Method for manufacturing vaccine formulations based on meningococcal proteins

Abstract

The present invention provides a fusion protein in which H factor binding is desiredly reduced, and in particular, the present invention provides an optimized manufacturing process for the fusion protein and formulations containing the fusion protein. The present invention provides an efficient platform process for producing effective vaccine formulations against Neisseria meningitidis that meet multiple criteria with improved immunogenicity, safety, stability and availability.

Description

[Technical Field]

[0001] The present invention relates to vaccine formulations and methods for producing the same. In particular, the present invention relates to the upstream, downstream, and formulation development of Neisseria meningitidis, recombinant / chimeric protein antigens based on Neisseria meningitidis serotype B, methods for preparing formulations based on said chimeric protein, and the use of these formulations for the prevention and / or treatment of subjects infected with Neisseria meningitidis serotype B. [Background technology]

[0002] All publications herein are to the same extent that individual publications or patent applications are specifically and individually referenced. The following description contains information that may be useful in understanding the present invention. It is not acknowledged that any information provided herein is prior art or relating to the currently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0003] Neisseria meningitidis is a significant pathogen, particularly in children and young adults. Sepsis and meningitis are the most life-threatening forms of invasive meningococcal disease (IMD). While the case fatality rate for disseminated disease is approximately 10%, one-third of meningococcal disease survivors suffer significantly from debilitating long-term sequelae. The disease is a global health problem due to its high morbidity and mortality rates.

[0004] Toxin-based vaccines have virtually eliminated diphtheria and tetanus in affluent countries, while capsule vaccines have substantially reduced diseases caused by certain strains of Haemophilus influenzae, Streptococcus pneumoniae, and Neisseria meningitidis. However, there are challenges in developing vaccines against pathogens for which toxin-based and capsule vaccines are not feasible. These pathogens include unclassified strains of Haemophilus influenzae and Streptococcus pneumoniae, unencapsulated pathogens such as Neisseria gonorrhoeae and Moraxella catarrhalis, and encapsulated serotype B. Capsule vaccines are not feasible against Neisseria meningitidis. Given the increasing emergence of multidrug-resistant bacteria, a new approach to vaccine development is needed. The development of vaccines against serotype B Neisseria meningitidis presents specific difficulties, such as poor immunogenicity due to the immunological similarity of the polysaccharide capsule to human nerve cell adhesion molecules. Furthermore, the diversity of pathogens and the challenges of presenting epitopes to the immune system from membrane-embedded surface proteins hinder strategies for successful vaccine development.

[0005] Neisseria meningitidis is a Gram-negative bacterium that colonizes the human upper respiratory tract and is the most prominent cause of meningitis and sepsis, occurring sporadically and periodically worldwide. Neisseria meningitidis typically has an outer membrane that forms the bacterial wall along with a cell membrane, peptidoglycan layer, and capsular polysaccharides, and cilia that protrude into the external environment. Neisseria meningitidis (Nm) remains a major cause of sepsis and bacterial meningitis in children and young adults. Approximately 500,000 cases of meningococcal disease are reported annually, resulting in approximately 50,000 deaths. In developed countries, this bacterium is a leading cause of death among children, and outbreaks in schools and universities have significant public health implications, potentially leading to serious physical disabilities in survivors. Encapsulated strains of Neisseria meningitidis are the primary cause of bacterial meningitis and sepsis in children and young adults. Meningococcal disease can be classified into at least 12 serotypes (including serotypes A, B, C, H, I, K, L, 29E, W135, X, Y, and Z). The most common serotypes are A, B, and C, which account for 90% of the disease worldwide. Serotype B is the most common cause of meningococcal disease in Europe, the United States, and some countries in Latin America, and outbreaks occur every 5-10 years in sub-Saharan Africa. Meningococcal disease is a devastating disease in which children and young adults can die within hours, despite the availability of antibiotics. Therefore, prophylactic immunization is the best way to protect individuals from meningococcal infection.

[0006] While vaccines based on bacterial polysaccharide capsules can be used, the polysaccharide capsule of Neisseria meningitidis serotype B is structurally identical to that of human glycoproteins in nerve tissue, resulting in poor immunogenicity. When used as a vaccine, it may induce autoimmunity. Therefore, the "humanization" of serotype B and the development of safe and immunogenic conjugate polysaccharide vaccines against type B were initially put on hold. This is because polysaccharides do not induce serum bactericidal antibodies, and in vitro anti-capsule B antibodies recognize nerve cell adhesion molecules in fetal brain tissue.

[0007] To develop vaccines against serotype B, two main approaches have been employed: meningococcal outer membrane vesicle vaccines (OMVVs) and recombinant protein subunit vaccines. OMVVs were developed in the early 1980s. The immunodominant antigen in meningococcal OMVVs is PorA, which is an abundant outer membrane porin with eight surface-exposed loops. Loops 1 and 4 elicit an immune response and undergo antigenic changes, and are therefore called variable regions 1 and 2 (VR1 and VR2), respectively. The VR2 loop governs PorA-specific immunity induced by OMVVs, and VR2 provides limited or no cross-protection against strains expressing different PorA. When evaluated and studied with longer follow-up periods, including 20 months in Chile, efficacy was shown to decrease by 50%, and the response lifetime was short (Non-Patent Literature 1).

[0008] To broaden the scope of application, OMVVs containing multiple PorA sequences have been developed and selected for the proliferation of PorA sequences in circulating strains. However, OMVVs present complex manufacturing and regulatory challenges. Prior art vaccines often utilize the purification of so-called "blebs," which represent vesicles detached from the cell surface of a specific organism of interest. However, these crude products present numerous problems. For example, the composition of these blebs is diverse. There is no reliable way to control which proteins are present in which blebs or excluded from which. These blebs may or may not contain the polysaccharide coating factors of the organism of interest. The various component ratios of the blebs cannot be reliably determined. The composition of these blebs cannot be easily determined or controlled.

[0009] OMV in Bexsero® (GSK vaccine) is highly reactive, and because parents are concerned about side effects, the vaccine is routinely administered with paracetamol (Non-Patent Literature 2). Bexsero® has the following drawbacks: 1) the antigen is derived from a single meningococcal strain (Non-Patent Literature 3); 2) immunogenicity studies were conducted on a 3+1 schedule rather than the 2+1 schedule planned in almost all UK (doses at 2, 4, and 12 months); 3) indirect correlations of protection, rather than direct correlations, were measured at approximately 13 months of age and not measured in the most high-risk infants.

[0010] Meningococcal serotype B vaccines based on outer membrane vesicles (OMVs) have been conveniently used to prevent outbreaks (most recently in New Zealand), but they only provide protection against strains that express PorA (outer membrane porin) variants, similar to OMVs. OMVs as immunogens are undesirable due to potential consistency and toxicity issues during manufacturing. For example, OMVs may contain toxic lipopolysaccharide (LPS).

[0011] PorA is the most abundant outer membrane protein (OMP) of Neisseria meningitidis and is the primary target of the immune response induced by OMV vaccines, which have been successfully used in pandemics, and induces subcutaneous balanitis (SBA). PorA variants differ in the variable region (VR), which is the surface-exposed loop targeted by the immune response. Notably, VR2 is responsible for most SBAs induced by OMV vaccines. However, PorA is an endogenous OMP with multiple hydrophobic domains. This makes it difficult to produce PorA as a recombinant protein in its native three-dimensional structure, thereby limiting its use as an antigen in subunit vaccines.

[0012] Factor H-binding protein (fHbp) is also an antigen that induces a serum bactericidal antibody response in immunized individuals and is a key component of research vaccines for the prevention of meningococcal diseases, particularly serotype B diseases, currently being evaluated in clinical trials. Factor H-binding protein (fHbp, also known in the industry as lipoprotein 2086 (Non-Patent Literature 4), genome-derived Neisseria antigen (GNA) 1870 (Non-Patent Literature 5), or "741") is a surface-exposed lipoprotein expressed in Neisseria meningitidis. Based on differences in nucleotides and predicted amino acid sequences, fHbp from different Neisseria meningitidis strains have been classified using several schemes. These include two subfamilies (A and B) (Non-Patent Literature 6) or three variant groups (V1, V2, and V3) (Non-Patent Literature 5), with subfamily A corresponding to V2 and V3, and subfamily B corresponding to V1 (the most abundant).

[0013] The recombinant subunit vaccines Bexcelo® (GSK Vaccines) and Trumenba® (Wyeth Pharmaceuticals) contain factor H-binding protein (fHbp), an important meningococcal antigen. fHbp is a lipoprotein composed of two β-barrels that strongly bind to domains 6 and 7 of human complement factor H (CFH). fHbp is antigenically variable, and genome sequence databases contain over 900 different fHbp peptides classified into three variant groups or two subfamilies: V1 (subfamily B), V2, and V3 (both subfamily A). Generally, immunization with a specific fHbp induces cross-protection against strains expressing fHbp belonging to the same but distinct variant groups, although cross-protection may exist between fHbp variant groups 2 and 3 (subfamily A). Vexero® contains a single fHbp peptide (V1.1) along with two further recombinant antigens and OMV, while Tormenba® consists of only two fHbp peptides (V1.55 and V3.45). Despite strains expressing this variant accounting for approximately 20-30% of all isolates (38% of UK cases), no research has been conducted on vaccines containing V2 fHbp. Since v2 fHbp is an inherently unstable antigen, no vaccines containing v2 fHbp exist. On Neisseria meningitidis, fHbp is a 27 kDa lipoprotein consisting of two beta barrels (N-terminal and C-terminal barrels) linked by a short amino acid linker. The reason why V2 fHbp is absent in currently approved vaccines is the instability of its N-terminal β-barrel (Non-Patent Literature 2 and 7). Furthermore, protein stability is important for vaccine manufacturing because it affects yield and is a critical issue for quality control. Furthermore, concerns have been raised about whether the antigens in Bexcelo® and Tormenba® can provide broad coverage against antigenically diverse pathogens, as 36% and 4.8% of their sequences are identical to the respective serotype B meningococcal disease isolates currently circulating in the UK.Furthermore, modified V2 fHbp, which is more stable than wild-type V2 fHbp, has been described, including modified V2 fHbp containing at least six mutations in Ser35, Asp 07, Val 112, Leu 114, Ser 137, and Gly 138. Since a single fHbp does not provide universal protection against meningococcal disease, immunization with a vaccine containing representatives of each of the three variants V1, V2, and V3 is necessary for protection against a wide range of meningococcal serotype B.

[0014] Both the Bexcelo® and Tormenba® vaccines were developed before it was recognized that complement factor H (CFH) binds to Neisseria meningitidis with high affinity via fHbp, and that this can impair the immune response (Non-Patent Literature 8). Although fHbp has been demonstrated to be an important protective antigen, the extent to which fHbp interacts with fH during immunization and what fHbp-fH interactions affect the overall immunogenicity of fHbp in humans are currently unknown. Studies in hfH transgenic mice and infant rhesus monkeys (the latter having polymorphisms in the fH gene that can bind highly or lowly to fHbp (Non-Patent Literature 9)) have demonstrated that binding of fH to fHbp reduces the immunogenicity of fHbp (Non-Patent Literature 10-14).

[0015] Furthermore, upstream, downstream, and formulation development can often be rate-limiting steps in the early introduction of biopharmaceuticals to the market and the fulfillment of population demand.

[0016] Upstream process development involves scaling up fermentation processes to ensure similar product yields, where the quality of large-scale production is comparable to that of small-scale production. Various culture parameters, such as media composition, pH, agitation, aeration, temperature, cell density, inducer concentration, induction time, and supply strategy, affect protein expression levels depending on the expression system. Therefore, it is essential to evaluate the culture conditions for the expression of any recombinant protein and for the development of effective bioprocesses.

[0017] Escherichia coli is the most widely used bacterial host for recombinant protein production because (1) it has a rapid growth rate with a generation time of 20 minutes under optimized conditions (Non-Patent Literature 15); (2) it has well-developed molecular manipulation tools based on deep knowledge of the organism; and (3) it can achieve high cell concentrations using inexpensive culture reagents. However, numerous obstacles must be overcome in the actual pipeline. These include poor host strain growth, protein instability or toxicity, aggregation and inclusion body formation, unsuitability of environmental conditions (temperature, pH, salt concentration, etc.), and non-amplification of expression. If the target protein is undetectable or detected at extremely low levels (less than micrograms per liter of culture), this is often due to slower growth rates, low final cell density, and cell death. E. coli cannot perform post-translational modification, limiting the range of products that can be produced in a soluble and active form in this host organism. Furthermore, E. coli cannot secrete recombinant proteins. Therefore, recombinant E. coli cells must be destroyed in order to bring them into contact with intracellular products, and then typically purified by a filtration process.

[0018] Furthermore, when recombinant proteins are expressed at high levels in E. coli, they aggregate into inclusion bodies (IBs). This necessitates large-scale processing, including isolation, solubilization, refolding, and purification from cells, to produce bioactive proteins, posing a significant challenge for the industrial-scale production of soluble recombinant proteins with appropriate biological function. Modifying culture conditions is usually presented as the simplest solution to reduce IB formation in E. coli. However, the preferred culture conditions for soluble protein production vary depending on the target protein involved and the E. coli host strain used, requiring experimental optimization. Factors such as expression strains, fermentation media, and operating conditions all play a crucial role in scaling up the process to provide large quantities of protein through a cost-effective and commercially viable manufacturing process, ultimately maintaining or improving yields at industrial scale. Therefore, it is important to identify the appropriate parameters or sets of parameters that are critical to a particular process.

