 were subjected to purification using following general steps in any order:

[0800] - Cell lysis by chemical and mechanical means;

[0801] - Chromatographic separation;

[0802] - One or more washing steps;

[0803] - Elution of protein;

[0804] - Concentration and diafiltration of protein​

[0805] - Store the protein until further use.

[0806] In case of expression of the marker protein as inclusion bodies (IB), additional steps of ammonium sulphate precipitation, urea unfolded protein and on-column refolding of the protein were performed.

[0807] The purification steps are explained in detail in the following section. Studies were performed to optimize various parameters (e.g. concentration of the chimeric protein, excipients etc.) which are explained in detail below.

[0808] Optimization studies:

[0809] Optimization of parameters such as mechanical lysis of the cells and TEV protease cleavage was performed.

[0810] i) Homogenizer optimization:

[0811] The harvested E. coli cell mass (for expression of the marker fHbp protein and TEV protease) was lysed in lysis buffer (25 mM Na-phosphate buffer + 100 mM NaCI + 20 mM imidazole, pH 7.4) and the cells were lysed with a Panda homogenizer at a pressure of 1000-1200 bar for 6 cycles. The 1st cycle was run without pressure and the next 5 cycles were run at a pressure of 1000-1200 bar.

[0812] The lysate was collected after each cycle and the optical density was determined at 600 nm using a spectrophotometer. The results are shown in Table 25.

[0813]

[0814] Conclusion - The optical density (OD) of the resuspended cells decreased only after the 1st cycle where pressure was applied. A further decrease in OD was observed up to the 3rd cycle. The 4th and 5th cycles did not show a significant difference in OD.

[0815] Taking into account that the resuspended sample has 100% viable cells and 0% lysis, the percentage lysis was calculated for all homogenization cycles and the results obtained are shown in Table 26 and Figure 13 .

[0816]

[0817] Conclusion - As can be seen from Table 26 and Figure 13 , about 80% lysis was achieved in the 1st cycle and was further decreased to about 98% at the end of the 3rd cycle. Therefore, at least / minimum 3 homogenization cycles at a pressure of 1000-1200 bar are necessary for efficient lysis of E. coli cells.

[0818] It is to be noted that in the present method, chemical lysis is used along with mechanical lysis for cell disruption. Chemical lysis is performed using lysis buffer with higher osmolality and mechanical lysis is performed using cell homogenizer.

[0819] Homogenizers are used for large scale (industrial / production scale; model capacity - 10 L / hour to thousands of L / hour) cell disruption and for providing consistency in lysis, whereas sonication is used for small scale (less than 100 ml) cell disruption.

[0820] ii) TEV protease cleavage process:

[0821] - Enzyme: substrate concentration optimization:

[0822] His-MBP tagged protein (FHBP V1.1: PorA 307-311 / exP1.4 ) was used for optimization of enzyme: substrate concentration of TEV protease (substrate here is tagged fHbp protein). For 1 part of TEV protease, different parts of substrate 5, 10, 20, 30 and 40 parts were studied. Incubation was performed at 30 °C for 1 hour with gentle mixing and samples were collected. Further identical reactions were incubated at 30 °C for 18 hours with gentle mixing as per Example-2C. TEV protease cleavage reactions were loaded on SDS-PAGE and results obtained are shown in Figure 14a and 14b .

[0823] Further, densitometry analysis was performed using Biorad gel documentation system where band intensity was measured and percentage of product formed was calculated considering 100% cleavage at 1:5 ratio. Results obtained are shown in Table 27 and Figure 14b . Figure 15

[0824]

[0825] Conclusion: From Table 27 and Figure 15 it can be seen that higher degree of cleavage was obtained using enzyme: substrate ratio of 1:5 and 1:10 after 1 hour of incubation. However, saturation was observed at 1:5, 1:10 and 1:20, i.e., about 95% and above product formation after 18 hours of incubation. Un-cleaved substrate was observed at higher enzyme: substrate ratio of 1:30 and 1:40 with product formation of about 81% and 76% respectively, indicating that longer duration might be required to achieve complete cleavage at these higher enzyme: substrate ratios. Therefore, for TEV cleavage, it was found that enzyme: substrate ratio of 1:20 or lower enzyme: substrate ratio incubated for 18 hours is optimal.

[0826] ​Similar findings were observed for the rest of the tagged fHbp proteins.

[0827] Temperature optimization and results of TEV protease cleavage experiments

[0828] His-MBP tagged protein (FHBP V1.1 : PorA 307-311 / exP1.4) was used to optimize TEV protease cleavage at various temperatures 5°C to 50°C. For 1 part TEV protease, 20 parts of substrate were used. Incubation was performed at various temperatures 5°C to 50°C for 1 hour with gentle mixing and samples were collected. As shown in Figure 16 TEV protease cleavage reactions at various temperatures were loaded on SDS-PAGE. In addition, densitometric analysis was performed using Biorad gel documentation system, where band intensities were measured and fold change of product formed was calculated considering the product intensity formed at initial temperature 5°C has value 1. The results obtained are shown in Table 28 and Figure 16 Figure 17

[0829]

[0830] Conclusion: From Table 28 and Figure 17 it can be seen that at 0 hour, no cleavage was observed. After 1 hour, product / chimeras formed at 30°C were higher, which was 2.5 times at 5°C. Thus, the optimum temperature for TEV protease cleavage was found to be around 30°C.

[0831] Example-2A: Purification of TEV protease

[0832] TEV protease harvested in Example-1B was purified using the process according to Figure 18 where the lysis buffer had pH 7.4.

[0833] Isoelectric point (pi) of a protein is defined as the pH at which the net charge of a protein molecule is zero. At solution pH above pi, the surface of the protein is predominantly negatively charged and thus molecules of the same charge will exhibit repulsive forces. Similarly, at solution pH below pi, the surface of the protein is predominantly positively charged and repulsion between proteins occurs. However, at pi, the negative and positive charges balance, reducing the repulsive electrostatic forces and attractive forces dominate, leading to aggregation and precipitation.

[0834] HIS-GST-TEV protease has pi 7.8, therefore, working pH of 7.4 did not result in the desired purity. Therefore, working pH was increased to 8.5, which resulted in improved purity of HIS-GST-TEV protease according to the process shown in Figure 19

[0835] ​​​Figure 20a and Figure 20b Purification profiles of TEV protease at pH 8.5 and pH 7.4 are shown in Figures 1 and 2, respectively. The lanes marked with a star symbol (★) represent the final elution fraction of TEV protease. The purity of TEV protease produced using the process at pH 8.5 is higher compared to the purity of TEV protease produced using the process at pH 7.4.

[0836] Example-2B: Purification of labeled fHbp proteins

[0837] According to Figure 21 The labeled fHbp proteins harvested in Example-1A were purified individually.

[0838] The labeled fHbp proteins were soluble at the working pH used for purification and the above process ( Figure 21 ) produced labeled fHbp proteins with the desired purity. Figure 21 The purification process shown was used for the purification of fHbp V3.45 M5:PorA316-320 / exP1.14, fHbp V2.19 M6:PorA316-320 / exP1.4, fHbp V1.14:PorA307-311 / exP1.9, fHbp V1.1:PorA307-311 / exP1.9 and fHbp V1.1:PorA307-311 / exP1.4.

[0839] However, during scale-up, the labeled fHbp proteins expressed at high rates can have solubility issues due to the aggregation of the proteins into inclusion bodies (IBs). IBs are formed due to the aggregation of partially folded and misfolded protein molecules.

[0840] To avoid such solubility issues and recover the expressed proteins from IBs, an alternative process ( Figure 22 ) involving urea can be used.

[0841] It is known that some inclusion bodies have protein molecules with native-like conformation and some inclusion bodies have significant biological activity.

[0842] In the alternative method ( Figure 22 ), the labeled proteins expressed in IBs are solubilized with 8M urea and subsequently bound to a Ni-Sepharose 6FF column for on-column refolding and purification.

[0843] The urea is removed from the column by reducing its concentration from 8M to 1M in the first step. Subsequently, the column is washed with 25mM Na-Phosphate buffer + 100mM NaCl, pH 7.4 for 5-6 column volumes to completely remove the urea from the column and the recombinant protein / modified fHbp fusion protein bound to the column.

[0844] Example-2C: Process for removal of tag from purified tagged fHbp protein

[0845] According to Figure 23 His-MBP tag from the purified protein (tagged fHbp protein) using TEV protease (SEQ ID NO. 12 or encoded by SEQ ID NO. 11) following the process outlined in

[0846] Following removal of the tag from the fHbp protein, the chimeric protein obtained in the digestion mixture was further purified by using two step chromatography of ion exchange chromatography Figure 24 followed by affinity chromatography Figure 25

[0847] Characterization of chimeric proteins produced using the process of the present disclosure is provided in Table 29.

[0848]

[0849] fHbp specifically binds to human factor H (fH), which downregulates complement activation and enhances the ability to resist bactericidal activity. Modifications introduced in the fHbp antigen result in reduced fH binding and can increase the protective antibody response.

[0850] As can be seen from Table 29, the recombinant fHbp has >85% reduced fH binding compared to wild type fHbp, which would help to improve the immunogenicity of the vaccine formulation containing these recombinant fHbp.