[0019] High-level production of recombinant proteins contributes to the approval of therapeutic products for human use, but subsequently, efficient purification processes on an industrial scale are required. Cell disruption is necessary to recover the desired protein expressed as intracellular IB. While cell disruption may be highly effective for small-scale operations, it is extremely inadequate for scale-up. Sonication has high energy requirements and significant health and safety issues due to noise. It is not continuous. Chemical cell lysis poses significant health and safety risks to the user, and the cost of using the large quantities of reagents required for large-scale production can be prohibitively high. Furthermore, salts and surfactants may not be compatible with protein assays. They may also affect the results of downstream applications (e.g., mass spectrometry). Therefore, the appropriate cell lysis method must consider the optimal results for scale-up.

[0020] It is well established that higher product concentrations in upstream processes lead to higher volumes of chromatographic resin and higher buffer requirements. Host cell proteins (HCPs) and DNA are the main sources of impurities, and the HCPs in each process differ significantly from one another in terms of molecular weight, charge, hydrophobicity, and structure. Therefore, they pose a challenge for chromatographic purification.

[0021] When purified, soluble, and active recombinant proteins are required, it is extremely valuable to have (i) means for detection by expression and purification schemes, (ii) means for achieving maximum solubility, and (iii) means for easy purification from an E. coli environment. These three goals can be directly achieved by expressing a tandem amino acid stretch (peptide tag) or a large polypeptide (fusion partner) with the desired protein to form a chimeric protein. Because peptide tags are small, they are less likely to interfere when fused to a protein. However, in some cases, they can have a negative effect on the tertiary structure or biological activity of the fusion chimeric protein. Therefore, peptide tags should also be removed because they can affect the three-dimensional structure of the protein, inhibit interactions with partner molecules, or reduce biological activity. In fact, if the tag is removed, the final solubility of the desired product is unpredictable. In the case of tag removal by enzymatic digestion, the expression vector has a sequence encoding a protease cleavage site downstream of the gene encoding the tag. The selection from different proteases is based on specificity, cost, the number of amino acids remaining in the protein after cleavage, and the ease of removal after digestion. Tobacco Etch Virus (TEV) type C cysteine ​​protease is one of the most widely used. However, TEV protease expression in E. coli presents challenges in terms of protein yield (reduced product yield) or low protein solubility at industrial scales, meaning that efficient cleavage requires large quantities and often long incubation times.

[0022] Vaccines based on recombinant protein antigens generally require adjuvants to achieve protection from related diseases. Aluminum salts are the most common adjuvants in vaccines approved for human use by the US Food and Drug Administration. The zero charge point (PZC) of an adjuvant is the point at which the net surface charge is zero; the PZC of aluminum oxyhydroxide is about 11, while the PZC of aluminum phosphate is about 4 - 5. Protein adsorption to the adjuvant surface generally maximizes when the sign of the protein's net charge is opposite that of the adjuvant surface, allowing for electrostatic attraction. Thus, protein vaccine formulations are formulated with adsorption buffers to improve adsorption to the surface. However, when a protein binds to a liquid - solid interface, the protein's conformation can change. Furthermore, conformational changes induced by binding to the adjuvant can change the stability of the protein during long - term storage. For example, when adsorption is essentially complete, aggregation via pathways occurring in the bulk solution does not seem to occur. Conversely, unfolding upon binding can expose residues that are normally buried to the solvent, promoting degradation processes such as oxidation (Non - patent Document 16).

[0023] The physical and covalent stabilization of recombinant protein antigens adsorbed to the adjuvant surface during long - term storage is a fundamental concern due to the potential impact of degradation on the immunogenicity, toxicity, and efficacy of the recombinant protein antigen.

[0024] There are various factors that affect the stability of recombinant protein antigens, contribute to storage stability, such as the proportion of antigen adsorption to the adjuvant surface, zeta potential, viscosity, solute concentration, pH changes, and temperature.

[0025] Zeta potential is one such physical property essential for optimizing the formulation of suspensions, emulsions, and protein solutions and for predicting surface interactions. Zeta potential is exhibited by any particles, macromolecules, or material surfaces in a suspension. Knowledge of zeta potential can reduce the time required to prepare test formulations. It can also be used to predict long-term stability. In some cases, particles in a dispersion may adhere to each other, forming aggregates whose size continuously increases and which then settle under the influence of gravity. Furthermore, it is found that zeta potential depends on the properties of the buffer used.

[0026] Various factors affect the zeta potential, including the excipients used in the formulation, the compatibility of the excipients, the concentration of the formulation components, pH, and conductivity.

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

[0028] The effects of pH and temperature on the stability and activity of recombinant protein antigens are considered in light of possible changes in the characteristics of the recombinant protein antigens, including degradation processes such as gradual aggregation and oxidation, which affect both physical and chemical stability.

[0029] Therefore, formulation development becomes increasingly important overall for the long-term storage of recombinant protein antigens. It is known that the effective concentration of available immunogen decreases as aggregation increases. Therefore, formulations and methods are needed to overcome the aggregation problem by stabilizing recombinant proteins against aggregation.

[0030] Therefore, there is a need for a meningococcal vaccine that exhibits maximum adsorption of recombinant protein antigens to the adjuvant surface, has low viscosity, does not aggregate, is stable over a wide temperature range, has improved immunogenicity, and is simultaneously affordable and safe.

[0031] Furthermore, there is a need for efficient platform processes to manufacture effective vaccines against Neisseria meningitidis that have improved immunogenicity and meet multiple criteria, including safety and availability, particularly improved formulations that are low viscosity, non-aggregating, and long-term stable over a wide temperature range.

[0032] Furthermore, it is necessary to prepare chimeric proteins that are soluble, produced in high yield, are stable, and all required protein antigens are immunogenic. [Prior art documents] [Non-patent literature]

[0033] [Non-Patent Document 1] AL Wilkins, MD Snape et al 2017 [Non-Patent Document 2] Prymula, R. 2014 [Non-Patent Document 3] Tan, L., et al. 2010 [Non-Patent Document 4] Fletcher et al (2004) Infect Immun 72:2088―2100 [Non-Patent Document 5] Masignani V, et al. (2003) The Journal of Experimental Medicine 197:789―799 [Non-Patent Document 6] Murphy E, et al. (2009) The Journal of Infectious Diseases 200:379―389 [Non-Patent Document 7] Johnson, S 2012 [Non-Patent Document 8] Schneider, M 2009 [Non-Patent Document 9] Konar M 2015 [Non-Patent Document 10] Beemink PT 2011 [Non-Patent Document 11] Costa 2014 [Non-Patent Document 12] Giuntini S 2015 [Non-Patent Document 13] Granoff DM 2015 [Non-Patent Document 14] Rossi R 2013 [Non-Patent Document 15] Clark and Maaloee, 1967 [Non-Patent Document 16] J Pharm Sci. 2009 Sep;98(9):2970―2993 [Overview of the project] [Problems that the invention aims to solve]

[0034] To overcome the limitations of the prior art described above, the applicant proposes improved upstream and downstream methods and formulation development for chimeric protein antigens based on meningococcal serotype B, and a method for preparing formulations based on said chimeric protein. [Means for solving the problem]

[0035] The applicant provides: 1) a chimeric antigen (CHA) against meningococcal serotype B (MenB). The CHA utilizes fHbp (non-lipidized) as a molecular scaffold for presenting a surface-exposed PorA VR2 loop, which is achieved by inserting the VR2 loop (a "10-20 amino acid" PorA VR2 loop instead of the "whole PorA protein") into the β-turn region of fHbp. The CHA has been found to carry epitopes derived from fHbp and PorA and to induce a functional immune response to both antigens. The incorporation 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) The soluble, high-yield chimeric protein is stable, and both fHbp and PorA VR2 loops are immunogenic. 3) To maximize vaccine coverage, chimeras are generated from the most common fHbp and PorA antigens. This is because the chimeric composition reflects the common fHbp and PorA antigens circulating within a given geographical region. 4) By inserting the PorA loop at a specific position of fHbp, H factor binding can be reduced as desired (at least 10%, at least 50%, preferably >70% reduction compared to the wild type), and the molecular weight can be in the range of 20kDa to 40kDa while preserving both the immunogenic epitopes of fHbp and PorA.

[0036] Table 1; fHbp-PorA chimera

[0037] [Table 1]

[0038] 5) Insert one PorA loop into one fHbp (instead of two PorA loops on one fHbp), and insert a maltose-binding protein (MBP) tag together with the His tag to ensure optimal expression, solubility, and stability. 6) Insert a TEV cleavage site between the His-MBP tag and the chimera to facilitate tag removal during downstream processing, and optimize the substrate (tagged recombinant protein / modified fHbp fusion protein) to enzyme ratio (20:1 compared to 5:1 and 10:1), and temperature / incubation (30°C, 15-18 hours). 7) Retention of plasmids for at least 50 generations without the need for antibiotics. 8) For large-scale cell lysis (pressure -1000-1500 bar; 3-8 cycles), the combined use of chemical lysis and homogenizer (instead of sonication or chemical lysis alone) is advantageous, considering the benefits of using a homogenizer, which include cell wall disruption at 4-15°C, effectiveness in neutral lipid extraction, ease of use in smaller volumes, faster processing, reliable operation, constant shear rate, and full expandability. 9) The yield of tagged fHbp protein may be at least 700 mg of purified tagged protein from 100 grams of moist cell aggregate (collection), resulting from the use of an optimal concentration of inducer (IPTG (1 mM~10 mM) or lactose (1 g / L~50 g / L)); the use of modified M9 salt medium (chemically defined medium) supplemented with L-methionine (maintaining the L-methionine concentration at 1~10 mM, preferably 2~5 mM, compared to Luria Broth (LB) medium (compound medium) / fed-batch culture mode); and inducing the culture by stopping glucose supply and starting glycerol supply when the OD at 590 / 600 nm is approximately 20~100, and adding and / or maintaining lactose at 1~50 g / L in fed-batch culture mode. 10) Instead of known three-step chromatography (multi-step affinity chromatography, ion exchange chromatography, and subsequent size exclusion), use two-step chromatography (ion exchange, followed by affinity chromatography).11) Use V2 fHbp stabilized by substituting amino acids in the N-terminal β-barrel (i.e., changing less than 5% of the residues in this barrel). 12) A formulation comprising an adjuvant (aluminum hydroxide), phosphate buffer, mannitol, and polysorbate (polysorbate 20), thereby conferring optimal pH (7-8), osmotic pressure (200-500, preferably 200-400 mOsmol / kg), and zeta potential (-16--30 mV), resulting in maximum adsorption and maintaining the integrity / stability of the chimeric protein. 13) For TEV cleavage, an enzyme:substrate ratio of 1:20 incubated for 18 hours was found to be optimal.

[0039] The present invention will be described with reference to the accompanying drawings. [Brief explanation of the drawing]

[0040] [Figure 1] This figure shows the vector map of the recombinant protein / modified fHbp fusion protein according to an embodiment of the present invention. [Figure 2] This figure shows the vector map of the recombinant protein / modified fHbp fusion protein according to an embodiment of the present invention. [Figure 3] This figure shows the vector map of the recombinant protein / modified fHbp fusion protein according to an embodiment of the present invention. [Figure 4] This figure shows the vector map of the recombinant protein / modified fHbp fusion protein according to an embodiment of the present invention. [Figure 5] This figure shows the vector map of the recombinant protein / modified fHbp fusion protein according to an embodiment of the present invention. [Figure 6] This figure shows a vector map of a TEV protease according to an embodiment of the present invention. [Figure 7] This is a flowchart for seed development in a 10 L scale fermentation batch for the production of tagged fHbp protein. [Figure 8] This is a flowchart for the generation of tagged fHbp protein in a 10 L scale fermenter. [Figure 9] Figures a-h show the growth profiles of tagged fHbp protein in a 10 L scale fermentation batch, along with SDS-PAGE gel images. [Figure 10] A flowchart for seed development in a 10 L scale fermentation batch for TEV protease production is shown. [Figure 11] This figure shows the production of TEV protease in a 10 L scale fermenter. [Figure 12] Figures a and b show the growth profiles of TEV protease in a 10 L scale fermentation batch, along with SDS-PAGE gel images. [Figure 13] This figure shows the solubility obtained using homogenization. [Figure 14] Images a and b are SDS-PAGE images of TEV protease cleavage reactions using different substrate:enzyme ratios. [Figure 15] This figure shows densitometry analysis of TEV protease cleavage reactions using different substrate:enzyme ratios. [Figure 16] These are SDS-PAGE images of TEV protease cleavage reactions using different temperatures. [Figure 17] This figure shows the densitometry analysis of TEV protease cleavage reactions using different temperatures. [Figure 18] This is a flowchart for the purification of TEV protease using Ni-Sepharose resin (pH 7.4). [Figure 19] This is a flowchart for the purification of TEV protease using Ni-Sepharose resin (pH 8.5). [Figure 20] Figures a and b show the purification profiles of TEV protease at pH 8.5 and pH 7.4, respectively. [Figure 21] This is a flowchart for purifying tagged proteins using Ni-Sepharose resin. [Figure 22] This figure shows the purification of the tagged protein expressed in the inclusion body shown in Figure 1. [Figure 23] This is a flowchart for removing tags from purified proteins. [Figure 24] This is a flowchart of the ion exchange chromatography process for purifying recombinant protein / modified fHbp fusion protein. [Figure 25] This is a flowchart of the affinity chromatography process for purifying recombinant protein / modified fHbp fusion protein. [Figure 26] This graph highlights individual data points and GMT for each bleeding day. [Figure 27] This figure shows representative data on the binding of human complement factor H to wild-type fHbp compared to Men B chimeric protein. [Figure 28] This graph shows the GMT values ​​for each time point (serum collection date) for the MenFive serotype. [Figure 29] This graph shows the GMT values ​​for each time point (serum bleeding day) for the MenB serotype. [Figure 30] This figure shows a calibration curve for protein estimation using the microbicinchoninic acid (BCA) method. [Modes for carrying out the invention]

[0041] [the purpose] One object of the present invention, according to at least one embodiment herein, is as follows:

[0042] The purpose of this disclosure is to provide an efficient platform process for manufacturing effective vaccine formulations against meningococcal bacteria that meet multiple criteria for improved immunogenicity, safety, and cost-effectiveness.