[0851] Previously, the digestion mixture was purified using multi-step chromatography including metal affinity chromatography, cation exchange, anion exchange and gel filtration chromatography (GFC). Whereas the present process uses 2-step chromatography (anion exchange and metal affinity chromatography) following TEV protease mediated tag removal. The chimeric proteins produced using the 2-step chromatography of the present disclosure show similar profile of characteristics and purity to the chimeric proteins produced using multi-step chromatography.

[0852] Example-3: Formulations comprising recombinant protein / modified fHbp fusion protein / chimeric protein

[0853] Different formulations of recombinant protein / modified fHbp fusion protein were prepared and evaluated to obtain a formulation with optimal physicochemical characteristics, stability and immunogenicity.

[0854] i) Adjuvant optimization:

[0855] ​To enhance the immunogenic response and physico-chemical properties, formulation development started with adsorption of proteins on different adjuvants such as aluminium hydroxide (Alhydrogel), aluminium phosphate (Adjuphos), double mutant heat labile toxin (dmLT) etc. These formulations were evaluated based on different physico-chemical parameters. The summary of the experiments, their results and conclusions are provided below.

[0856]

[0857] Conclusion: Three adjuvants, namely dmLT, Alhydrogel and Adjuphos were evaluated. As can be seen from Table 30, the formulations adsorbed with Alhydrogel and Adjuphos gave satisfactory zeta potential as compared to the formulation adsorbed with dmLT.

[0858] Alhydrogel has an isoelectric point (pi) of about 11.4, whereas Adjuphos has a pi value between 4.5-6.0. All fHbp proteins have a pi in the range of 5.5 to 6.5. Therefore, to achieve optimal adsorption of all proteins on adjuvant in the vaccine formulation, further studies were carried out using Alhydrogel.

[0859] ii) Optimization of buffer and sugar:

[0860]

[0861] As can be seen from Table 31, when sodium chloride and sucrose were used in the formulation, the desired zeta potential was not obtained for lot 051021-A and lot 051021-B. Sucrose was used in the initial phase of formulating the composition. However, the composition containing sucrose did not result in optimal physico-chemical characteristics and hence was not included in the subsequent compositions.

[0862] When phosphate buffer was added to the formulation (lot 061021), improvement in zeta potential was observed, but the pH value was not in the expected range. Further studies were carried out using mannitol and phosphate buffer. The details of the formulation and the results obtained are summarized in Table 32.

[0863]

[0864] As can be seen from Table 32, when mannitol and phosphate buffer were used in the formulation, the pH, osmolality and zeta potential were in the expected range. It was also observed that increasing the concentration of mannitol from 4% to 5% resulted in optimal osmolality of the formulation.

[0865] iii) Single chimeric protein formulation:

[0866] Similar studies were performed for the remaining proteins. The formulation details and the results obtained are summarized in Table 33.

[0867]

[0868] It can be seen that the use of formulation comprising mannitol (5% v / v), polysorbate 20 (0.05 mg / mL) as provided in Table 33 resulted in optimal physicochemical parameters for all individual proteins.

[0869] iv) Formulations comprising multiple chimeric proteins:

[0870] Similar to the individual protein formulations, the physicochemical properties of the formulations comprising combinations of proteins were determined. The formulation details and the results obtained are summarized in Table 34. The proteins were individually adsorbed on the adjuvant and then added to the excipient mixture to obtain the formulation.

[0871]

[0872] It can be seen from Table 34 that the formulation was stable when stored at 2-8 °C for about 7 days. Further, the pH, zeta potential and % adsorption remained stable. Therefore, this combination of excipients in the formulation was used for further scale-up.

[0873] v) Optimization of chimeric protein concentration:

[0874] Studies were performed using higher concentrations of chimeric protein to determine its effect on pH, osmolality and zeta potential. The formulation details and the results obtained are summarized in Tables 35a and 35b.

[0875]

[0876]

[0877] It can be seen from Tables 35a and 35b that the formulation of higher concentration of protein in combination with phosphate buffer, polysorbate 20 and 5% mannitol conferred optimal pH, osmolality and zeta potential.

[0878] vi) Optimization of excipient concentration:

[0879] Studies were performed to optimize the concentration of excipients. The formulation details and the results obtained are summarized in Tables 36 to 39.

[0880]

[0881]

[0882]

[0883]

[0884] As can be seen from Tables 36 to 39, the combination of 5% v / v mannitol; 0.05 mg / mL polysorbate 20; 5 mM phosphate buffer (pH 7.0) and 0.5% w / v 2-phenoxyethanol with 120 pg / mL of each chimeric protein resulted in the most optimal profile characteristics.

[0885] vii) Optimization of pH:

[0886] Studies were conducted to check the effect of pH on adsorption. The formulation details and the results obtained are summarized in Tables 40 and 41.

[0887]

[0888]

[0889] As can be seen from Tables 40 to 41, increasing the pH of the phosphate buffer to 7.4 resulted in an increase in the pH of the final formulation (7.83 to 7.86). Further, the increase in the pH of the phosphate buffer resulted in a decrease in protein adsorption from 94% to 85%.

[0890]

[0891]

[0892] As can be seen from Tables 42 to 43, when formulated using phosphate buffer with a pH of 7.0, a final pH in the range of 7.3 to 7.5 was achieved along with an increase in protein adsorption.

[0893] Example-4: Stability studies on vaccine formulation comprising recombinant protein / modified fHbp fusion protein

[0894] Stability studies were conducted for 2 batches at 2-8°C for 6 months and 10 months. The formulation details and the results obtained are summarized in Table 44.

[0895]

[0896] Conclusion: As can be seen from Table 44, the % protein adsorption, pH, zeta potential and PSD of the vaccine formulation comprising recombinant protein / modified fHbp fusion protein were within the desired range at 2-8°C for up to 6 months.

[0897] Example-5: Immunogenicity studies on formulations comprising recombinant / chimeric proteins

[0898] Details of the biological materials used are provided below:

[0899] Neisseria meningitidis serogroup B strain: Neisseria meningitidis serogroup B strain:

[0900] A glycerol stock of N. meningitidis was received from Professor Chris Tang (Sir William Dunn School of Pathology, UK). Four N. meningitidis serogroup B strains were received. Strain details are provided below and are further summarised in Table 45.

[0901] 1. N. meningitidis M08.240157 (fHbp V1.1 : PorA VR2 16)

[0902] 2. N. meningitidis M17.240832 (fHbp 1.4 : PorA VR2 4)

[0903] 3. N. meningitidis M17.240156 (fHbp 3.45 : PorA VR2 14)

[0904] 4. N. meningitidis M18 240043 (fHbp 1.15 : PorA VR2 15-11)

[0905]

[0906] Details of the formulations used for immunogenicity studies are summarised in Table 46.

[0907]

[0908] Serum bactericidal assay

[0909] Procedure for hSBA against Men B strains

[0910] Preparation of SBA from working stocks

[0911] Take the master stock vial from the deep freeze and streak the culture rapidly onto a blood agar plate (Columbia blood agar with 5% horse blood) that has been dried. Incubate the plate overnight at 37°C, 5% C02. Pick approximately 50 colonies with a sterile loop and resuspend (to create a dense suspension) in a sterile 50ml falcon containing 15ml of BHI broth with glycerol. Aliquot the suspension into 0.25ml volumes into 1.8ml cryovials. Label the vials and store at -70°C in the deep freeze.

[0912] Assay procedure for serum bactericidal assay for antibody titration against Men B strains:

[0913] Neisseria meningitidis B strain (working stock) was streaked onto individual blood agar plates and incubated overnight at 37°C, 5% C02. The culture was streaked again onto fresh blood agar plates to achieve confluent growth as a square in the center of the plate. For all Men B strains, the plates were incubated at 37°C, 5% C02 in a C02 incubator for 4 hours (+ 15 minutes). Growth scraped from the center of the plate was suspended in 5 ml of Ca+2 and Mg+2 containing Hanks Balanced Salt Solution / Buffer (Invitrogen) to make a suspension. Using 1 ml of the suspension, the absorbance was read at 650 nm. The suspension was adjusted by dilution to 650 nm = 0.1 OD, then diluted 1 / 10 with buffer, then diluted 1 / 250. 20 μΐ of buffer was added to all wells of a 96-well microtiter plate up to column eleven. 20 μΐ (diluted or undiluted) test serum sample was added to the first column of the plate; the last two rows were reserved for quality control sera. 20 μΐ was serially diluted from the first column to the second column, and so on up to the ninth column, discarding the 20 μΐ from the ninth column. The tenth and eleventh columns were reserved for complement controls. 10 μΐ of the bacterial suspension that had been prepared was added to all wells up to the eleventh column. 10 μΐ of human complement was added to all wells up to the tenth column. 10 μΐ of heat inactivated (30 minutes at 56°C) human complement was added to all wells in the eleventh column. The contents of the wells were mixed well, and the plate was incubated at 37°C for 1 hour. After incubation, 10 μΐ of the contents of each well was spotted onto appropriately labeled blood agar plates. The blood agar plates were incubated in a C02 incubator at 37°C, 5% C02. The next day, bacterial colonies were counted using an automated colony counter (Synbiosis - ProtoCOL3).

[0914] Study details:

[0915] An immunogenicity study was performed in rabbits (New Zealand white rabbits) using 3 formulations / group, G1 - Trumenba (Pfizer), G2 - Formulation 1, G3 - Formulation 2, with each formulation injected into 4 male and 4 female rabbits. Injections were given on days 0, 14, and 28, and bleeds were performed on DO and D37. Serum samples were analyzed for immunogenicity by hSBA (serum bactericidal assay using human complement). All rabbits were given a full human dose. Drug product: Meningococcal B vaccine (MenB) (250423 - Formulations 1 and 2)

[0916] Study design: The study design is summarized in Table 47.

[0917]

[0918] Criteria for significant differences between groups:

[0919] A 2-fold change in titer was considered assay variability, and a >4-fold difference in titer between comparison groups was considered significant.

[0920] Objectives and scope:

[0921] The objective of this study was to compare the immunogenic response of individual Men B formulations - Formulation 1 and Formulation 2, and Trumenba - against Men B strains - M 08240157 (fhbp 1.1), M 17240832 (fhbp 1.4), M 17240156 (fhbp 3.45), and M 18240043 (fhbp 1.15) for hSBA. The results obtained are shown in Table 48.

[0922]

[0923] In Figure 26 A graphical representation of each day of blood collection with individual data points and highlighted GMTs is provided in

[0924] From the hSBA data, it can be seen that the formulations comprising the recombinant protein / modified fHbp fusion proteins of the disclosure (Formulation 1 / Quadrivalent 1 and Formulation 2 / Quadrivalent 2) are highly immunogenic and capable of eliciting protective SBA titers against different (M 08240157, M 17240832, M 17240156, and M 18240043) strains of N. meningitides.

[0925] i) ELISA for assessing human complement factor H binding to fHbp-PorA chimeric proteins compared to recombinant wild type of Men B samples

[0926] Objective:

[0927] The objective of this study was to describe a method for assessing human complement factor H binding to fHbp-PorA chimeric proteins compared to recombinant wild type of Men B samples.

[0928] Procedure:

[0929] The required chemicals and reagents are provided in Table 49.

[0930]

[0931] For all the above chemicals, equivalent models from other brands can also be used.

[0932] Required equipment:

[0933] - ELISA plate reader capable of measuring wavelengths at 450 nm and 630 nm.

[0934] - Microplate washer

[0935] - Incubator capable of achieving and maintaining 37°C

[0936] - Refrigerator

[0937] Required consumables:

[0938] - NUNC-Immuno Plates, model: Thermo Scientific; Catalog No: 442404 or equivalent.

[0939] - Plate sealing tape

[0940] - 50 mL reagent reservoirs

[0941] - Appropriate glassware

[0942] - Micropipettes (single and multichannel) and their corresponding tips

[0943] - Vortex mixer

[0944] - Microcentrifuge tubes - 15 mL and 50 mL

[0945] Preparation of reagents and solutions:

[0946] - Coating buffer: 1X PBS pH 7.4

[0947] Dissolve 2 PBS tablets in 800 μL of WFI and then make up the volume to 1 L. Store at room temperature (RT) and use within 1 month of preparation.

[0948] - Washing buffer: 1X PBST (0.05% Tween 20 in 1X PBS pH 7.4)

[0949] Prepare 1 L of PBS as described above. Add 0.5 mL of Tween 20 solution to 999.5 mL of 1X PBS and mix using a magnetic stirrer. Store at room temperature (RT) and use within 1 month of preparation. This 1X PBST is also used as blocking solution and diluent for antibody preparation.

[0950] - Blocking: 4% BSA in PBST

[0951] Weigh accurately 4.0 g of BSA and dissolve it in 100 mL of 1X PBST.

[0952] TMB solution: 2-component mixture

[0953] Accurately measure equal volumes of Solution A and B (provided as chromogenic reagents). Equilibrate to RT (store in the dark). Prepare fresh, mix gently before use.

[0954] Note: The procedure given above for the preparation of reagents and solutions is for illustration purpose only. Actual volume / quantity of chemicals / reagents should be calculated based on the number of assay plates.

[0955] Method:

[0956] - Antigen coating:

[0957] - Label the side of the 96 well plate with antigen details, analyst initials and date of coating the plate.

[0958] - Determine the volume of respective antigen and coating buffer required for the required number of plates to be coated.

[0959] - Coat each well of the ELISA plate with 5.0 μg of antigen (fHbp-PorA chimera) diluted in Phosphate Buffered Saline (1X PBS) to achieve a final concentration of 0.1 μg / μL and add 50 μL of the diluted antigen to each well of the 96 well Nunc flat bottom plate by means of a multichannel pipette.

[0960] - As a control, add 50.0 μL of PBS alone to the wells.

[0961] - Incubate the sealed plate at 2-8 °C overnight.

[0962] - Washing:

[0963] - Take the coated 96 well microtiter plate and place the plate in the microplate washer and run the washing program by selecting the pre-programmed procedure for 3 washes.

[0964] - For manual washing, empty all the wells by discarding the contents into the sink, add 300 μL / well of wash buffer / 1X PBST, immerse for a few seconds and empty all the wells into the sink. Tap gently on a paper towel free surface to ensure all the wells are completely emptied. Repeat the washing process two times.

[0965] - Blocking:

[0966] - Remove unbound antigen by washing the plate with 300 μL / well of 1X PBST three times the next day.

[0967] - Block the wells with 200 μL of 4% BSA in PBST. Incubate the plate at 37 °C for 1 hour.

[0968] - Addition of human complement factor H:

[0969] - Remove unbound BSA by washing the plate three times with 300 pL / well of IX PBST.

[0970] - Human Complement Factor H dilution scheme is summarized in Table 50:

[0971]

[0972] - Add 50.0 pL of Factor H diluted to the appropriate final concentration (follow dilution scheme as shown in Table 50, below are the final concentrations of Factor H, 0.0 pg, 0.001 pg, 0.005 pg, 0.01 pg, 0.05 pg, 0.1 pg, 0.2 pg, 0.3 pg, 0.4 pg, 0.5 pg, and 1.0 pg of protein in 50.0 pL), and incubate the plate at 37°C for 1 hour.

[0973] - Primary antibody addition:

[0974] - Remove unbound Factor H by washing the plate three times with 300 pL / well of IX PBST.

[0975] - Add 50.0 pL of OX24 mAb diluted to 1 / 10,000 in PBST, and incubate the plate at 37°C for 1 hour.

[0976] - Secondary antibody addition:

[0977] - Remove unbound primary antibody (OX24 mAb) by washing the plate three times with 300 pL / well of IX PBST.

[0978] - Add 50.0 pL of secondary antibody diluted to a final concentration of 1 / 10,000 in PBST, and incubate the plate at 37°C for 1 hour.

[0979] - TMB substrate addition:

[0980] - Remove unbound secondary antibody by washing the plate three times with 300 pL / well of IX PBST.

[0981] - Develop the plate by adding 100.0 pL of color reagent (1:1 mix of color reagent solution A and solution B). Incubate the plate at 25°C / RT for 20 minutes and stop the reaction by adding 50.0 pL of stop solution, which turns the reaction yellow.

[0982] - Read the absorbance of the developed wells on a microplate reader at the absorbance of 450 nm & 630 nm. Plot the normalized absorbance (A450nm-A630nm) using excel and Graph-pad PRISM.

[0983] Figure 27Representative data for human complement factor H binding to wild type fHbp compared to Men B chimeric proteins is illustrated in Table 51.

[0984]

[0985] Conclusion: From Table 51 and Figure 27 It can be seen that the recombinant protein / modified fHbp fusion proteins of the present application show more than 80% reduction in human complement factor H binding compared to wild type fHbp proteins.

[0986] Example-6: Combination formulation comprising formulation of recombinant protein / modified fHbp fusion protein

[0987] Physico-chemical characterization of combination formulation:

[0988] It was found that the vaccine formulation of the present application comprising recombinant protein / modified fHbp fusion protein from serogroup B of Neisseria meningitidis was stable and immunogenic. Further, combination formulation comprising vaccine formulation and other serogroups of Neisseria meningitidis was studied to find out its effect on physico-chemical characterization, stability and immunogenicity.

[0989] Applicant's MenFive formulation against Neisseria meningitidis serogroup ACYWX was used for this study. Lyophilized MenFive vaccine was reconstituted with MenB formulation and stored at room temperature for 2 hours to study its effect on physico-chemical parameters of the overall reconstituted solution. This study was carried out to assess the stability of reconstituted solution during animal injection and to mimic routine clinical practice.

[0990] MenB formulation from batch no. 090823 and MenFive formulation (batch no. 2352M001: 5 dose lyophilized vials for this study, content per vial: each antigen (A, C, Y, W, X) = 32 μg; sucrose = 15 mg; trisodium citrate = 2.5 mg; Tris buffer = 0.61 mg) was used for this study. Test parameters and results obtained are summarized in Table 52.

[0991]

[0992] Conclusion: From Table 52 it can be seen that the reconstituted solution was physico-chemically stable for 2 hours and no particulate aggregation / settlement was seen visually also.

[0993] Therefore, further studies were planned to determine the immunogenicity and non-interference of MenFive + MenB combination formulation.

[0994] Immunogenicity and non-interference study of combination formulation:

[0995] Details of biological material used are provided below:

[0996] Neisseria meningitidis serogroup B strain:

[0997] The Neisseria meningitidis serogroup B strain used was the same as used in Example-5.