[0043] Another object of this disclosure is to provide an efficient platform process for producing an effective vaccine formulation comprising one or more recombinant protein / modified fHbp fusion proteins derived from Neisseria meningitidis serotype B and a pharmaceutically acceptable carrier or excipient.

[0044] Another objective of this disclosure is to develop and optimize upstream bioprocesses to increase the cell density of lead cell lines and the productivity of recombinant protein / modified fHbp fusion proteins.

[0045] Another objective of this disclosure is to develop and optimize downstream bioprocesses to produce recombinant protein / modified fHbp fusion proteins in high yield and high purity.

[0046] Another objective of this disclosure is to develop and optimize formulations containing recombinant protein / modified fHbp fusion proteins that have improved immunogenicity, low viscosity, are non-aggregating, and are long-term stable over a wide temperature range.

[0047] Another objective of this disclosure is to develop and optimize formulations containing a chimeric antigen (CHA) against meningococcal serotype B. The CHA utilizes fHbp (non-lipidized) as a molecular scaffold for presenting a surface-exposed PorA VR2 loop, which is achieved by inserting the VR2 loop (in place of the "whole PorA protein") into the β-turn region of fHbp. The CHA has been found to retain epitopes derived from fHbp and PorA and to induce a functional immune response to both antigens, and the incorporation of the VR2 loop does not alter the overall structure of fHbp, and the VR2 loop folds into a conformation that is recognized by bactericidal mAbs.

[0048] Another objective of this disclosure is to develop and optimize formulations containing a chimeric protein that is soluble, yields high yield, is stable, and both the fHbp and PorA VR2 loops are immunogenic.

[0049] Another objective of this disclosure is to develop and optimize formulations containing chimeric elements composed of the most common fHbp and PorA antigens to maximize the scope of vaccine application.

[0050] Another objective of this disclosure is to develop and optimize formulations containing chimeric proteins in the molecular weight range of 20 kDa to 40 kDa that preserve both fHbp and PorA immunogenic epitopes.

[0051] Table 2; fHbp-PorA chimera

[0052] [Table 2]

[0053] Another objective of this disclosure is to develop and optimize downstream processes, including inserting a TEV cleavage site between the His-MBP tag and the chimera, to facilitate tag removal during downstream processing and optimize substrate-to-enzyme ratio / temperature / incubation and the retention of the largest generation plasmid without the need for antibiotics.

[0054] Another object of this disclosure is the combined use of chemical lysis and homogenizers (using optimized conditions instead of sonication or chemical methods alone) for large-scale cell lysis.

[0055] Another objective of this disclosure is to develop high-yield chimeric fHbp-PorA proteins resulting from the use of optimal concentrations of inducers; the use of L-methionine supplemented modified M9 salt medium (chemically defined medium) / fed-batch culture mode; and inducing the culture by stopping glucose supply and starting glycerol supply when a specific OD is reached at 590 / 600 nm, and adding lactose or IPTG.

[0056] Another object of this disclosure is to develop a two-step chromatography method as an alternative to known methods that use at least three steps of chromatography.

[0057] Another objective of this disclosure is to develop and optimize formulations comprising a chimeric protein, an adjuvant (aluminum hydroxide), phosphate buffer, mannitol, and polysorbate (polysorbate 20) to impart optimal pH, osmotic pressure, and zeta potential, thereby resulting in maximum adsorption and preservation of integrity / stability of the chimeric protein.

[0058] Another object of this disclosure is to provide a method for vaccinating a host.

[0059] Further purposes and benefits of this disclosure will become clearer from the following description, which is not intended to limit the scope of this disclosure.

[0060] [Embodiments of the Invention] Before describing the compositions and formulations of the present invention, it should be understood that the present invention is not limited to the specific compositions and formulations described. Furthermore, it should be understood that the scope of the present invention is limited only by the appended claims, and therefore the terms used herein are not intended to be limiting.

[0061] While this disclosure may accept different aspects and embodiments, it can be considered illustrative of the principles of this disclosure and is not intended to limit the scope of this disclosure to those illustrated and disclosed herein. Specific embodiments are shown in the drawings and in the following detailed description.

[0062] The embodiments and models are provided to thoroughly and completely convey the scope of this disclosure to those skilled in the art. Numerous details are given with respect to specific components and processes in order to provide a complete understanding of the embodiments of this disclosure. It will be obvious to those skilled in the art that the details provided in the examples should not be construed as limiting the scope of the invention. In some embodiments, well-known compositions, well-known processes and well-known techniques are not described in detail.

[0063] The terms used in this disclosure are for the purpose of describing specific embodiments only, and should not be considered to limit the scope of this disclosure. As used in this disclosure, “a,” “an,” and “the” (original text) may be intended to include the plural form unless the context explicitly suggests otherwise.

[0064] The terms “equipped,” “containing,” “including,” and “possessing” are non-restrictive transitional phrases that thus specify the presence of the described features, integers, processes, operations, elements, modules, units, and / or components, but do not prohibit the presence or addition of one or more further features, integers, processes, operations, elements, components, and / or groups thereof. The specific sequence of processes disclosed in the process of this disclosure should not be construed as necessarily requiring their performance as described or illustrated. It should also be understood that additional or alternative processes may be used.

[0065] As used herein, the terms “comprising,” “comprises,” and “comprised of” are synonymous with “including,” “includes,” or “containing,” and are comprehensive or non-exclusive, and do not exclude further unlisted members, elements, or process steps. As used herein, the terms “comprising,” “comprises,” and “comprised of” will be understood to include the terms “consisting of,” “consists,” and “consists of.” More specifically, as used herein, “comprising” means that the claims encompass all listed elements or process steps, but may also include further, unlisted elements or process steps. For example, a method comprising steps a), b), and c) encompasses, in its narrowest sense, a method comprising steps a), b), and c). The term "consists of" means that a composition (or apparatus or method) has the listed elements (or steps), but nothing more. In contrast, the term "includes" may also include methods that include additional steps, such as steps d) and e), in addition to steps a), b) and c).

[0066] The terms 1, 2, 3, etc., should not be construed as limiting the scope of this disclosure, and such terms may be used only to distinguish one element, component, area, layer, or section from other elements, areas, layers, or sections. Where used herein, the terms 1, 2, 3, etc., do not imply any particular order or sequence unless expressly suggested by this disclosure.

[0067] Furthermore, terms such as “first,” “second,” “third,” or “(a),” “(b),” “(c),” “(d),” etc., in the description and claims are used to distinguish similar elements and are not necessarily used to describe a continuous or temporal order. Terms used in this manner are interchangeable under appropriate circumstances, and it should be understood that embodiments of the invention described herein may operate in an order other than those described or illustrated herein. In the case of terms such as “first,” “second,” “third,” or “(A),” “(B),” and “(C),” or “(a),” “(b),” “(c),” “(d),” “i,” “ii,” etc., there is no consistency in time or time intervals between steps with respect to the steps of the method, use, or assay; that is, unless otherwise indicated in this application, as described above or below herein, steps may be performed simultaneously or there may be time intervals of seconds, minutes, hours, days, weeks, months, or even years between such steps.

[0068] The following sections define different embodiments of the present invention in more detail. Each of the embodiments defined in this way may be combined with any further embodiments unless expressly indicated otherwise. In particular, any feature indicated as preferred or advantageous may be combined with any further feature indicated as preferred or advantageous.

[0069] Throughout this specification, any reference to “one embodiment” or “embodiment” means that a particular feature, structure, or characteristic described in relation to the embodiment is included in at least one embodiment of the present invention. Therefore, where the phrase “in one embodiment” or “in one embodiment” appears in various places throughout this specification, it may refer to the same embodiment, although not all instances of this refer to the same embodiment. Furthermore, features, structures, or characteristics may be combined in any suitable manner in one or more embodiments, as will be apparent to those skilled in the art from this disclosure. Moreover, some embodiments described herein include some features included in further embodiments, while others do not. Combinations of features from different embodiments, as will be understood to those skilled in the art, fall within the scope of the present invention and form different embodiments. For example, in the appended claims, any of the claimed embodiments may be used in any combination.

[0070] Each feature, embodiment, or combination described herein is a non-limiting exemplary example of any aspect of the Invention and is therefore meant to be combinable with any further features, embodiments, or combinations described herein. For example, where a feature is described in terms such as “one embodiment,” “a certain embodiment,” “a particular embodiment,” “a further embodiment,” “a particular exemplary embodiment,” and / or “another embodiment,” each of these types of embodiments is a non-limiting example of a feature intended to be combined with any further features or combinations of features described herein, without the need to enumerate all possible combinations. Such features or combinations of features are applicable to any aspect of the Invention.

[0071] Furthermore, the ranges defined throughout this specification also include the terminal values, i.e., the range from 1 to 10 and between 1 and 10 mean that both 1 and 10 are included in the range. To avoid any doubt, the applicant has the right to any equivalents in accordance with applicable law.

[0072] As used herein, the term "about" means, when modifying the value of a listed item, number, ratio, or term, a range of plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the value of the listed item, number, ratio, or term. A range of plus or minus 10% is preferred.

[0073] Where numerical ranges such as "at a concentration of 1 to 5 micromoles" are used herein, the range includes not only 1 to 5 micromoles but also any numerical values ​​between 1 and 5 micromoles, such as 2, 3, and 4 micromoles. The term "in vitro" as used herein means outside or external to the body of an animal or human. The term "in vitro" as used herein should be understood to include "ex vivo." The term "ex vivo" usually refers to tissue or cells that have been removed from the body of an animal or human and maintained or grown outside the body, for example, in a culture vessel. The term "in vivo" as used herein means inside or internal to the body of an animal or human.

[0074] [Definition] To facilitate understanding of this disclosure, certain terms are first defined below. Further definitions of the following terms and other terms may be provided throughout this specification.

[0075] As used herein, the terms “protein,” “polypeptide,” “(poly)peptide,” or “peptide” (all terms are interchangeable unless otherwise indicated) encompass isolated and / or purified and / or recombinant (poly)peptides that essentially do not contain further host cell polypeptides. As used herein, “peptide” comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 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 more amino acid residues, where one α-carboxyl group is bonded to another α-amino group. The post-translational modifications of proteins or peptides used and envisioned herein are modifications of newly formed proteins or peptides and may include deletion, substitution or addition of amino acids, chemical modification of specific amino acids, such as amidation, acetylation, phosphorylation, glycosylation, formation of pyroglutamic acid, oxidation / reduction of a sulfo group on methionine, or addition of a similar small molecule to a specific amino acid.

[0076] As used herein, the term "homologous organism" refers to bacterial, fungal, plant, or animal homologs, preferably plant homologs, of oxidase enzymes or rubredoxin or rubredoxin reductase useful in the present invention, but also includes shortened sequences of coding and non-coding DNA sequences, single-stranded DNA, or RNA.

[0077] 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, and is determined by comparing these sequences. Typically, sequence identity or similarity is compared over the entire length of the sequences, but may be compared only over portions of sequences that align with each other. Preferably, sequence identity or similarity is compared over the entire length of the sequences herein. In the art, “identity” or “similarity” also sometimes means the degree of sequence relevance between polypeptide sequences or nucleic acid sequences, determined by the agreement between such sequences. Sequence alignments can be generated using several software tools, such as the following: See 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. This algorithm is implemented, for example, in the "NEEDLE" program, which performs global alignment of two sequences. The NEEDLE program is included, for example, in the European Molecular Biology Open Software Suite (EMBOSS). EMBOSS—a collection of various programs. The European Molecular Biology Open Software Suite (EMBOSS), Trends in Genetics 16 (6), 276 (2000). BLOSUM (BLOCKS substitution matrix) is typically generated based on the alignment of conserved regions of protein domains, for example (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 the many inputs is "BLOSUM62," which is often the "default" setting for many programs when aligning protein sequences. BLAST (Basic Local Alignment Search Tool) consists of several individual programs (BlastP, BlastN) primarily used to search for similar sequences in large sequence databases. The BLAST program also creates local alignments. Typically, the "BLAST" interface provided by NCBI (National Centre for Biotechnology Information) is used, which is an improved version ("BLAST2"). "Original" BLAST: Altschul, SF, Gish, W., Miller, W., Myers, EW & Lipman, DJ (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.