[0998] Neisseria meningitidis serogroups A, C, W, Y and X strains:

[0999] Glycerol stocks of Neisseria meningitidis were received from Professor Ray Borrow (Health Protection Agency, Manchester Laboratory UK) in July 2012. The five serogroups of Neisseria meningitidis received were as follows:

[1000] 1. Neisseria meningitidis serogroup A, F8238

[1001] 2. Neisseria meningitidis serogroup C 11, M05 240852

[1002] 3. Neisseria meningitidis serogroup W135, M01 0240070

[1003] 4. Neisseria meningitidis serogroup X, BF2 / 97

[1004] 5. Neisseria meningitidis serogroup Y, M03 0241125

[1005] Procedure for hSBA against Men B strains:

[1006] Preparation of SBA from stock by working

[1007] Take the master stock vial from the deep freeze and streak the culture rapidly onto a blood agar plate (Columbia blood agar containing 5% horse blood) which has been dried. Incubate the plate overnight at 37°C, 5% C02. Pick approximately 50 colonies with a sterile loop and resuspend (to create a dense suspension) in a sterile 50ml centrifuge tube containing 15ml of BHI broth with glycerol. Aliquot the suspension into 0.25ml volumes into 1.8ml cryovials. Label the vials and store at -70°C in the deep freeze.

[1008] Procedure for serum killing assay for antibody titration against Men B strains:

[1009] Neisseria meningitidis B strain (working stock) was streaked onto individual blood agar plates and incubated overnight at 37°C, 5% C02. The culture was streaked again onto fresh blood agar plates to achieve confluent growth as a square in the center of the plate. For all Men B strains, the plates were incubated for 4 hours (+ 15 minutes) at 37°C, 5% C02 in a C02 incubator. Growth scraped from the center of the plate was suspended in 5 ml of Ca+2 and Mg+2 containing Hanks Balanced Salt Solution / Buffer (Invitrogen) to make a suspension. Using 1 ml of the suspension, the absorbance was read at 650 nm. The suspension was adjusted by dilution to 650 nm = 0.1 OD, then diluted 1 / 10 with buffer, then diluted 1 / 250. 20 μl of buffer was added to all wells of a 96 well microtiter plate up to column eleven. 20 μl (diluted or undiluted) test serum sample was added to the first column of the plate; the last two rows were reserved for quality control sera. 20 μl was serially diluted from the first column to the second column, and so on up to the ninth column, discarding the 20 μl from the ninth column. The tenth and eleventh columns were reserved for complement controls. 10 μl of the bacterial suspension that had been prepared was added to all wells up to the eleventh column. 10 μl of human complement was added to all wells up to the tenth column. 10 μl of heat inactivated (30 minutes at 56°C) human complement was added to all wells in the eleventh column. The contents of the wells were mixed well, and the plate was incubated at 37°C for 1 hour. After incubation, 10 μl of the contents of each well was spotted onto appropriately labeled blood agar plates. The blood agar plates were incubated in a C02 incubator at 37°C, 5% C02. The next day, bacterial colonies were counted using an automated colony counter (Synbiosis - ProtoCOL3).

[1010] Procedure for hSBA against MenFive serogroups (A, C, W, Y and X):

[1011] Preparation of SBA from working assay stock

[1012] The master stock vial was removed from the deep freeze and the culture was streaked rapidly onto a blood agar plate (Columbia blood agar containing 5% horse blood) that had been dried. The plate was incubated overnight at 37°C, 5% C02. Approximately 50 colonies were picked with a sterile loop and resuspended (to create a concentrated suspension) in a sterile 50 ml centrifuge tube containing 15 ml of BHI broth containing glycerol. The suspension was aliquoted into 0.25 ml volumes into 1.8 ml cryovials. The labeled vials were stored in a -70°C deep freeze.

[1013] Assay procedure for serum bactericidal assay for antibody titration for MenFive serogroups (A, C, W, Y and X):

[1014] Working stocks of N. meningitidis serogroups A, C, W, Y and X were streaked onto individual blood agar plates and incubated overnight at 37°C, 5% C02. Cultures were streaked again onto fresh blood agar plates to achieve confluent growth as a square in the center of the plate. Plates were incubated for 4 hours (± 15 minutes) at 37°C, 5% C02 in a C02 incubator for serogroups C, W, Y, X and 3 hours (± 15 minutes) for serogroup A. For serogroups A, Y and X, growth was scraped from the center of the plate into 5ml of Ca +2 and Mg +2 containing Hanks balanced buffer (Hanks Balanced Salt Solution, Invitrogen) for serogroups A, Y and X and Ca- and Mg- containing Hanks balanced buffer with BSA for serogroups C and W to make a suspension. Using 1ml of suspension, the absorbance was read at 650nm. The suspension was adjusted to 650nm = 0.1 OD by dilution, then diluted 1 / 10 with buffer, then 1 / 250. 20μ1 of buffer was added to all wells of a 96 well microtitre plate up to column 11. 20μ1 (diluted or undiluted) test serum sample was added to the first column of the plate; the last two rows were reserved for quality control sera. 20μ1 was serially diluted from the first column to the second, and so on up to the ninth column, discarding the 20μ1 from the ninth column. The tenth and eleventh columns were reserved for complement controls. 10μ1 of the bacterial suspension that had been prepared was added to all wells up to column 11. 10μ1 of human complement was added to all wells up to column 10. 10μ1 of heat inactivated human complement was added to all wells in column 11. The contents of the wells were mixed thoroughly and the plate was incubated at 37°C for 1 hour. After incubation, 10μ1 of the contents of each well was spotted onto a suitably labelled blood agar plate. The blood agar plates were incubated in a C02 incubator at 37°C, 5% C02. The following day, bacterial colonies were counted using an automated colony counter (Synbiosis - ProtoCOL3).

[1015] Study details:

[1016] An immunogenicity study was performed in rabbits (New Zealand White rabbits) using 5 formulations per group (G1 - placebo, G2 - MenFive, G3 - MenFive + MenB - formulation 1, G4 - MenFive + MenB - formulation 2 and G5 - Menfive + Trumenba), with 4 male and 4 female rabbits injected per formulation. Injections were given on day 0, day 14 and day 28, and blood was taken on DO, D28 and D41 for analysis of the immunogenicity of the serum samples by hSBA (serum killing assay using human complement). All rabbits were given a full human dose.

[1017] Drug product: Meningococcal (A-TT, C-CRM, Y-CRM, W-CRM, X-TT) polysaccharide conjugate vaccine - 5 dose vials (Men5) (2352M001) reconstituted with Meningococcal B vaccine (MenB) (090823 - Formulations 1 and 2)

[1018] Study design:

[1019] The study design is shown in Table 53.

[1020]

[1021] Criteria for significant differences between groups:

[1022] A 2-fold change in titer was considered assay variability, and a >4-fold difference in titer between groups was considered significant.

[1023] Purpose and scope:

[1024] The purpose of this study was to compare the immunogenic response to the individual MenFive + MenB, mixed formulations against MenFive serogroups (A, C, W, Y and X) and MenB strains - M 08240157 (fhbp 1.1), M 17240832 (fhbp 1.4), M 17240156 (fhbp 3.45) and M18240043 (fhbp 1.15) using hSBA. The scope also included checking for any interference / cross-protection in the response between the two individual vaccine formulations (MenFive and MenB).

[1025] The results obtained are summarized in Tables 54-55 and Figures 28-29 .

[1026]

[1027]

[1028] Conclusions: Based on the results obtained in Tables 54-55 and Figures 28-29 , the following observations were made.

[1029] Conclusions for MenFive serogroups: The titers obtained with the combined formulations (MenFive + Formulation 1 and MenFive + Formulation 2) against all five serogroups (A, C, W, Y and X) were comparable (within 2-fold) to the individual MenFive formulations, indicating no loss of immunogenic response when mixed with the Men B component. There was a slight increase in the SBA titers in quantity, but no decrease was observed for any of the serogroups.

[1030] Implications for Men B serogroup: The titers of MenFive + MenB formulation against Men B strains showed a titer increase of >4 fold when comparing pre- (D0) and post- (D41) titers, thus indicating an improved and acceptable immune response against Men B for 4 different strains.

[1031] The results indicated that the combination of MenFive with Men-B formulation did not result in any loss of immunogenic response, thus indicating no interference for any of the vaccine components of the combination formulation.

[1032] Example-7: Quantification of recombinant protein / modified fHbp fusion proteins using ELISA

[1033] Bicinchoninic acid (BCA) method is routinely used to estimate the total recombinant protein / modified fHbp fusion protein content in the final vaccine formulation.

[1034] The vaccine formulation of the present application comprises more than one recombinant protein / modified fHbp fusion protein. BCA provides quantification of total recombinant protein / modified fHbp fusion protein and cannot provide individual content of each protein present in the vaccine formulation.

[1035] As an alternative, ELISA is used to quantify the individual recombinant protein / modified fHbp fusion protein content in the vaccine formulation. This test can also be used as a batch release test.

[1036] The materials used in the ELISA for quantification of individual recombinant protein / modified fHbp fusion proteins are summarized in Table 56.

[1037]

[1038] Procedure for assay:

[1039] Step 1 (capture antibody coating): JAR-41 or JAR-5 antibody will be diluted using 0.1% BSA in PBS, and used in the range of 0.5 μg to 10 μg per well for coating, and 100 μL will be loaded per well on 96 well plate. Coating incubation will be carried out at room temperature or 2-8 °C for 2.5 hours to 18 hours.