[0078] As used herein, the term “sequence identity” is preferably a value determined by the EMBOSS Pairwise Alignment Algorithm “Needle”. In particular, the NEEDLE program from the EMBOSS package can be used (version 2.8.0 or later, EMBOSS: The European Molecular Biology Open Software Suite—Rice, P., et al. Trends in Genetics (2000) 16:276-277; http: / / emboss.bioinformatics.nl) using the NOBRIEF option ("Simple Identity and Similarity to NO") to calculate “longestidentity"). Identity, homology, or similarity between two aligned sequences is calculated as follows: the number of corresponding positions in the alignment that show the same amino acid in both sequences, divided by the total length of the alignment after subtracting the total number of gaps in the alignment. Default parameters for amino acid sequence alignment are matrix=Blosum62; open gap penalty=10.0; gap elongation penalty=0.5. Matrix = DNAfull; Open gap penalty = 10.0; Gap extension penalty = 0.5.

[0079] Sequence identity is typically provided as "% sequence identity" or "% identity". In the first step, a pairwise sequence alignment (i.e., a pairwise global alignment) is generated between the two sequences, where the two sequences are aligned over their full length, full length, or entire length, in order to determine the identity ratio between the two amino acid sequences. The alignment is generated using a program or software described herein. A preferred alignment for the purposes of the present invention is one that can determine the highest sequence identity.

[0080] As used herein, the term "sequence identity" refers to the identity ratio between two sequences aligned using standard NCBI BLAST parameters (http: / / blast.ncbi.nlm.nih.gov).

[0081] The nucleic acid (or polynucleotide) of the present invention comprises a nucleic acid sequence encoding the fusion protein of the present invention. The nucleic acid sequence encoding the fusion protein of the present invention is preferably a recombinant and / or isolated and / or purified nucleic acid sequence. The nucleic acid sequence encoding the fusion protein of the present invention can be generated and isolated using known molecular biological standards, sequence information provided herein, and biological organisms.

[0082] As used herein, the term “nucleic acid” includes references to single-stranded or double-stranded deoxyribonucleotides or ribonucleotide polymers, i.e., polynucleotides, and, unless otherwise specified, includes known analogues that possess the essential properties of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids). Polynucleotides may be full-length or partial sequences of natural or heterologous structural or regulatory genes. Unless otherwise specified, the term includes references to specific sequences and their complementary sequences. Thus, DNA or RNA whose backbone has been modified for stability or further reasons is a “polynucleotide” when the term is intended herein. Furthermore, to give two examples, DNA or RNA containing unusual bases such as inosine or modified bases such as tritylated bases is a “polynucleotide” as used herein. It will be understood that a wide variety of modifications are made to DNA and RNA to serve many useful purposes known to those skilled in the art. As used herein, the term “polynucleotide” encompasses chemically, enzymatically, or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells (including, in particular, simple and complex cells). All nucleic acid sequences herein encoding polypeptides such as oxidase enzymes or rubredoxin or rubredoxin reductase also describe all possible silent variations of the nucleic acid by reference to the genetic code. The term “conservatively modified variant” applies to both amino acid sequences and nucleic acid sequences. When the term “conservatively modified variant” is used in reference to a particular nucleic acid sequence, it may refer to a nucleic acid encoding an identical variant of the amino acid sequence or a conservatively modified variant, due to the degeneracy of the genetic code. The term “degeneracy of the genetic code” refers to the fact that a number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Therefore, at all positions where alanine is identified by the codon, the codon can be changed to any of the corresponding codons described herein without altering the encoded polypeptide.The nucleic acid mutation in question is a "silent mutation" and represents a type of conservatively modified mutation. In this specification, the terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to polymers of amino acid residues.

[0083] The terms “polypeptide,” “peptide,” and “protein” also apply to amino acid polymers, which are artificial chemical analogs of naturally occurring amino acids, in which one or more amino acid residues correspond to naturally occurring amino acids. The essential property of such analogues of naturally occurring amino acids is that, when incorporated into a protein, that protein is specifically reactive to antibodies that are induced against the same protein but consist entirely of naturally occurring amino acids. The terms “polypeptide,” “peptide,” and “protein” also include modifications, including but not limited to glycosylation, lipid binding, sulfation, γ-carboxylation, hydroxylation, and ADP-ribosylation of glutamate residues. In the context of this application, oligomers (oligonucleotides, oligopeptides, etc.) are considered a species of polymer. Oligomers have a relatively small number of monomer units, generally 2 to 100, particularly 6 to 100, including primer sequences, such as those used in the examples for cloning oxidase enzymes or rubredoxin or rubredoxin reductase useful in the present invention.

[0084] As used in relation to nucleic acids (DNA or RNA) or proteins of the present invention, the term "heterogeneous" means a nucleic acid or protein found in a cell or location(s) within a genome or DNA or RNA sequence that is not naturally present or found in nature as part of the organism, cell, genome, or DNA or RNA sequence in which it exists. Heterogeneous nucleic acids or proteins of the present invention are not endogenous to the cell into which they are introduced, but are derived from other cells or produced synthetically or recombinantly. Generally, though not necessarily, such nucleic acids encode proteins that are not normally produced by the cell in which the DNA is expressed. A gene that is endogenous to a particular host cell but has been modified from its natural form, for example using DNA shuffling, is also called heterogeneous. The term "heterogeneous" also includes multiple non-natural copies of a naturally occurring DNA sequence. Thus, the term "heterogeneous" may be a DNA segment that is foreign or heterogeneous to the cell, or homogeneous to the cell but is located in a location and / or number within the host cell nucleic acid where its segment is not normally found. Exogenous DNA segments are expressed to produce exogenous polypeptides.

[0085] The “homologous” DNA sequences of this invention are DNA sequences that are naturally associated with the host cell into which they are introduced. Any nucleic acid or protein that a person skilled in the art would recognize as heterologous or exogenous to the cell in which it is expressed is included herein in the term heterologous nucleic acid or protein.

[0086] The terms “modified,” “modified,” “mutated,” and “mutated” as used herein in relation to a protein or polypeptide compared to another protein or polypeptide are applied mutatis mutandis to nucleotide or nucleic acid sequences. These terms are used to indicate that a modified nucleotide or nucleic acid sequence encoding a protein or polypeptide has at least one difference in its nucleotide or nucleic acid sequence compared to the nucleotide or nucleic acid sequence of the protein or polypeptide being compared. These terms are used regardless of whether the modified or mutant protein was actually obtained by mutagenesis of the nucleic acid encoding these amino acids, modification of the polypeptide or protein, or in other ways, for example, using artificial gene synthesis methodologies. Mutagenesis is a well-known method in the art and includes, for example, PCR-mediated site-directed mutagenesis or oligonucleotide-mediated mutagenesis, as described in Sambrook, J., and Russell, DW Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (2001). As used herein with respect to genes, the terms “modified,” “mutated,” or “mutated” are used to indicate that at least one nucleotide in the nucleotide sequence of that gene or its regulatory sequence is different from the nucleotide sequence being compared. Modification or mutation may, in particular, be a substitution of a nucleotide by a different nucleotide, a deletion of a nucleotide, or an insertion of a nucleotide.

[0087] The terms "modified fHbp / modified fHbp fusion protein / recombinant protein / chimera / chimeric protein / chimeric molecule / chimeric antigen / fusion protein / clone" are used interchangeably herein and refer to proteins produced through the ligation of two or more genes that originally encoded separate or identical proteins, resulting in a polypeptide containing a combination of sequences derived from different gene products or sources. Translation of this recombinant / chimeric / fusion gene yields a single polypeptide possessing the functional properties derived from each of the original proteins, along with a few additional features.

[0088] As used herein, the term "tagged protein" refers to a protein that has a specific peptide sequence (also known as a tag) genetically grafted onto it. Tags serve a variety of purposes and can be attached to either end of a target protein, being specific to either the C-terminus, the N-terminus, or both. Some tags can also be inserted into sites within the target protein; these are known as internal tags.

[0089] Affinity tags are attached to proteins using affinity technology to purify them from their crude biological source. Examples of affinity tags include chitin-binding proteins (CBPs), maltose-binding proteins (MBPs), Strep tags, and glutathione-S-transferase (GST). Poly(His) tags are commonly used protein tags that bind to a matrix containing immobilized metal ions.

[0090] As used herein, the term "peptide loop" is intended to refer to a single-chain polypeptide sequence fixed at both ends (e.g., fixed to a scaffold such as fHbp). The term "loop" does not imply or require any specific secondary structure adopted by the polypeptide.

[0091] In the context of "exogenous peptide loops," the term "exogenous" as used herein is understood to mean that the peptide loop originates from a source different from the fHbp protein (i.e., not fHbp or a fragment thereof). However, it may originate from the same organism as fHbp. For example, modified fHbp may include meningococcal fHbp modified with an (exogenous) peptide loop derived from Neisseria meningitidis PorA.

[0092] The term "isolated," when applied to the modified fHbp of the present invention, means a protein that is (i) encoded by nucleic acids using recombinant DNA or a viral vector, or (ii) synthesized by, for example, chemical synthesis, or (iii) isolated from a biological material and then purified. The isolated polypeptides of the present invention include proteins expressed from a protein-coding nucleotide sequence or from a recombinant vector containing a protein-coding nucleotide sequence.

[0093] As used herein, the terms "immunogenic" or "antigen" refer to molecules that can induce an immune response in the body of a human or animal. The immune response may be defensive.

[0094] As used herein, the term “protective” means the prevention of disease, the reduction of the risk of infection, transmission and / or progression of disease, the reduction of the severity of disease, the cure of a condition or disease, the alleviation of symptoms, or the reduction of the severity of disease or disease symptoms.

[0095] In this specification, “prevention” means preventive or protective measures against disease. Prevention may include reducing the risk of infection, transmission and / or progression, or reducing the severity of the disease.

[0096] As used herein, the term "treatment" means the cure of a condition or disease, the alleviation of symptoms, or the reduction of the severity of a disease or its symptoms.

[0097] The term "freeze-drying" refers to a process in which a suspension or solution is frozen, and then water is removed by sublimation at low pressure.

[0098] The term "sublimation" refers to a change in the physical properties of a composition, where the composition changes directly from a solid state to a gaseous state without becoming a liquid.

[0099] This disclosure envisions an efficient platform process for producing an effective vaccine against Neisseria meningitidis that meets multiple criteria for improved immunogenicity, safety, and cost-effectiveness, particularly an improved formulation that is low viscosity, does not aggregate, and exhibits long-term stability over a wide temperature range.

[0100] In one aspect of the present invention, a modified factor H binding protein (fHbp) is provided. In one embodiment, the modified factor H binding protein (fHbp) is a fusion protein comprising a wild-type fHbp variant and at least one exogenous peptide loop.

[0101] In one embodiment, fHbp is meningococcal fHbp. In another embodiment, fHbp is gonorrhea fHbp.

[0102] In one embodiment, the modified factor H binding protein (fHbp) has an amino acid sequence that is at least 75% identical to one of the sequences of sequence numbers 6 to 10.

[0103] In one embodiment, the modified factor H binding protein (fHbp) contains an amino acid sequence that is at least 75% identical to one of the sequences of sequence numbers 6 to 10.

[0104] Those skilled in the art will understand that one, two, three, or four or more amino acid substitutions, deletions, or additions can be made to the modified fHbp of the present invention without substantially impairing its immunogenic function or affecting its stability. Substitutions may be made, for example, to similar amino acid residues or synthetic analogs that have similar MW, charge, hydrophobicity, or moiety. Such modifications are envisioned as part of the present invention.

[0105] In one embodiment, the modified fHbp may 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 with any one of the modified fHbp sequences described herein.

[0106] In one embodiment, the modified factor H binding protein (fHbp) is selected from an amino acid sequence that has at least 75.0%, 80.0%, 85.0%, 90.0%, 95.0%, 95.0%, 98.0%, 99.0%, or 99.5% identity with any one of the sequences of sequence numbers 6 to 10.

[0107] In one embodiment, the modified factor H binding protein (fHbp) is selected from an amino acid sequence that has at least 75.0% identity with 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 that has at least 75.0%, 80.0%, 85.0%, 90.0%, 95.0%, 95.0%, 98.0%, 99.0%, or 99.5% identity with the sequence of SEQ ID NO: 6.

[0108] In one embodiment, the modified factor H binding protein (fHbp) is selected from an amino acid sequence that has at least 75.0% identity with 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 that has at least 75.0%, 80.0%, 85.0%, 90.0%, 95.0%, 95.0%, 98.0%, 99.0%, or 99.5% identity with the sequence of SEQ ID NO: 7.

[0109] In one embodiment, the modified factor H binding protein (fHbp) is selected from an amino acid sequence that has at least 75.0% identity with 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 that has at least 75.0%, 80.0%, 85.0%, 90.0%, 95.0%, 95.0%, 98.0%, 99.0%, or 99.5% identity with the sequence of SEQ ID NO: 8.

[0110] In one embodiment, the modified factor H binding protein (fHbp) is selected from an amino acid sequence that has at least 75.0% identity with 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 that has at least 75.0%, 80.0%, 85.0%, 90.0%, 95.0%, 95.0%, 98.0%, 99.0%, or 99.5% identity with the sequence of SEQ ID NO: 9.

[0111] In one embodiment, the modified factor H binding protein (fHbp) is selected from an amino acid sequence that has at least 75.0% identity with 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 that has at least 75.0%, 80.0%, 85.0%, 90.0%, 95.0%, 95.0%, 98.0%, 99.0%, or 99.5% identity with the sequence of SEQ ID NO: 10.

[0112] In embodiments of the present invention, at least one exogenous peptide loop is immunogenic and derived from a bacterial membrane protein.

[0113] In one embodiment, the fHbp variant is selected from v1, v2, and v3 and modified with at least one PorA loop inserted into the β-turn region of fHbp. In one embodiment, fHbp may include fHbp v1. In another embodiment, fHbp may include fHbp v2. In yet another embodiment, fHbp may include fHbp v3.

[0114] In one embodiment, a variant of wild-type fHbp includes a wild-type Neisseria meningitidis ortholog of fHbp. For example, a variant of fHbp may include Ghfp, a Neisseria gonorrhoeae homolog of fHbp. Ghfp is non-functional and closely related to v3 fHbp (over 95% aa identity, dissociation constant KD > 100 μM with factor H).

[0115] In other embodiments, the PorA loop is selected from VR1 and VR2. In one embodiment, the PorA loop is VR1. In other embodiments, the PorA loop is VR2.

[0116] In one embodiment, the modified H factor-binding protein exhibits reduced H factor-binding activity. In a preferred embodiment, the modified fHbp shows more than 80% less binding to human H factor (fH) compared to wild-type fHbp.

[0117] In another aspect of the present invention, nucleic acids that essentially or at least encode the modified fHbp according to the present invention are provided.

[0118] In one embodiment, the present invention is a nucleic acid sequence encoding a modified fHbp, which has at least 75.0%, 80.0%, 85.0%, 90.0%, 95.0%, 95.0%, 98.0%, 99.0%, or 99.5% identity with any one of the sequences of sequence numbers 1 to 5.

[0119] In one embodiment, the present invention is a nucleic acid sequence encoding a modified fHbp that has at least 75.0% identity with the sequence of SEQ ID NO: 1. In a preferred embodiment, the present invention is a nucleic acid sequence encoding a modified fHbp that has at least 75.0%, 80.0%, 85.0%, 90.0%, 95.0%, 95.0%, 98.0%, 99.0%, or 99.5% identity with the sequence of SEQ ID NO: 1.

[0120] In one embodiment, the present invention is a nucleic acid sequence encoding a modified fHbp that has at least 75.0% identity with the sequence of SEQ ID NO: 2. In a preferred embodiment, the present invention is a nucleic acid sequence encoding a modified fHbp that has at least 75.0%, 80.0%, 85.0%, 90.0%, 95.0%, 95.0%, 98.0%, 99.0%, or 99.5% identity with the sequence of SEQ ID NO: 2.

[0121] In one embodiment, the present invention is a nucleic acid sequence encoding a modified fHbp that has at least 75.0% identity with the sequence of SEQ ID NO: 3. In a preferred embodiment, the present invention is a nucleic acid sequence encoding a modified fHbp that has at least 75.0%, 80.0%, 85.0%, 90.0%, 95.0%, 95.0%, 98.0%, 99.0%, or 99.5% identity with the sequence of SEQ ID NO: 3.

[0122] In one embodiment, the present invention is a nucleic acid sequence encoding a modified fHbp that has at least 75.0% identity with the sequence of sequence number 4. In a preferred embodiment, the present invention is a nucleic acid sequence encoding a modified fHbp that has at least 75.0%, 80.0%, 85.0%, 90.0%, 95.0%, 95.0%, 98.0%, 99.0%, or 99.5% identity with the sequence of sequence number 4.

[0123] In one embodiment, the present invention is a nucleic acid sequence encoding a modified fHbp that has at least 75.0% identity with the sequence of sequence number 5. In a preferred embodiment, the present invention is a nucleic acid sequence encoding a modified fHbp that has at least 75.0%, 80.0%, 85.0%, 90.0%, 95.0%, 95.0%, 98.0%, 99.0%, or 99.5% identity with the sequence of sequence number 5.

[0124] In one embodiment, a nucleic acid sequence encoding the modified fHbp of the present invention is provided. In one embodiment, the nucleic acid is a vector such as a viral vector.

[0125] In other embodiments, the present invention is an immunogenic composition comprising at least one modified fHbp described herein.

[0126] In one embodiment, the present invention is an immunogenic composition comprising at least one modified fHbp described herein or encoded by a nucleic acid sequence encoding a modified fHbp described herein.

[0127] In one embodiment, an immunogenic composition comprising at least one modified fHbp of the present invention is provided.

[0128] In one embodiment, the present invention is an immunogenic composition comprising at least one modified fHbp, wherein the modified fHbp is as follows: —At least one modified fHbp represented by an amino acid sequence that is at least 75% identical to any one of the amino acid sequences represented by SEQ ID NOs: 6-10, or any combination thereof. —At least one modified fHbp that encodes a nucleic acid sequence that has at least 75% identity with any one of the nucleic acid sequences represented by sequence numbers 1 to 5, or any combination thereof.

[0129] In one embodiment, the present invention is an immunogenic composition comprising at least one modified fHbp, wherein the modified fHbp is as follows: ―fHbpV3.45 M5:PorA316―320 / exP1.14 (represented by sequence number 6 or coded by sequence number 1) or ―fHbpV2.19 M6:PorA316―320 / exP1.4 (represented by sequence number 7 or coded by sequence number 2) or ―fHbpV1.14:PorA307―311 / exP1.9 (represented by sequence number 8 or coded by sequence number 3) or ―fHbpV1.1:PorA307―311 / exP1.4 (represented by sequence number 9 or coded by sequence number 4) or ―fHbpV1.1:PorA307―311 / exP1.9 (coded by sequence number 10 or sequence number 5) or It's a combination of those.

[0130] In other embodiments, the immunogenic composition comprises two or more different modified fHbp.

[0131] In yet another embodiment, the immunogenic composition comprises three or more different modified fHbp.

[0132] In yet another embodiment, the immunogenic composition comprises four different modified fHbp.

[0133] In one embodiment, the immunogenic composition comprises a pharmaceutically acceptable carrier. In further embodiments, the immunogenic composition comprises an adjuvant. In other embodiments, the immunogenic composition further comprises at least one additional prophylactic or therapeutically active molecule.

[0134] At least one further prophylactic or therapeutically active molecule is one of the following: Monovalent protein:capsular polysaccharide vaccine; or A conjugate vaccine is a vaccine in which an antigen containing an fHbp scaffold with an exogenous peptide loop is incorporated as a protein carrier molecule in the conjugate vaccine.

[0135] The modified fHbp, nucleic acids, or compositions of the present invention may be used as pharmaceuticals or for the treatment or prevention of pathogenic infection or colonization in subjects.

[0136] The present invention further envisions an immunogenic composition comprising a combination of modified fHbp, nucleic acid, and at least one further prophylactic or therapeutically active molecule.

[0137] In one embodiment, at least one further prophylactic or therapeutically active molecule comprises a protein:capsular polysaccharide complex vaccine.

[0138] In one embodiment, the protein:capsular polysaccharide vaccine includes any of the following: a serotype C or A capsule containing a bacterial toxin, a bivalent vaccine (containing serotype C and A capsular polysaccharides combined with the bacterial toxin), a tetravalent vaccine (containing serotype A, C, Y, and W polysaccharides combined with the bacterial toxin), or a pentavalent vaccine (containing serotype A, C, Y, W, and X polysaccharides combined with the bacterial toxin).

[0139] In other embodiments, at least one further prophylactic or therapeutically active molecule comprises a complex vaccine in which an antigen comprising an fHbp scaffold having an exogenous peptide loop is incorporated into the complex vaccine as a protein carrier molecule, and optionally the complex vaccine comprises one of the serotype capsular polysaccharides selected from strains A, C, Y, W, or X or combinations thereof.

[0140] In one embodiment, factor H binding protein (fHbp) is used as an epitope display scaffold.

[0141] fHbp, found on the surface of Neisseria meningitidis, is a 27 kDa lipoprotein consisting of two beta barrels (N-terminal and C-terminal barrels) linked by a short amino acid linker. Charged carbohydrates on the surface of vascular endothelium bind to fH, and charged amino acids in fHbp bind to fH at the same site of this complement regulator with nanomolar affinity. Based on differences in nucleotides and predicted amino acid sequences, fHbp from different Neisseria meningitidis strains are classified using several schemes. Factor H-binding protein (fHbp, also known in the art as lipoprotein 2086 (Non-Patent Literature 4), genome-derived Neisseria antigen (GNA) 1870 (Masignani et al. (2003) J Exp Med 197:789-99) or "741") is a surface-exposed lipoprotein expressed in Neisseria meningitidis. Based on differences in nucleotides and predicted amino acid sequences, fHbp from different Neisseria 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), with subfamily A corresponding to V2 and V3, and subfamily B corresponding to V1 (the most abundant).

[0142] While fHbp belonging to the same mutant family share over 85% amino acid similarity, the similarity between the three mutant families is only 60-70%. fHbp is also an antigen that elicits a serum bactericidal antibody response in immunized individuals and is a key component of research vaccines for the prevention of meningococcal diseases, particularly serotype B, currently being evaluated in clinical trials. However, immunization with a protein belonging to one mutant fHbp family does not result in cross-reactivity to further mutant families, leading to a mutant-specific response. Therefore, a single fHbp does not provide universal protection against meningococcal diseases; consequently, broad vaccines must immunize with representatives of each of the three mutants, V1, V2, and V3.

[0143] Meningococcal outer membrane vesicles (OMVs) are PorA, the endogenous outer membrane protein (OMP) of Neisseria meningitidis. However, the sequence of this protein is diverse, and the prevalence of specific variants varies geographically. Variants of PorA are identified in sequences within the protein's variable region (VR), which is located in the surface-exposed peptide loops and is a target of the immune response. PorA has seven extracellular peptide loops, the fourth of which is variable region 2 (VR2), and is the target of most of the serological bactericidal activity (SBA) produced by PorA after spontaneous infection and after immunization with OMVs. SBA is known to be associated with protection against meningococcal disease. Despite the sequence diversity, approximately 70% of UK isolates are covered by vaccines containing six PorA proteins (http: / / pubmlst.org / neisseria / PorA / ).

[0144] This specification demonstrates that immunogenic peptides, such as those derived from PorA, can be introduced into factor H-binding protein (fHbp), which acts as a molecular scaffold. The peptides introduced into fHbp can be presented to the immune system and can induce defensive responses such as SBA. Advantageously, the fHbp molecule provides an ideal molecular scaffold for the presentation of epitopes, particularly for the stable inclusion of peptide loops for endogenous OMP-derived loops such as PorA, which are difficult to stabilize and present in their native three-dimensional structures. In particular, many OMPs, including PorA, are difficult to express due to the insolubility of their transmembrane domains. PorA has been demonstrated to be the most effective antigen, possessing a 16-beta chain barrel structure with surface-exposed loops between chains 1 and 2 (loop 1), chains 7 and 8 (loop 4), chains 9 and 10 (loop 5), and chains 11 and 12 (loop 7). fHbp contains two beta barrels, and therefore, by inserting an OMP-derived peptide loop sequence into the tip of the loop between the beta chains of fHbp, it is possible to present an extracellular loop fragment derived from endogenous OMP in the native three-dimensional structure of immunization. Thus, the modified fHbp skeletal molecule of the present invention can be used as a prophylactic or therapeutic vaccine against Neisseria gonorrhoeae or Neisseria gonorrhoeae, in which a single protein presents important epitopes from two different antigens.

[0145] In one embodiment, modified fHbp is a fusion protein such as a recombinant fusion protein. In another embodiment, modified fHbp is an isolated modified fHbp molecule. In yet another embodiment, the modified fHbp molecule of the present invention is included as a single protein in a polyvalent vaccine. In yet another embodiment, modified fHbp is included in an OMV vaccine.

[0146] In embodiments in which one or more exogenous peptide loops are inserted into fHbp or its variants, the exogenous peptide loops are the same, for example, identical or substantially similar in sequence. For example, certain epitopes, such as the PorA epitope, may not elicit a sufficient functional response when presented alone on fHbp. In this case, the present invention can be used to enhance the immunogenicity recognition of the epitope by providing the same epitope to multiple sites on the same modified fHbp molecule. Alternatively, the exogenous peptide loops are different from one another. For example, if the exogenous peptide loops originate from a single protein such as PorA, then different exogenous peptide loops originate from distinct regions of the protein such as PorA. In one embodiment, different exogenous peptide loops originate from overlapping distinct regions of the protein such as PorA. In embodiments in which one or more exogenous peptide loops are inserted into fHbp or its variants, the exogenous peptide loops originate from different species or strains. For example, this may be the case when a multivalent vaccine is desired against multiple different antigens, including those of different organisms.

[0147] In another aspect of the present invention, 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.