[1040] Step 2 (blocking): 300 to 320 μL of 1% BSA in PBS will be added to the 96 well plate and incubated at room temperature for 1 hour to 2.5 hours.

[1041] Step 3 (standard / test sample addition): Reference standard chimera is serially diluted using a solution of 0.1% BSA in PBS from 0.5 mg / mL to 1 ng / mL and 50 μL to 150 μL is added to the corresponding well. Test samples are diluted using a solution of 0.1% BSA in PBS so that they will fall within the quantification range by loading from 50 μL to 150 μL. Incubation is carried out at room temperature for 0.5 hours to 2.0 hours after both standard and test sample addition.

[1042] Step 4 (detection antibody addition): Detection antibody (anti-POR-A P1.14 mAb / anti-POR-A P1.4 mAb / anti-POR-A P1.9 mAb / JAR-11 mAb) is diluted anywhere between 100-fold to 100000-fold and loaded 100 μL per well on a 96-well plate. Detection antibody incubation is carried out at room temperature for 0.5 hours to 2.0 hours.

[1043] Step 5 (second detection antibody addition): Second detection antibody (goat anti-mouse HRP antibody) is diluted anywhere between 100-fold to 100000-fold and loaded 100 μL per well on a 96-well plate. Second detection antibody incubation is carried out at room temperature for 0.5 hours to 2.0 hours.

[1044] Step 6 (substrate addition and stop): 100 μL of TMB substrate is added to each well and incubated at room temperature for 10 to 45 minutes and then stopped by adding 100 μL of 1 N HC1 to each well. The absorbance of the plate is immediately read at 450 / 630.

[1045] Example-8: Estimation of protein in recombinant protein / modified fHbp fusion proteins using BCA

[1046] Objective:

[1047] The objective of this study was to provide a procedure for estimation of protein in Men B samples by 96-well plate micro bicinchoninic acid (BCA) method.

[1048] BCA is a method for quantifying protein concentration in a sample. Cu 2+ reacts with protein to produce Cu + . The produced Cu + reacts with bicinchoninic acid to form a purple colored complex. The color develops after incubation at 37°C. The colored complex is detected at 562 nm using a spectrophotometer.

[1049] Materials and apparatus:

[1050] - Micro BCA Protein Assay Kit (Thermoscientific, 23235) containing

[1051] a. Trace amounts of BCA reagent A (MA)

[1052] b. Micro-batch BCA reagent B (MB)

[1053] c. Trace amounts of BCA reagent C (MC)

[1054] d. Bovine serum albumin standard ampoules (2 mg / ml)

[1055] - Water for Injection (WFI)

[1056] - Microcentrifuge tubes (1.5mL Eppendorf tubes)

[1057] - Pipettes and pipette tips

[1058] - 96-well plate

[1059] - 96-well microplate reader (TECAN, Infinite M200)

[1060] - Plate incubator (Biosan Thermoshaker)

[1061] Test program:

[1062] Preparation of standards and working reagents:

[1063] - Set the incubator to 37°C.

[1064] - Prepare a stock solution of 200 μg / ml from BSA standard (2 mg / ml).

[1065] - Prepare stock solutions using 200 μg / ml as shown in Table 57 and Figure 30 The standard curve is given in the text. A standard curve is prepared each time a measurement is to be performed.

[1066] - The working reagent is prepared by mixing 25 parts of trace BCA reagent MA and 24 parts of reagent MB with 1 part of reagent MC (25:24:1).

[1067]

[1068] Sample preparation:

[1069] The MEN-B samples were prepared and analyzed as a) the uncentrifuged sample, b) the supernatant after centrifugation, and c) the precipitate after centrifugation.

[1070] a. Uncentrifuged samples: Formulation samples should either be used as is or diluted to fit the standard curve.

[1071] b. Supernatant: 500 μΐ of each sample was taken and centrifuged at 8000 rpm for 5 minutes. After centrifugation, the supernatant was carefully transferred to a new tube. The collected supernatant was taken as the original sample or diluted to fit the curve.

[1072] c. Precipitate: The precipitate formed after centrifugation was resuspended by adding 500 μΐ of WFI. These samples were diluted identically to the samples themselves (a).

[1073] - Dilutions were prepared in duplicate for each sample and standard.

[1074] - 150 μΐ of the standard and sample were transferred to the plate wells.

[1075] - 150 μΐ of working reagent was added to each well and the plate was mixed thoroughly on a plate shaker for 30 seconds.

[1076] - The plate was covered and incubated at 37 °C for 2 hours.

[1077] - The absorbance at 562 nm was measured and the results obtained are provided in Table 58.

[1078]

[1079] The percentage of adsorption was calculated by subtracting the percentage of the concentration of the sample in the supernatant relative to the concentration of the same sample that was not centrifuged from 100. The percentage of adsorption is provided in Table 59.

[1080]

Claims

1. A modified factor H-binding protein (fHbp) comprising a wild-type fHbp variant and at least one exogenous loop, wherein - The modified factor H-binding protein (fHbp) is selected from any amino acid sequence that has at least 75% identity with any of the sequences in SEQ ID No. 6 to 10. -At least one exogenous peptide ring is immunogenic. -At least one exogenous peptide ring is derived from a bacterial membrane protein. - The modified fHbp is a fusion protein. The -fHbp variants are selected from v1, v2, and v3, and are modified by inserting at least one PorA ring into the β-turn region of fHbp, the PorA ring containing at least 10 amino acids; and -PorA rings are selected from VR1 and VR2.

2. The modified factor H binding protein of claim 1, wherein the modified fHbp is modified to reduce factor H binding activity.

3. A nucleic acid sequence encoding a modified fHbp, wherein the nucleic acid sequence has at least 75% identity with any one of the sequences in SEQ ID NO. 1 to 5.

4. An immunogenic composition comprising at least one modified fHbp as described in any one of claims 1 to 2 or a nucleic acid sequence encoding a modified fHbp as described in claim 3.

5. The immunogenic composition of claim 4, wherein the composition comprises two or more different modified fHbp.

6. The immunogenic composition of any one of claims 4 to 5, wherein the composition comprises a pharmaceutically acceptable carrier.

7. The immunogenic composition according to any one of claims 4 to 6, wherein the composition further comprises an adjuvant.

8. The immunogenic composition of any one of claims 4 to 7, wherein the composition further comprises at least one other prophylactic or therapeutic active molecule comprising a monovalent carrier protein:capsular polysaccharide conjugate vaccine.

9. The immunogenic composition of any one of claims 4-8, wherein the fHbp scaffold carrying the exogenous peptide ring is incorporated as a protein carrier molecule in the conjugated vaccine.

10. The immunogenic composition of any one of claims 4 to 9, comprising a recombinant protein / modified fHbp fusion protein, and in combination with at least one additional antigen selected from: - Protein antigens from PorB, Fet A, OmpC, NHBA, NadA, meningococcal antigen 287, NspA, HmbR, NhhA, App, and 936. - Sugar or conjugated antigens from Neisseria meningitidis serogroups A, C, W, Y and / or X, - Sugars or conjugated antigens from Streptococcus pneumoniae, - Diphtheria antigens, such as diphtheria toxoids, such as the CRM197 mutant, -Tetanus antigens, such as tetanus toxoid. - Antigens from Bordetella pertussis, acellular or whole-cell pertussis antigens. - Sugar or conjugated antigens from Haemophilus influenzae B, - Polio antigens, such as IPV, - Measles, mumps and / or rubella antigen - Influenza antigens, such as hemagglutinin and / or neuraminidase surface proteins, - Antigens (proteins, sugars, or conjugates) from Streptococcus agalactiae (Group B streptococci). - Antigens (proteins, sugars, or conjugates) from Streptococcus pyogenes (Group A streptococci). - Antigens (proteins, sugars, or conjugates) from Staphylococcus aureus. - Antigens (proteins, sugars, or conjugates) from certain species of Salmonella (Salmonella Spp).

11. The immunogenic composition of any one of claims 4 to 10, wherein the fHbp scaffold carrying the exogenous peptide ring is incorporated as a protein carrier molecule in the polysaccharide conjugated vaccine selected from monovalent (A, X, C, W, Y), bivalent (AC, AB, XB, CB), trivalent (AC-B, AC-Hib), tetravalent (AC-Hib-B), pentavalent (ACWYX) or hexavalent (ACWYX-B).

12. The modified fHbp of any one of claims 1 to 2, the nucleic acid of claim 3, or the composition of any one of claims 4 to 8, used as a drug, or for the treatment or prevention of pathogen infection or colonization in a subject.

13. The modified fHbp of any one of claims 1 to 2, the nucleic acid of claim 3, or the composition of any one of claims 4 to 8, combined with at least one other preventive or therapeutic active molecule.

14. The combination of claim 13 or the composition of claim 8, wherein at least one other prophylactic or therapeutic active molecule comprises a conjugated vaccine containing any serogroup capsular polysaccharide selected from strains A, C, Y, W or X or combinations thereof.