[0148] In one embodiment, a vaccine formulation comprising at least one recombinant protein / modified fHbp fusion protein further comprises an antigen selected from antigen peptide submutants fHbp 3.45, fHbp 1.55, fHbp-fHbp-fHbp or any further fHbp fusion protein, fHbp-cholera toxin, multiple Por A fused to a single fHbp, recombinant meningococcal group B NHBA fusion protein, recombinant meningococcal group B NadA protein, recombinant meningococcal group B fHbp fusion protein or outer membrane vesicles (OMVs) derived from meningococcal group B strain NZ98 / 254.

[0149] In one embodiment, the recombinant protein may include transferrin-binding protein, Neisseria heparin-binding protein, Neisseria surface protein A, PorA, meningococcal enterobactin receptor FetA, Neisseria adhesin A, or factor H-binding protein (fHbp).

[0150] More preferably, the recombinant protein / modified fHbp fusion protein comprises a modified factor H binding protein (fHbp) containing fHbp or a variant thereof, and can act as a molecular scaffold by modification through the addition of at least one exogenous peptide loop derived from a different antigen.

[0151] More preferably, the recombinant protein / modified fHbp fusion protein comprises a modified factor H binding protein (fHbp) containing fHbp or a variant thereof, and can act as a molecular scaffold by modification through the addition of at least two exogenous peptide loops derived from different antigens.

[0152] For example, the vaccine formulation may contain an fHbp variant selected from v1, v2, or v3. The formulation may contain an fHbp variant selected from v1 and v2. The formulation may contain an fHbp variant selected from v2 and v3. The formulation may contain an fHbp variant selected from v1, v2, and v3.

[0153] The recombinant protein / modified fHbp fusion protein is an fHbp variant selected from v1, v2, and v3, modified by at least one PorA loop containing at least 10 amino acids inserted into the β-turn region of fHbp. In one embodiment, the PorA loop is selected from VR1 and VR2, and its binding to human H factor (fH) is reduced by more than 80% compared to wild-type fHbp.

[0154] All activities related to the production of recombinant modified H factor-binding protein (fHbp) were carried out by Oxford University Innovation Limited. The contents of PCT / GB2013 / 052215 and PCT / GB2017 / 052535 are incorporated herein by reference in their entirety.

[0155] In one embodiment, the vaccine formulation is developed against Neisseria meningitidis, and the vaccine formulation may comprise one or more recombinant protein / modified fHbp fusion proteins derived from Neisseria meningitidis and a pharmaceutically acceptable carrier or excipient.

[0156] In one embodiment, the recombinant protein / modified fHbp fusion protein is derived from meningococcal serotypes A, B, C, H, I, K, L, 29E, W135, X, Y, and Z.

[0157] In one preferred embodiment, the recombinant protein / modified fHbp fusion protein is derived from Neisseria meningitidis serotype B.

[0158] In one embodiment, the vaccine formulation may include one or more different variants of modified factor H binding protein (fHbp) as antigens selected from the group including:

[0159] [Table 3] TIFF2026511179000004.tif188170

[0160] In one embodiment, the molecular weight of the recombinant protein / modified fHbp fusion protein is in the range of 10 kDa to 200 kDa, preferably up to 50 kDa.

[0161] In one embodiment, the molecular weight of the recombinant protein / modified fHbp fusion protein is in the range of 20 kDa to 40 kDa. In one embodiment, the formulation may contain four different variants of modified factor H binding protein (fHbp) selected from the group summarized in Table 4.

[0162] [Table 4]

[0163] In one embodiment, the formulation may include four different variants of modified factor H-binding protein (fHbp) as antigens selected from Table 1.

[0164] In one embodiment, the recombinant protein / modified fHbp fusion protein is adsorbed onto an adjuvant to improve the immunogenicity of the antigen and maximize protection against related diseases.

[0165] In one embodiment, the adsorption of recombinant protein / modified fHbp fusion protein onto the adjuvant is evaluated and optimized in terms of its adsorption ratio onto the adjuvant. In embodiments of the present invention, an adsorption buffer is used to improve surface adsorption, reduce aggregation and unfolding during binding, and reduce the dose reduction effect of the recombinant protein / modified fHbp fusion protein as an antigen after adsorption.

[0166] In one embodiment, the recombinant protein / modified fHbp fusion protein is: IL-2, RANTES, GMCSF, TNF-α, Lipid A, Lipid A derivative, Monophosphoryl Lipid A, GLA, 3-Deacylated Monophosphoryl Lipid A, AS01, AS03, AF3 (including all three suppliers of MPLA being explored by the inventors, including synthesis, etc.; MPL derived from Salmonella enterica serotype Minnesota Re 595) (e.g., Sigma Aldrich Catalog#L6895)), oligonucleotides, oligonucleotides and / or liposomes containing at least one unmethylated CpG, Freund adjuvants, Freund complete adjuvants, Freund incomplete adjuvants, polymers, copolymers such as polyoxyethylene-polyoxypropylene copolymers including block copolymers, polymer p 1005, CRL-8300 adjuvant, muramyl dipeptide, e.g., TLR1 / 2 agonists (may be synthetic ligands) (e.g., Pam3Cys), TLR2 (e.g., CFA, Pam2Cys), TLR3 (e.g., polyFC, polyA:U), TLR-4 agonists (e.g., MPLA, Lipid A and LPS), TLR5 (e.g., flagellin), TLR7 (e.g., galdikimod, imiquimod, loxoribine, Resiquimod®), TLR7 / 8 (e.g., R0848), TLR8 (e.g., imidazoquionolines, ssPolyU, 3M-012), TLR9 (e.g., ODN 1826 (type B), ODN2216 (Type A) and / or TLR11 / 12 (e.g., Profilin), TLR-4 agonist, Flagellin, TLR7 (e.g., Galdikimod, Imiquimod, Loxoribine, Resiquimod®), TLR7 / 8 (e.g., R), TLR8 (e.g., Galdikimod, Imiquimod, Loxoribine, Resiquimod®), TLR9 (e.g., ODN (Type B), ODN (Type A) and / or TLR11 / 12 (e.g., Profilin), TLR-4 agonist, Flagellin, Flagellin derived from Gram-negative bacteria, TLR-5 agonist, Fragments of Flagellin capable of binding to the TLR-5 receptor, Alpha-C-galactosylceramide, Chitosan, Interleukin-2, QS-21, ISCOMS, Squalene mixture (SAF-1), Quil The adjuvant is adsorbed onto an adjuvant selected from the group consisting of A, cholera toxin B subunit, polyphosphazene and derivatives, mycobacterial cell wall preparations, mycolic acid derivatives, nonionic block copolymer surfactants, OMV, fHbp, sterols and lipids, and saponins in combination, TLR agonists (MPL, CpG, poly-IC, imiquimod), dmLT, 125-dihydroxyvitamin D3 CAF01, poly[di(carboxylatofenoxy)-phosphazene] (PCPP), and Venezuelan encephalitis (VEE) replicon particles or combinations thereof.

[0167] In one embodiment, the amount of adjuvant is in the range of 0.5 mg / ml to 4.5 mg / ml.

[0168] In one embodiment, the adjuvant is aluminum hydroxide with a particle size greater than 500 nm.

[0169] In one embodiment, the adsorption ratio of recombinant protein / modified fHbp fusion protein onto the adjuvant is in the range of 70% to 100%.

[0170] In other embodiments, fHbp V3.45 M5 PorA is applied to the adjuvant. 316―320 exP1.14 The adsorption ratio is in the range of 80% to 100%.

[0171] In other embodiments, fHbp V1.14 PorA is applied to the adjuvant. 307―311 exP1.9 The adsorption ratio is in the range of 80% to 90%.

[0172] In other embodiments, fHbp V2.19 PorA is applied to the adjuvant. 316―320 exP1.4 The adsorption ratio is in the range of 80% to 90%.

[0173] In other embodiments, fHbp V1.1 PorA is applied to the adjuvant. 307―311 exP1.4 The adsorption ratio is in the range of 80% to 90%.

[0174] In other embodiments, fHbp V1.1 PorA is applied to the adjuvant. 307―311 exP1.9 The adsorption ratio is in the range of 70% to 80%.

[0175] In one embodiment, pharmaceutically acceptable carriers or excipients may be selected from the group comprising buffers, sugars, sugar alcohols or polyols, surfactants, polymers, salts, amino acids or pH adjusters, hydrolyzed proteins, preservatives, and liquid carriers.

[0176] Examples of buffers are selected from the group comprising carbonates, phosphates, acetates, HEPES, succinates, TRIS, borates, citrates, lactates, glucons, and tartrates, as well as more complex organic buffers, including phosphate buffers containing sodium phosphate and / or potassium phosphate in proportions selected to achieve a desired pH. In other examples, the buffer contains tris(hydroxymethyl)aminomethane, i.e., "TRIS," formulated to achieve a desired pH. In yet another example, the buffer may be a minimal essential medium containing Hanks salts. Further buffers such as HEPES, piperazine-N,N'-bis (PIPES), and 2-ethanesulfonic acid (MES) are also assumed by this disclosure. The buffers assist in the stabilization of the recombinant protein / modified fHbp fusion protein of this disclosure. In one embodiment, the amount of buffer is in the range of 0.1 mM to 300 mM.

[0177] Examples of sugars used as excipients are selected from the group consisting of trehalose, mannose, raffinose, lactobionic acid, glucose, maltulose, isomaltulose, maltose, lactose, dextrose, fructose, or combinations thereof. In one embodiment, the amount of sugar is in the range of 5 mg / ml to 100 mg / ml.

[0178] Examples of sugar alcohols or polyols used as excipients are selected from the group consisting of mannitol, lactitol, sorbitol, glycerol, xylitol, maltitol, lactitol, erythritol, isomalt, and hydrolyzed starch or combinations thereof. In one embodiment, the amount of sugar alcohol or polyol is in the range of 5 mg / ml to 100 mg / ml.

[0179] Surfactants can be classified by their "HLB" (hydrophilic / lipophilic balance). The preferred HLB of the surfactant of the present invention is at least 10, preferably at least 15, and more preferably at least 16. Examples of surfactants used as excipients include nonionic surfactants (0.001%~0.05%) such as polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene 660 hydroxystearate, polyoxyethylene 35 ricinolate, soy lecithin, and poloxamer; nonionic surfactants such as copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO) sold under the trademark DOWF AX (linear EO / PO block copolymers, etc.); octoxynol (especially octoxynol 9 (Triton)) with a changing number of repeating ethoxy(oxy-1,2-ethanediyl) groups. Examples include nonionic surfactants such as X-100 or t-octylphenoxypolyethoxyethanol, (octylphenoxy)polyethoxyethanol (IGEPAL CA-630 / NP-40); phospholipids (e.g., phosphatidylcholine (lecithin)); nonylphenol ethoxylates (e.g., Tergitol® NP series); polyoxyethylene fatty ethers derived from lauryl alcohol, cetyl alcohol, stearyl alcohol, and oleyl alcohol (known as Brij® surfactants) (e.g., triethylene glycol monolauryl ether (Brij® 30)); and sorbitan esters (generally known as SPAN) (e.g., sorbitan trioleate (generally known as SPAN) (Span 85) and sorbitan monolaurate). Nonionic surfactants are preferred. A preferred surfactant to include in the emulsion is Tween 20. In one embodiment, the amount of surfactant is in the range of 0.01 mg / ml to 2 mg / ml.Examples of polymers include dextran, carboxymethylcellulose, hyaluronic acid, and cyclodextrin.

[0180] Examples of salts include NaCl, KCl, KH2PO4, Na2HPO4.2H2O, CaCl2, and MgCl2.

[0181] Examples of amino acids used as excipients include tricine, leucine, isoleucine, glycine, glutamine, L-arginine, L-arginine hydrochloride, lysine, L-alanine, tryptophan, phenylalanine, tyrosine, valine, cysteine, glycine, methionine, proline, serine, and threonine.

[0182] Examples of hydrolyzed proteins include gelatin, lactalbumin hydrolysate, monosodium glutamate, collagen hydrolysate, keratin hydrolysate, peptides, casein hydrolysate and whey protein hydrolysate, and serum albumin.

[0183] Examples of preservatives include phenoxyethanol, benzethonium chloride (Phemerol), phenol, m-cresol, thiomersal, formaldehyde, paraben esters (e.g., methyl-, ethyl-, propyl-, or butylparaben), benzalkonium chloride, benzyl alcohol, chlorobutanol, p-chloro-m-cresol, or benzyl alcohol, or combinations thereof.

[0184] The vaccine composition may contain a preservative for a single immunization or materials for multiple immunizations (i.e., a “multi-dose” kit). In the multi-dose configuration, the inclusion of a preservative is preferred. Instead of (or in addition to) including a preservative in the multi-dose composition, the composition may be contained in a container with a sterile adapter for dispensing the materials. In one embodiment, the amount of preservative is in the range of 1 mg / mL to 10 mg / mL.

[0185] Suitable liquid carriers include water for injection (WFI) and physiological saline.

[0186] In one embodiment, the vaccine formulation is as follows: At least one recombinant protein / at least one modified fHbp; Aluminum hydroxide; Mannitol; Phosphate; and Contains polysorbate.

[0187] In other embodiments, the vaccine formulation is as follows: At least one modified fHbp represented by an amino acid sequence that is at least 75% identical to any one of the amino acid sequences represented by SEQ ID NOs: 6-10; or At least one modified fHbp encoding a nucleic acid sequence that is at least 75% identical to any one of the nucleic acid sequences represented by sequence numbers 1 to 5; and Aluminum hydroxide; Mannitol; Phosphate; and Contains polysorbate.

[0188] In embodiments of the present invention, the vaccine formulation is formulated so that the dose is in the range of 0.1 mL to 1 mL, for example, 0.2 mL to 0.8 mL or 0.4 mL to 0.6 mL. In one embodiment, the vaccine formulation is formulated so that the dose is 0.5 mL.

[0189] In embodiments of the present invention, the recombinant protein / modified fHbp fusion protein is 15 μg / ml to 200 μg / ml, for example, 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 It is present in the vaccine preparation in amounts ranging from 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, and 120 μg / ml to 150 μg / ml.

[0190] In embodiments of the present invention, recombinant protein / modified fHbp fusion protein is present in the vaccine formulation in amounts 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, and 200 μg / ml.

[0191] In one embodiment, 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.

[0192] In one 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 μg / ml to 150 μg / ml.

[0193] In one 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 μg / ml to 150 μg / ml.

[0194] In one 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 μg / ml to 150 μg / ml.

[0195] In one embodiment, the vaccine formulation is as follows: At least one modified fHbp represented by an amino acid sequence that is at least 75% identical to any one of the amino acid sequences represented by SEQ ID NOs: 6-10; or At least one modified fHbp encoding a nucleic acid sequence that is at least 75% identical to any one of the nucleic acid sequences represented by sequence numbers 1 to 5; and Aluminum hydroxide; Mannitol; Phosphate; and Contains polysorbate.

[0196] In one embodiment, the vaccine formulation is At least one modified fHbp represented by an amino acid sequence in the range of 15 μg / ml to 150 μg / ml that is at least 75% identical to any one of the amino acid sequences represented by SEQ ID NOs: 6 to 10; or At least one modified fHbp encoding a nucleic acid sequence in the range of 15 μg / ml to 150 μg / ml that is at least 75% identical to any one of the nucleic acid sequences represented by sequence numbers 1 to 5; and Aluminum hydroxide in amounts ranging from 0.5 mg / ml to 4.5 mg / ml; Mannitol in amounts ranging from 5 mg / ml to 100 mg / ml; A phosphate buffer solution in the range of 1 mM to 10 mM; and Contains polysorbate 20 in amounts ranging from 0.01 mg / ml to 2 mg / ml.

[0197] In one embodiment, the vaccine formulation is fHbpV3.45 M5:PorA316―320 / exP1.14 (represented by sequence number 6 or coded by sequence number 1) in quantities in the range of 15 μg / ml to 150 μg / ml; or fHbpV2.19 M6:PorA316―320 / exP1.4 (represented by sequence number 7 or coded by sequence number 2) in quantities in the range of 15 μg / ml to 150 μg / ml; or fHbpV1.14:PorA307―311 / exP1.9 (represented by sequence number 8 or coded by sequence number 3) in quantities in the range of 15 μg / ml to 150 μg / ml; or fHbpV1.1:PorA307―311 / exP1.4 (represented by sequence number 9 or coded by sequence number 4) in quantities in the range of 15 μg / ml to 150 μg / ml; and Aluminum hydroxide in amounts ranging from 0.5 mg / ml to 4.5 mg / ml; Mannitol in amounts ranging from 5 mg / ml to 100 mg / ml; A phosphate buffer solution in the range of 1 mM to 10 mM; and Contains polysorbate 20 in amounts ranging from 0.01 mg / ml to 2 mg / ml.

[0198] In one embodiment, the vaccine formulation is fHbpV3.45 M 5:PorA 316-320 / ExP 1.14 (represented by sequence number 6 or coded by sequence number 1) in quantities ranging from 15 μg / ml to 150 μg / ml; fHbpV2.19 M 6:PorA 316-320 / ExP 1.4 (represented by sequence number 7 or coded by sequence number 2) in quantities ranging from 15 μg / ml to 150 μg / ml; fHbpV1.14:PorA 307-311 / ExP 1.9 (represented by sequence number 8 or coded by sequence number 3) in quantities ranging from 15 μg / ml to 150 μg / ml; fHbpV1.1:PorA 307-311 / ExP 1.4 (represented by sequence number 9 or coded by sequence number 4) in quantities ranging from 15 μg / ml to 150 μg / ml; and Aluminum hydroxide in amounts ranging from 0.5 mg / ml to 4.5 mg / ml; Mannitol in amounts ranging from 5 mg / ml to 100 mg / ml; A phosphate buffer solution in the range of 1 mM to 10 mM; and Contains polysorbate 20 in amounts ranging from 0.01 mg / ml to 2 mg / ml.

[0199] In one embodiment, the vaccine formulation is fHbpV3.45 M5:PorA316―320 / exP1.14 (represented by Sequence ID 6 or coded by Sequence ID 1) in quantities in the range of 15 μg / mL to 150 μg / mL; or fHbpV2.19 M6:PorA316―320 / exP1.4 (represented by Sequence ID 7 or coded by Sequence ID 2) in quantities in the range of 15 μg / mL to 150 μg / mL; or fHbpV1.1:PorA307―311 / exP1.9 (represented by sequence number 10 or coded by sequence number 5) in the range of 15 μg / mL to 150 μg / mL; or fHbpV1.14:PorA307―311 / exP1.9 (represented by sequence number 8 or coded by sequence number 3) in quantities in the range of 15 μg / mL to 150 μg / mL; and Aluminum hydroxide in amounts ranging from 0.5 mg / mL to 4.5 mg / mL; Mannitol in amounts ranging from 5 mg / mL to 100 mg / mL; A phosphate buffer solution in the range of 1 mM to 10 mM; and Contains polysorbate 20 in an amount ranging from 0.01 mg / mL to 2 mg / mL.

[0200] In one embodiment, the vaccine formulation is fHbpV3.45 M 5: PorA316-320 / ExP 1.14 (represented by SEQ ID NO: 6 or coded by SEQ ID NO: 1) in the range of 15 μg / mL to 150 μg / mL; fHbpV2.19 M 6: PorA316-320 / ExP 1.4 (represented by SEQ ID NO: 7 or coded by SEQ ID NO: 2) in the range of 15 μg / mL to 150 μg / mL; HbpV1.1: PorA 307-311 / ExP 1.9 in the range of 15 μg / mL to 150 μg / mL (represented by SEQ ID NO: 10 or coded by SEQ ID NO: 5); fHbpV1.14: PorA 307-311 / ExP 1.9 (represented by SEQ ID NO: 8 or coded by SEQ ID NO: 3) in the range of 15 μg / mL to 150 μg / mL; Aluminum hydroxide in amounts ranging from 0.5 mg / mL to 4.5 mg / mL; Mannitol in amounts ranging from 5 mg / mL to 100 mg / mL; A phosphate buffer solution in the range of 1 mM to 10 mM; and Contains polysorbate 20 in an amount ranging from 0.01 mg / mL to 2 mg / mL.

[0201] In one embodiment, the vaccine formulation contains 2-phenoxyethanol in an amount ranging from 1 mg / mL to 10 mg / mL.

[0202] In embodiments of the present invention, the vaccine formulation comprises (i) at least one fusion protein comprising a stable non-functional / non-lipidized fHbp and a PorA VR2 loop, and (ii) at least one polysaccharide protein complex.

[0203] In one embodiment, the vaccine formulation comprises (i) at least one fusion protein comprising a stable non-functional / non-lipidized fHbp and a PorA VR2 loop, and (a) a complex of (i) meningococcal serotype A capsular sugar and (ii) CRM197; (b) a complex of (i) meningococcal serotype C capsular sugar and (ii) CRM197; (c) a complex of (i) meningococcal serotype Y capsular sugar and (ii) CRM197; (d) a complex of (i) meningococcal serotype W135 capsular sugar and (ii) CRM197; and (d) a complex of (i) meningococcal serotype X capsular sugar and (ii) tetanus toxin.

[0204] In one embodiment, the vaccine formulation comprises (i) at least two fusion proteins, each consisting of one fHbp mutant bound to one PorA VR2 loop, and (a) a complex of (i) meningococcal serotype A and (ii) CRM197; (b) a complex of (i) meningococcal serotype C and (ii) CRM197; (c) a complex of (i) meningococcal serotype Y and (ii) CRM197; (d) a complex of (i) meningococcal serotype W135 and (ii) CRM197; and (d) a complex of (i) meningococcal serotype X and (ii) tetanus toxin.

[0205] In one embodiment, the vaccine formulation comprises (i) at least three fusion proteins, each consisting of one fHbp mutant bound to two PorA VR2 loops, and (a)(i) a capsular sugar complex of meningococcal serotype A and (ii) CRM197; (b)(i) a capsular sugar complex of meningococcal serotype C and (ii) CRM197; (c)(i) a capsular sugar complex of meningococcal serotype Y and (ii) CRM197; (d)(i) a capsular sugar complex of meningococcal serotype W135 and (ii) CRM197; and (d)(i) a capsular sugar complex of meningococcal serotype X and (ii) tetanus toxin.

[0206] In one embodiment, the vaccine formulation comprises (i) at least four fusion proteins, each consisting of one fHbp mutant bound to three PorA VR2 loops, and (a) a complex of (i) meningococcal serotype A capsule sugar and (ii) CRM197; (b) a complex of (i) meningococcal serotype C capsule sugar and (ii) CRM197; (c) a complex of (i) meningococcal serotype Y capsule sugar and (ii) CRM197; (d) a complex of (i) meningococcal serotype W135 capsule sugar and (ii) CRM197; and (d) at least one complex selected from (i) meningococcal serotype X capsule sugar and (ii) tetanus toxin.

[0207] In one embodiment, the recombinant protein / modified fHbp fusion protein of the present invention is administered co-administered with one or more vaccines selected from Bexcelo®, MENVEO, MENACTRA, NIMENRIX, MenQuadFi, MENFIVE, MenAfriVac, Men AC, and Men ACHib.

[0208] In a preferred embodiment, the recombinant protein / modified fHbp fusion protein of the present invention is administered simultaneously with MENFIVE.

[0209] In one embodiment, the vaccine formulation contains 2-phenoxyethanol in an amount ranging from 1 mg / mL to 10 mg / mL.

[0210] In one embodiment, the zeta potential of the vaccine formulation is in the range of -16mV to -30mV.

[0211] In one embodiment, the osmotic pressure of the vaccine formulation is in the range of 200 mOsmol / kg to 500 mOsmol / kg.

[0212] The above vaccine formulations are as follows: Proliferating host cells containing the expression vector in a nutrient medium; Inducing the host cells to express the protein; To recover and isolate the host cells; The recovered cells are lysed to separate the host cell fragments and obtain tagged proteins; Purifying the aforementioned tagged protein; The tag is removed from the tagged protein to obtain a recombinant protein / modified fHbp fusion protein; Purifying the recombinant protein / modified fHbp fusion protein; and To prepare a vaccine formulation containing the purified recombinant protein / modified fHbp fusion protein. Includes; It is manufactured through a wide range of processes, which will be described in more detail in the following paragraphs.

[0213] In yet another aspect of the present invention, upstream bioprocesses are developed and optimized to increase the cell density of lead cell lines and the productivity of recombinant protein / modified fHbp fusion proteins.

[0214] In one embodiment, the upstream bioprocess may include batch, fed-add, continuous, or perfusion modes of culture for the production of recombinant protein / modified fHbp fusion protein.

[0215] In one embodiment, the upstream bioprocess is as follows: (a) Prepare aqueous fermentation tank nutrient medium and supply solution; (b) Inoculating the nutrient medium of the fermentation tank with the host cell line; (c) Continuous supply of the supply solution; (d) Inducing protein expression; (e) Recovery and cell separation; This may include additive flow methods.

[0216] In one embodiment, numerous experiments were conducted during upstream bioprocess development to achieve the high productivity required for future commercial processes. The culture process development efforts focused on four main areas: (1) verifying the performance of high-expression strains identified during clone screening and selection using an improved baseline culture process; (2) developing enhanced culture medium compositions capable of supporting high-productivity culture performance; (3) adjusting fermentation process parameters and in-process controls to achieve high cell density and expression of recombinant protein / modified fHbp fusion protein; and (4) optimizing supply strategies and amino acid supplementation to prevent stalling during transcription and translation.

[0217] In one embodiment, the aqueous fermentation tank nutrient medium may include an undefined medium, terrific broth (TB) medium, lysogeny broth, LB medium or Luria-Bertani medium, a chemically defined medium, M9 minimal medium, a chemically defined M9 modified salt medium, 2xYT medium or super optimal broth containing catabolite repression (SOC) medium and combinations thereof.

[0218] More preferably, the composition of the LB medium is summarized in Table 5.

[0219]

Table 5

[0220] NaCl - Sodium chloride More preferably, the composition of the chemically defined M9 modified salt medium is summarized in Table 6.

[0221]

Table 6

[0222] K2HPO4 - Dipotassium hydrogen phosphate KH2PO4 - Potassium dihydrogen phosphate (NH4)2SO4 - Ammonium sulfate MgSO4·7H2O - Magnesium sulfate heptahydrate TES Trace element solution Using a 14% liquid ammonia solution and 6% orthophosphoric acid, maintain the pH in the fermentation batch within the range of 6.8 - 8.2.