15. The combination of claim 14 or the composition of claim 8, wherein the protein:capsular polysaccharide vaccine comprises any serogroup C or A capsule conjugated with a bacterial toxoid, a bivalent vaccine (having serogroup C and A capsular polysaccharides conjugated to a bacterial toxoid), a tetravalent (serogroup A, C, Y, W polysaccharides conjugated to a bacterial toxoid), or a pentavalent (serogroup A, C, Y, W, X polysaccharides conjugated to a bacterial toxoid) conjugated vaccine.

16. The factor H binding protein (fHbp) of claims 1 to 2, which serves as an epitope display scaffold and is used as an epitope display scaffold.

17. A vaccine formulation comprising at least one recombinant protein / modified fHbp fusion protein, an adjuvant, and one or more pharmaceutically acceptable excipients, wherein in the recombinant protein / modified fHbp fusion protein, one or more exogenous rings are selected from, but not limited to, transferrin-binding protein, Neisseria heparin-binding protein, Neisseria surface protein A, PorA, meningococcal enterobacterin receptor FetA, Neisseria adhesin A, the fHbp-fHbp fusion protein of any one of the preceding claims, or combinations thereof.

18. The vaccine formulation of claim 17, wherein the recombinant protein / modified fHbp fusion protein is the nucleic acid sequence encoding the modified fHbp of any one of claims 1-2 or the modified fHbp of claim 3, and wherein the fHbp is derived from Neisseria meningitidis serogroups A, B, C, H, I, K, L, 29E, W135, X, Y and Z.

19. The vaccine formulation of claim 17 or 18, wherein the recombinant protein / modified fHbp fusion protein is derived from Neisseria meningitidis serogroup B.

20. The vaccine formulation of any one of claims 17-19, wherein the recombinant protein / modified fHbp fusion protein is fHbp having a molecular weight in the range of 10 kDa to 200 kDa, preferably up to 50 kDa.

21. The vaccine formulation of any one of claims 17-20, wherein the adjuvant is selected from aluminum hydroxide, aluminum phosphate, aluminum hydroxide, and potassium aluminum sulfate, MF-59, liposomes, lipopolysaccharides, saponins, lipid A, lipid A derivatives, monophospholipid A, GLA, 3-deacylated monophospholipid A, AS01, AS03, AF3, IL-2, RANTES, GM-CSF, TNF-α, IFN-g, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L and 41BBL, oligonucleotides, oligonucleotides and / or liposomes comprising at least one unmethylated CpG, Freund's adjuvant, Freund's complete adjuvant, Freund's incomplete adjuvant, polymers, copolymers such as polyoxyethylene-polyoxypropylene copolymers, including block copolymers, polymer p 1005, CRL-8300 adjuvant, muramyl dipeptide, agonists of TLR1 / 2, TLR2, TLR3, TLR-4 agonists, TLR5, TLR7, TLR7 / 8, TLR8, TLR9, ODN 2216 (type A), TLR11 / 12, TLR-4 agonists, flagellin, flagellin derived from Gram-negative bacteria, TLR-5 agonists, fragments of flagellin capable of binding to TLR-5 receptors, Alpha-C-galactosylceramide, chitosan, interleukin-2, QS-21, squalene, Quil A. Cholera toxin B subunit, polyphosphazene and its derivatives, mycobacterial cell wall preparations, mycolic acid derivatives, nonionic block copolymer surfactants, OMV, fHbp, combinations of saponins with sterols and lipids, dmLT, 1,25-dihydroxyvitamin D3, CAF01, poly[di(carboxyphenoxy)phosphazene] (PCPP) and Venezuelan equine encephalitis (VEE) replicon particles or combinations thereof.

22. The vaccine formulation of any one of claims 17-21, wherein the adjuvant is aluminum hydroxide with a particle size >500 nm.

23. The vaccine formulation of any one of claims 17-20, wherein one or more pharmaceutically acceptable excipients are a. Buffers selected from carbonates, phosphates, acetates, HEPES, succinates, TRIS, borates, citrates, lactates, gluconates, tartrates, or combinations thereof; b. Sugars selected from trehalose, mannose, raffinose, lactobionic acid, glucose, maltitol, isomaltitol, maltose, lactose, dextrose, fructose, or combinations thereof; c. Sugar alcohols or polyols selected from mannitol, lactitol, sorbitol, glycerol, xylitol, maltitol, lactitol, erythritol, isomaltitol and hydrogenated starch hydrolysate or combinations thereof; d. Surfactants selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonylphenyl alcohol ether-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 castor oil oleate, soybean lecithin, poloxamer, copolymers of ethylene oxide (EO), propylene oxide (PO) and / or butylene oxide (BO), octoxynol, phospholipids, nonylphenol ethoxylates, polyoxyethylene fatty ethers derived from lauryl alcohol, cetyl alcohol, stearyl alcohol and oleyl alcohol, sorbitan esters or combinations thereof; e. Polymers selected from dextran, carboxymethyl cellulose, hyaluronic acid, cyclodextrin, or combinations thereof; f. Salts selected from NaCl, KCl, KH2PO4, Na2HPO4·2H2O, CaCl2, MgCl2, or combinations thereof; g. An amino acid selected from tris(hydroxymethyl)methylglycine, leucine, isoleucine, glycine, glutamine, L-arginine, L-arginine hydrochloride, lysine, L-alanine, tryptophan, phenylalanine, tyrosine, valine, cysteine, glycine, methionine, proline, serine, threonine, or combinations thereof. h. Hydrolyzed protein selected from gelatin, whey protein hydrolysate, monosodium glutamate, collagen hydrolysate, keratin hydrolysate, peptide, casein hydrolysate, whey protein hydrolysate, serum albumin or combinations thereof; i. Preservatives selected from phenoxyethanol, benzyl chloride (Phemerol), phenol, m-cresol, thimerosal, formaldehyde, p-hydroxybenzoate, benzalkonium chloride, benzyl alcohol, chlorobutanol, p-chloro-m-cresol, benzyl alcohol, or combinations thereof; and j. A liquid carrier selected from water for injection (WFI) or saline.

24. The vaccine formulation according to any one of claims 17-23, wherein the vaccine formulation comprises: - At least one recombinant protein according to any one of the preceding claims / at least one modified fHbp; - Aluminum hydroxide; - Mannitol; -phosphates; and - Polysorbate.

25. The vaccine formulation of any one of claims 17-24, wherein the vaccine formulation comprises: - at least one modified fHbp of an amino acid sequence having at least 75% identity with any one of the amino acid sequences in SEQ ID NO. 6 to 10; or - fHbp encoded by at least one modified nucleic acid sequence having at least 75% identity with any one of the nucleic acid sequences SEQ ID NO. 1 to 5; and - Aluminum hydroxide; - Mannitol; -phosphate; and - Polysorbate.

26. The vaccine formulation of any one of claims 17-25, comprising (i) at least one recombinant protein / modified fHbp fusion protein; (ii) aluminum hydroxide in an amount ranging from 0.5 mg / ml to 4.5 mg / ml; (iii) mannitol in an amount ranging from 5 mg / ml to 100 mg / ml; (iv) phosphate buffer in an amount ranging from 1 mM to 10 mM; and (v) polysorbate 20 in an amount ranging from 0.01 mg / ml to 2 mg / ml; wherein each recombinant protein / modified fHbp fusion protein is present in an amount ranging from 15 μg / ml to 150 μg / ml.

27. The vaccine formulation of any one of claims 17-25, comprising (i) at least two recombinant protein / modified fHbp fusion proteins; (ii) aluminum hydroxide in an amount ranging from 0.5 mg / ml to 4.5 mg / ml; (iii) mannitol in an amount ranging from 5 mg / ml to 100 mg / ml; (iv) phosphate buffer in an amount ranging from 1 mM to 10 mM; and (v) polysorbate 20 in an amount ranging from 0.01 mg / ml to 2 mg / ml; wherein each recombinant protein / modified fHbp fusion protein is present in an amount ranging from 15 μg / ml to 150 μg / ml.

28. The vaccine formulation of any one of claims 17-25, comprising (i) at least three recombinant protein / modified fHbp fusion proteins; (ii) aluminum hydroxide in an amount ranging from 0.5 mg / ml to 4.5 mg / ml; (iii) mannitol in an amount ranging from 5 mg / ml to 100 mg / ml; (iv) phosphate buffer in an amount ranging from 1 mM to 10 mM; and (v) polysorbate 20 in an amount ranging from 0.01 mg / ml to 2 mg / ml; wherein each recombinant protein / modified fHbp fusion protein is present in an amount ranging from 15 μg / ml to 150 μg / ml.

29. The vaccine formulation of any one of claims 17-25, comprising (i) at least four recombinant protein / modified fHbp fusion proteins; (ii) aluminum hydroxide in an amount ranging from 0.5 mg / ml to 4.5 mg / ml; (iii) mannitol in an amount ranging from 5 mg / ml to 100 mg / ml; (iv) phosphate buffer in an amount ranging from 1 mM to 10 mM; and (v) polysorbate 20 in an amount ranging from 0.01 mg / ml to 2 mg / ml; wherein each recombinant protein / modified fHbp fusion protein is present in an amount ranging from 15 μg / ml to 150 μg / ml.

30. The vaccine formulation of any one of claims 17-24, wherein the vaccine formulation comprises: - at least one modified fHbp of an amino acid sequence having at least 75% identity with any one of the amino acid sequences in SEQ ID NO. 6 to 10; or - fHbp encoded by at least one modified nucleic acid sequence having at least 75% identity with any one of the nucleic acid sequences SEQ ID NO. 1 to 5; and - Amounts of aluminum hydroxide ranging from 0.5 mg / ml to 4.5 mg / ml; - Amounts of mannitol ranging from 5 mg / ml to 100 mg / ml; - Phosphate buffer in amounts ranging from 1 mM to 10 mM; and - Amounts of polysorbate 20 in the range of 0.01 mg / ml to 2 mg / ml.

31. The vaccine formulation according to any one of claims 17-30, comprising: a. Amounts (of SEQ ID NO. 6 or encoded by SEQ ID NO. 1) of fHbpV3.45 M5:PorA316-320 / exP1.14 in the range of 15 μg / ml to 150 μg / ml; or b. Amounts (of SEQ ID NO. 7 or encoded by SEQ ID NO. 2) of fHbpV2.19 M6:PorA316-320 / exP1.4 in the range of 15 μg / ml to 150 μg / ml; or c. Amounts (of SEQ ID NO. 8 or encoded by SEQ ID NO. 3) of fHbpV1.14:PorA307-311 / exP1.9 in the range of 15 μg / ml to 150 μg / ml; or d. Amounts (of SEQ ID NO. 9 or encoded by SEQ ID NO. 4) of fHbpV1.1:PorA307-311 / exP1.4 in the range of 15 μg / ml to 150 μg / ml; and e. Amounts of aluminum hydroxide ranging from 0.5 mg / ml to 4.5 mg / ml; f. Amounts of mannitol ranging from 5 mg / ml to 100 mg / ml; g. Phosphate buffer in amounts ranging from 1 mM to 10 mM; and h. Amounts of polysorbate 20 in the range of 0.01 mg / ml to 2 mg / ml.

32. The vaccine formulation according to any one of claims 17-30, comprising: a. Amounts (of SEQ ID NO. 6 or encoded by SEQ ID NO. 1) of fHbpV3.45 M5:PorA316-320 / exP1.14 in the range of 15 μg / ml to 150 μg / ml; b. Amounts (of SEQ ID NO. 7 or encoded by SEQ ID NO. 2) of fHbpV2.19 M6:PorA316-320 / exP1.4 in the range of 15 μg / ml to 150 μg / ml; c. Amounts (of SEQ ID NO. 8 or encoded by SEQ ID NO. 3) of fHbpV1.14:PorA307-311 / exP1.9 in the range of 15 μg / ml to 150 μg / ml; d. Amounts (of SEQ ID NO. 9 or encoded by SEQ ID NO. 4) of fHbpV1.1:PorA307-311 / exP1.4 in the range of 15 μg / ml to 150 μg / ml; e. Amounts of aluminum hydroxide ranging from 0.5 mg / ml to 4.5 mg / ml; f. Amounts of mannitol ranging from 5 mg / ml to 100 mg / ml; g. Phosphate buffer in amounts ranging from 1 mM to 10 mM; and h. Amounts of polysorbate 20 in the range of 0.01 mg / ml to 2 mg / ml.

33. The vaccine formulation according to any one of claims 17-30, comprising: a. Amounts (of SEQ ID NO. 6 or encoded by SEQ ID NO. 1) of fHbpV3.45 M5:PorA316-320 / exP1.14 in the range of 15 μg / ml to 150 μg / ml; or b. Amounts (of SEQ ID NO. 7 or encoded by SEQ ID NO. 2) of fHbpV2.19 M6:PorA316-320 / exP1.4 in the range of 15 μg / ml to 150 μg / ml; or c. Amounts (of SEQ ID NO. 10 or encoded by SEQ ID NO. 5) of fHbpV1.1:PorA307-311 / exP1.9 in the range of 15 μg / ml to 150 μg / ml; or d. Amounts (of SEQ ID NO. 8 or encoded by SEQ ID NO. 3) of fHbpV1.14:PorA307-311 / exP1.9 in the range of 15 μg / ml to 150 μg / ml; and e. Amounts of aluminum hydroxide ranging from 0.5 mg / ml to 4.5 mg / ml; f. Amounts of mannitol ranging from 5 mg / ml to 100 mg / ml; g. Phosphate buffer in amounts ranging from 1 mM to 10 mM; and h. Amounts of polysorbate 20 in the range of 0.01 mg / ml to 2 mg / ml.

34. The vaccine formulation according to any one of claims 17-30, comprising: a. Amounts (of SEQ ID NO. 6 or encoded by SEQ ID NO. 1) of fHbpV3.45 M5:PorA316-320 / exP1.14 in the range of 15 μg / ml to 150 μg / ml; b. Amounts (of SEQ ID NO. 7 or encoded by SEQ ID NO. 2) of fHbpV2.19 M6:PorA316-320 / exP1.4 in the range of 15 μg / ml to 150 μg / ml; c. Amounts (of SEQ ID NO. 10 or encoded by SEQ ID NO. 5) of fHbpV1.1:PorA307-311 / exP1.9 in the range of 15 μg / ml to 150 μg / ml; d. Amounts (of SEQ ID NO. 8 or encoded by SEQ ID NO. 3) of fHbpV1.14:PorA307-311 / exP1.9 in the range of 15 μg / ml to 150 μg / ml; e. Amounts of aluminum hydroxide ranging from 0.5 mg / ml to 4.5 mg / ml; f. Amounts of mannitol ranging from 5 mg / ml to 100 mg / ml; g. Phosphate buffer in amounts ranging from 1 mM to 10 mM; and h. Amounts of polysorbate 20 in the range of 0.01 mg / ml to 2 mg / ml.

35. The vaccine formulation of any one of claims 17-34, wherein the formulation comprises an amount of 2-phenoxyethanol in the range of 1 mg / mL to 10 mg / mL.

36. The vaccine formulation of any one of claims 17-35, wherein the vaccine composition is stable at 2-8°C, 25°C and 40°C for a period of more than six months.

37. The vaccine formulation according to any one of claims 17-35, having a zeta potential in the range of -16 mV to -30 mV; and a weight molar osmolality in the range of 200 mOsmol / kg to 500 mOsmol / kg.

38. The vaccine formulation of any one of claims 17-35, further comprising a mixture selected from diphtheria toxoid (D), tetanus toxoid (T), whole-cell pertussis (wP), hepatitis B surface antigen (HBsAg), Haemophilus influenzae b PRP-carrier protein conjugate (Hib), Haemophilus influenzae (serotypes a, c, d, e, f and uncapsulated strains), Neisseria meningitidis A antigen, Neisseria meningitidis C antigen, Neisseria meningitidis W-135 antigen, Neisseria meningitidis Y antigen, Neisseria meningitidis X antigen, Streptococcus pneumoniae antigen, Neisseria meningitidis B vesicle or purified antigen, Staphylococcus aureus antigen, anthrax, BCG, hepatitis (strains A, C, D, E, F and G) antigen, human papillomavirus, HIV, Salmonella typhi. The antigens include typhi antigen, acellular pertussis, modified adenylate cyclase, malaria antigen (RTS, S), measles, mumps, rubella, dengue fever, Zika, Ebola, chikungunya, Japanese encephalitis, rotavirus, diarrhea antigen, flavivirus, smallpox, yellow fever, herpes zoster, varicella virus antigen, and one or more combinations thereof.

39. The vaccine formulation of any one of claims 17-35, 38, comprising (i) at least one fusion protein comprising a stable nonfunctional / non-esterified fHbp and a PorA VR2 ring, and (ii) at least one polysaccharide-protein conjugate.

40. The vaccine formulation of any one of claims 17-39, wherein the recombinant protein / modified fHbp fusion protein is co-administered with one or more vaccines selected from BEXSERO, MENVEO, MENACTRA, NIMENRIX, MenQuadFi, MENFIVE, MenAfriVac, Men AC, and MenACHib.

41. The vaccine formulation of any one of claims 17-40, wherein the recombinant protein / modified fHbp fusion protein is co-administered with MENFIVE.

42. The vaccine formulation of any one of claims 17-41, comprising i) at least one fusion protein comprising a stable nonfunctional / non-esterified fHbp and a PorA VR2 ring, and at least one conjugate selected from: (a)(i) a conjugate of capsular polysaccharide of serogroup A Neisseria meningitidis and (ii) tetanus toxoid; (b)(i) a conjugate of capsular polysaccharide of serogroup C Neisseria meningitidis and (ii) CRM197; (c)(i) a conjugate of capsular polysaccharide of serogroup Y Neisseria meningitidis and (ii) CRM197; (d)(i) a conjugate of capsular polysaccharide of serogroup W135 Neisseria meningitidis and (ii) CRM197; and (d)(i) a conjugate of capsular polysaccharide of serogroup X Neisseria meningitidis and (ii) tetanus toxoid.

43. The vaccine formulation of any one of claims 17-41, comprising i) at least two fusion proteins, each fusion protein consisting of an fHbp variant type ring-coupled with a PorA VR2, and at least one conjugate selected from: (a)(i) a conjugate of capsular polysaccharide of serogroup A Neisseria meningitidis and (ii) tetanus toxoid; (b)(i) a conjugate of capsular polysaccharide of serogroup C Neisseria meningitidis and (ii) CRM197; (c)(i) a conjugate of capsular polysaccharide of serogroup Y Neisseria meningitidis and (ii) CRM197; (d)(i) a conjugate of capsular polysaccharide of serogroup W135 Neisseria meningitidis and (ii) CRM197; and (d)(i) a conjugate of capsular polysaccharide of serogroup X Neisseria meningitidis and (ii) tetanus toxoid.

44. The vaccine formulation of any one of claims 17-41, comprising i) at least three fusion proteins, each fusion protein consisting of an fHbp variant type ring-coupled with two PorA VR2, and at least one conjugate selected from: (a)(i) a conjugate of capsular polysaccharide of serogroup A Neisseria meningitidis and (ii) tetanus toxoid; (b)(i) a conjugate of capsular polysaccharide of serogroup C Neisseria meningitidis and (ii) CRM197; (c)(i) a conjugate of capsular polysaccharide of serogroup Y Neisseria meningitidis and (ii) CRM197; (d)(i) a conjugate of capsular polysaccharide of serogroup W135 Neisseria meningitidis and (ii) CRM197; and (d)(i) a conjugate of capsular polysaccharide of serogroup X Neisseria meningitidis and (ii) tetanus toxoid.

45. The vaccine formulation of any one of claims 17-41, comprising i) at least four fusion proteins, each fusion protein consisting of an fHbp variant type ring-coupled with three PorA VR2, and at least one conjugate selected from: (a)(i) a conjugate of capsular polysaccharide of serogroup A Neisseria meningitidis and (ii) tetanus toxoid; (b)(i) a conjugate of capsular polysaccharide of serogroup C Neisseria meningitidis and (ii) CRM197; (c)(i) a conjugate of capsular polysaccharide of serogroup Y Neisseria meningitidis and (ii) CRM197; (d)(i) a conjugate of capsular polysaccharide of serogroup W135 Neisseria meningitidis and (ii) CRM197; and (d)(i) a conjugate of capsular polysaccharide of serogroup X Neisseria meningitidis and (ii) tetanus toxoid.

46. ​​The vaccine formulation of any one of claims 17-45, for the treatment or prevention of infection and / or disease caused by Neisseria meningitidis serogroup B.

47. The vaccine formulation of any one of claims 17-45, wherein the vaccine formulation demonstrates cross-protection against Neisseria gonorrhea strains and Neisseria meningitidis serogroup ACWYX.

48. The vaccine formulation of any one of claims 17-45, wherein the percentage of adsorption of the recombinant protein / modified fHbp fusion protein on the adjuvant is in the range of 70% to 100%.

49. The vaccine formulation of claim 48, wherein fHbp V3.45 M5 PorA 316-320 exP1.14 The adsorption percentage on the adjuvant is in the range of 80% to 100%.

50. The vaccine formulation of claim 48, wherein fHbp V1.14 PorA 307-311 exP1.9 The adsorption percentage on the adjuvant is in the range of 80% to 90%.

51. The vaccine formulation of claim 48, wherein fHbp V2.19 PorA 316-320 exP1.4 The adsorption percentage on the adjuvant is in the range of 80% to 90%.

52. The vaccine formulation of claim 48, wherein fHbp V1.1 PorA 307-311 exP1.4 The adsorption percentage on the adjuvant is in the range of 80% to 90%.

53. The vaccine formulation of claim 48, wherein fHbp V1.1 PorA 307-311 exP1.9 The adsorption percentage on the adjuvant is in the range of 70% to 80%.

54. A method for manufacturing a vaccine formulation according to any one of claims 17-53, the method comprising the following steps: (a) Culture host cells containing the expression vector in a nutrient medium; (b) Inducing host cells to express proteins; (c) Harvesting and separating the host cells; (d) Lyse the harvested cells and separate host cell debris to obtain the marker proteins; (e) Purify the labeled protein; (f) Remove the tag from the labeled protein to obtain the recombinant protein / modified fHbp fusion protein; (g) Purify recombinant protein / modified fHbp fusion protein; and (h) Prepare a vaccine formulation containing purified recombinant protein / modified fHbp fusion protein.

55. The method of claim 54, wherein the host cell is a bacterial expression host system.

56. The method of claim 55, wherein the bacterial expression host system is an *Escherichia coli* strain selected from BL21(DE3), BL21(DE3)pLysS*, BL21(DE3)pLysE*, BL21 star(DE3), BL21-A1, BLR(DE3), HMS174(DE3)**, Turner(DE3), Origami2(DE3)**, Rosetta2(DE3)*, Rosettaami(DE3), Lemo21(DE3)*, T7 Express, m15 pREP4*, C41(DE3), C43(DE3), or B834(DE3).

57. The method of claim 54, wherein the nutrient medium is selected from undefined medium, Terrific Broth (TB) medium, Lysogenia Broth, Luria Broth or Luria-Bertani medium, chemically defined medium, M9 minimal medium, chemically defined M9 modified salt medium, 2xYT medium or Super Optimal broth (SOC) medium with catabolite inhibition, and combinations thereof.

58. The method of claim 54, wherein the concentration of L-methionine during host cell growth in step (a) is maintained in the range of 1 mM to 10 mM, wherein fermentation is in fed-batch mode.

59. The method of claim 54, wherein the host cells are grown at a temperature in the range of 35°C to 39°C; a pH in the range of 5.0 to 9.0; dissolved oxygen in the range of 10% to 100%; stirring in the range of 100 to 1800 rpm; and a gas flow rate in the range of 0 to 2 gas volumes per unit volume of liquid per minute (VVM).

60. The method of claim 54, wherein the host cells are induced using an inducer selected from lactose and its non-hydrolyzed analogue isopropyl β-D-1-thiogalactoside (IPTG).

61. The method of claim 60, wherein the concentration of lactose is in the range of 1 g / L to 50 g / L and the concentration of IPTG is in the range of 1 mM to 10 mM.

62. The method of claim 54, wherein the host cell is lysed using a method selected from chemical, biological, physical, mechanical, and combinations thereof.

63. The method of claim 62, wherein a combination of chemical and mechanical methods is used to lyse the host cell.

64. The method of any one of claims 62-63, wherein a lysis buffer having a pH in the range of 7-9 is used, and then the host cells are lysed by mechanical lysis for 3-8 cycles at a pressure in the range of 1000-1500 bar.

65. The method of claim 64, wherein a homogenizer is used for mechanical pyrolysis.

66. The method of claim 54, wherein the labeled protein in step (e) is purified using a chromatographic step, followed by concentration and percolation.

67. The method of claim 54, wherein a TEV protease having a protein:TEV protease ratio in the range of 5:1 to 30:1 is used to remove the tag.

68. The method of claim 54, wherein the recombinant protein / modified fHbp fusion protein in step (g) is purified using a chromatographic step, followed by concentration and percolation.

69. The method of claim 68, wherein the chromatogram is selected from column chromatography, ion exchange chromatography, anion exchange chromatography, cation exchange chromatography, column chromatography, rapid chromatography, gel filtration / size exclusion / gel permeation (molecular sieve) chromatography, affinity chromatography, paper chromatography, thin layer chromatography, gas chromatography, dye ligand chromatography, hydrophobic interaction chromatography, pseudo affinity chromatography, liquid chromatography, high performance liquid chromatography (HPLC), fixed metal affinity chromatography, anion exchange chromatography, cation exchange chromatography, multimode chromatography, multimode anion exchange chromatography, electrostatic interaction chromatography, hydrogen bonding chromatography, reversed phase chromatography, and combinations thereof.

70. The method of any one of claims 54-69, wherein the labeled protein is expressed as an inclusion body (IB) and purified by urea unfolding protein and column-refolding protein.

71. The method of claim 54, wherein the vaccine formulation is prepared by adsorbing the individual recombinant protein / modified fHbp fusion protein onto an adjuvant and then adding it to an excipient mixture comprising a sugar alcohol, a buffer, a stabilizer, and a liquid carrier.

72. The method of claim 71, wherein the excipient mixture comprises a preservative.

73. The method of claim 67, wherein the TEV protease is produced by a method comprising the following steps: (a) Culture host cells containing the expression vector in a nutrient medium; (b) Inducing host cells to express TEV protease; (c) Harvesting and separating the host cells; (d) Lyse the harvested cells and separate the host cells to obtain TEV protease; (e) Purification of TEV protease; and (f) Concentration, percolation and storage of purified TEV protease.

74. The method of claim 73, wherein the host cell for expressing the TEV protease is an *E. coli* strain selected from BL21(DE3), BL21(DE3) pLysS*, BL21(DE3) pLysE*, BL21 star(DE3), BL21-A1, BLR(DE3), HMS174(DE3)**, Turner(DE3), Origami2(DE3)**, Rosetta2(DE3)*, Rosettataami(DE3), Lemo21(DE3)*, T7 Express, m15 pREP4*, C41(DE3), C43(DE3), Rosetta™(DE3)pLysS, or B834(DE3).

75. The method of any one of claims 54-74, wherein glucose feeding is stopped when the OD at 590 / 600 nm is 20-100 and glycerol feeding is started, and the culture is induced by adding lactose in a fed-batch mode and / or maintaining lactose at 1-50 g / L.

76. A method for inducing an immune response against Neisseria meningitidis serogroup B strain by administering to an individual the vaccine formulation of any of the preceding claims, wherein the administration step induces an immune response against Neisseria meningitidis serogroup B strain.

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