[0223] More preferably, the composition of the trace element solution is summarized in Table 7.

[0224]

Table 7

[0225] More preferably, the composition of the TB medium is summarized in Table 8.

[0226] [Table 8]

[0227] In one embodiment, the aqueous fermenter nutrient medium may further contain an antibiotic selected from the group including kanamycin, neomycin, streptomycin, tobramycin, paromomycin, amphotericin B, ampicillin, erythromycin, gentamicin, nystatin, penicillin-streptomycin, polymyxin B, tetracycline, thiabendazole, or tylosin, or a combination thereof.

[0228] In one embodiment, the host cell line may include a bacterial expression host system.

[0229] More preferably, the bacterial expression host system is Escherichia coli, and the Escherichia coli strain is selected from the group consisting of 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)*, T7 Express, m15 pREP4*, C41(DE3), C43(DE3), or B834(DE3).

[0230] * indicates the presence of further plasmids. Most preferably, the bacterial expression host system is the E. coli strain B834(DE3).

[0231] In one embodiment, the fermentation conditions after inoculation of the host cell line may 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%; stirring in the range of 100 to 1800 rpm; and a gas flow rate of 0 to 2 VVM (volts of gas per unit volume of liquid per minute).

[0232] In one embodiment, the inoculated fermentation tank nutrient medium may include continuously supplying a feed solution after depletion of the carbon source.

[0233] Preferably, the composition of the feed solution containing glucose is summarized in Table 9.

[0234] [Table 9]

[0235] (Glucose / dextrose / dextrose monohydrate are used interchangeably in this application) Preferably, the composition of the feed solution containing glycerol is summarized in Table 10.

[0236] [Table 10]

[0237] In one embodiment, the concentration of glycerol in the glycerol feed composition is in the range of 30% to 80%. In one embodiment, the concentration of glycerol in the glycerol feed composition is 50%.

[0238] In one embodiment, the feed rate of the glucose feed is in the range of 0.3 to 3.0 mL / min / L culture.

[0239] In one embodiment, after replacing the glucose feed with a glycerol feed solution, the host cell line can be induced to express a recombinant protein / modified fHbp fusion protein.

[0240] In one embodiment, the feed rate of the glycerol feed is in the range of 0.3 to 3.0 mL / min / L culture.

[0241] In one embodiment, the host cell line is selected from the inducer lactose or its non-hydrolyzable analog isopropyl β-D-1-thiogalactopyranoside (IPTG).

[0242] In one embodiment, the induction parameter may be induced in the range of 20 to 100 OD measured at 590 / 600 nm. In one embodiment, the inducing factor is IPTG in the range of 1 mM to 10 mM. In another embodiment, the inducing factor is IPTG in the range of 5 mM to 10 mM. In one embodiment, the inducing substance is lactose in the range of 1 g / L to 50 g / L. In another embodiment, the inducing substance is lactose in the range of 5 g / L to 50 g / L.

[0243] In one embodiment, the induction temperature is in the range of 17°C to 37°C, and the induction time is in the range of 3 to 24 hours.

[0244] In one embodiment, several experiments were performed during induction to optimize induction density and length, culture temperature, culture pH, temperature shift, and glucose / glycerol supply rate.

[0245] In one embodiment, three different fed-add methods / protocols for the production of chimeric proteins in a fermenter are summarized in Table 11.

[0246] [Table 11]

[0247] In one embodiment, the culture is harvested 3 to 7 hours after induction.

[0248] In one embodiment, after harvesting, the cells are separated from the fermentation tank nutrient medium by centrifugation.

[0249] Preferably, the centrifugal separation parameters may include a relative centrifugal force (rcf) of 6000 to 8000 for 30 to 60 minutes at 1°C to 5°C.

[0250] In one embodiment, the moist cell mass (g / L) obtained after harvesting may contain 150 to 350 g / L of harvested broth.

[0251] In yet another aspect of the present invention, downstream bioprocesses are developed and optimized to produce recombinant protein / modified fHbp fusion proteins in high yield and high purity.

[0252] In one embodiment, the downstream bioprocess is as follows: (a) Cell lysis / cell destruction; (b) Cell isolation and clarification; (c) Purification of tagged proteins; (d) Affinity tag removal; (e) Protein purification; (f) Concentration / dialysis / buffer exchange It may include any one of the following steps.

[0253] In one embodiment, during the development of a downstream bioprocess, several experiments were conducted to achieve the high yield and high purity required for future commercial processes.

[0254] In one embodiment, intracellular products can be utilized by lysing or destroying the cell culture after recovery and cell separation.

[0255] In one embodiment, cell lysis / disruption is carried out by a method selected from the group including chemical, biological, physical, or mechanical modes, or a combination thereof. Here, the chemical method of cell lysis may include surfactants, solvents, acids, or bases, or a combination thereof. Here, the biological method of cell lysis may include lysozyme. Here, the physical method of cell lysis may include freeze-thaw, acoustic cavitation, hydrodynamic cavitation, or osmotic shock, or a combination thereof. Here, the mechanical mode of cell lysis may include grinding (e.g., bead milling) or high-pressure homogenization, or a combination thereof.

[0256] In one embodiment, cell lysis / disruption is performed in a mechanical mode, including high-pressure homogenization.

[0257] In other embodiments, cell lysis / destruction is performed by chemical dissolution using a lysis buffer.

[0258] In yet another embodiment, cell lysis / destruction is carried out in combination with a mechanical mode including chemical lysis followed by high-pressure homogenization. In one embodiment, high-pressure homogenization includes a pressure range of 1000-1500 bar, a cycle range of 3-8, a flow rate of 9 L / hour + / - 5%, and a temperature range of 4°C-15°C.

[0259] In one embodiment, the flow rate increases as the process is scaled up.

[0260] In one embodiment, mechanical dissolution is performed using a homogenizer.

[0261] In one embodiment, the lysis buffer contains sodium phosphate in the range of 10 to 100 mM, imidazole in the range of 10 to 50 mM, and sodium chloride (NaCl) in the range of 100 to 500 mM, with a pH of 6.0 to 8.0.

[0262] Lysis buffer: Lysis buffers are buffers used to break down cells and open them up.

[0263] In one embodiment, after cell lysis / disruption, the disruption efficiency and potential product loss are evaluated by measuring any or all of the following parameters of intracellular products, including total protein release, cell viability, and particle size distribution.

[0264] In one embodiment, after cell lysis / disruption, the expression levels of recombinant protein / modified fHbp fusion protein in the intracellular product are evaluated by a method selected from the group including SDS-PAGE, Western blotting, ELISA, and enzyme assay.

[0265] Preferably, the recombinant protein / modified fHbp fusion protein may contain an fHbp-PorA chimeric protein.

[0266] In one embodiment, the fHbp-PorA chimeric protein may include an affinity tag selected from the group comprising a peptide tag and / or a large polypeptide.

[0267] Affinity tags: When designing a project requiring purified, soluble, active recombinant protein / modified fHbp fusion protein, it is (often) extremely valuable to have means to (i) detect it by expression and purification schemes, (ii) achieve maximum solubility, and (iii) easily purify it from the E. coli cell environment. These three goals can be directly achieved by expressing a tandem amino acid stretch (peptide tag) or a large polypeptide (fusion partner) with the desired protein to form a tagged fusion protein.

[0268] Preferably, the fHbp-PorA chimeric protein may include a small peptide tag selected from the group including poly-Arg-, FLAG-, poly-His-, c-Myc-, S-, and Strep II- tags, and a large polypeptide (fusion partner) selected from the group including maltose-binding protein (MBP), N-utilizing protein A (NusA), thioredoxin (Trx), glutathione S-transferase (GST), ubiquitin, and SUMO.

[0269] Most preferably, the fHbp-PorA chimeric protein may contain poly-His and MBP tags, and the expression vector may contain pET28a-His-MBP-TEV-fHbp-PorA.

[0270] In one embodiment, after cell lysis / disruption, the cell aggregate is subjected to cell separation and clarification.

[0271] Preferably, cell separation is performed by centrifugation, and intracellular products containing recombinant protein / modified fHbp fusion protein are separated into the supernatant.

[0272] In one embodiment, centrifugation is performed at 1°C to 5°C for 30 to 60 minutes at 4000 to 10000 RPM.

[0273] In one embodiment, after cell separation, the supernatant is clarified to remove lysed proteins.

[0274] In one embodiment, clarification is performed using a filter selected from the group including progressively decreasing pore sizes (e.g., 6 μm, 5 μm, 0.8 μm, 0.65 μm, 0.45 μm, 0.2 μm). Suitable commercially available filters and filtration devices are well known in the art and can be selected by those skilled in the art. Exemplary filtration devices may be made of polypropylene, cellulose acetate, or polyethersulfone, and commercially available filters may be Millipak (Millipore), Kleenpak (Pall), and Sartobran (trademark) P filtration devices.

[0275] In one embodiment, after cell separation and clarification, the supernatant containing the tagged recombinant protein / modified fHbp fusion protein is subjected to purification by a chromatographic purification method, ultrafiltration, dialysis, or a combination thereof.

[0276] In one embodiment, the chromatography-based purification method includes ion exchange chromatography, affinity chromatography, or a combination of both.

[0277] In other embodiments, a chromatography-based purification method includes affinity chromatography-based purification in which a tagged recombinant protein / modified fHbp fusion protein is bound to an affinity column and further subjected to washing with increasing concentrations of a buffer containing sodium phosphate + NaCl, + imidazole. The tagged protein is eluted with an elution buffer.

[0278] Preferably, the affinity column may contain an immobilized metal affinity column resin.

[0279] Immobilized metal affinity chromatography (IMAC) resins are highly binding resins for purifying His-tagged proteins, and their effectiveness depends on the affinity of His to the immobilized transition metal.

[0280] According to one embodiment, the immobilized metal affinity column resin contains metal ions selected from copper, zinc, nickel, and the like.

[0281] Preferably, the immobilized metal affinity column resin includes uncharged or pre-charged Ni2+ resin.

[0282] In one embodiment, the uncharged form is selectively charged with metal ions that are more flexible for further purification. In another embodiment, the immobilized metal affinity column resin may include a Ni Sepharose 6 Fast Flow column.

[0283] In one embodiment, the washing buffer comprises 20-50 mM sodium phosphate buffer, 50-300 mM sodium chloride (NaCl) pH 7.4, and the imidazole concentration is increased by 20-80 mM (5-6 CV each).

[0284] In one embodiment, target protein elution is performed using an elution buffer containing 20-50 mM sodium phosphate buffer, 50-120 mM sodium chloride (NaCl) pH 7.4, and 100-300 mM imidazole.

[0285] In one embodiment, after elution, the eluate containing the tagged recombinant protein / modified fHbp fusion protein is further subjected to concentration and dialysis.

[0286] In one embodiment, concentration and dialysis are typically performed using tangential flow filtration (TFF) through a molecular weight cutoff (MWCO) in the range of 5 kDa to 50 kDa and a filter in a 10-50 mM sodium phosphate buffer pH 7.4.

[0287] In one embodiment, concentration and dialysis are typically performed using tangential flow filtration (TFF) through a molecular weight cutoff (MWCO) in the range of 5 kDa to 50 kDa and a filter, with a sodium phosphate buffer pH 7.4 or Tris-HCl buffer pH 8.5, which is usually 10 to 50 mM.

[0288] In one embodiment, the tagged recombinant protein / modified fHbp fusion protein is subjected to TEV protease digestion to remove the affinity tag.

[0289] In one embodiment, a recombinant protein / modified fHbp fusion protein containing poly-His and MBP tags is subjected to TEV protease digestion for removal of the His-MBP tag, and the TEV protease digestion comprises incubating the recombinant protein containing poly-His and MBP tags with the TEV protease.

[0290] The tobacco etch virus (TEV) protease is a 27 kDa catalytic domain of polyprotein nuclear inclusion body a(NIa) in TEV, which recognizes a specific amino acid sequence ENLYFQG / S and cleaves between Q and G / S. In one embodiment, a tagged recombinant protein / modified fHbp fusion protein is subjected to TEV protease digestion to remove the affinity tag, and the HIS-GST-TEV protease is encoded by a nucleic acid sequence that is at least 75.0% identical to amino acid sequence number 12 or at least 75.0% identical to the sequence of sequence number 11.

[0291] In one embodiment, the molecular size of the His-GST-TEV protease is approximately 50 kDa.

[0292] In a preferred embodiment, the tagged recombinant protein / modified fHbp fusion protein is subjected to TEV protease digestion to remove the affinity tag, and the HIS-GST-TEV protease is encoded by a nucleic acid sequence that is at least 75%, 80.0%, 85.0%, 90.0%, 95.0%, 98.0%, 99.0%, or 99.5% identical to amino acid sequence number 12, or at least 75.0%, 80.0%, 85.0%, 90.0%, 95.0%, 98.0%, 99.0%, or 99.5% identical to the sequence of sequence number 11.

[0293] In one embodiment, the ratio of tagged recombinant protein / modified fHbp fusion protein to 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 one embodiment, the ratio of tagged recombinant protein / modified fHbp fusion protein (substrate) to TEV protease is 20:1.

[0294] In one embodiment, the incubation temperature range is 4°C to 35°C, and the incubation time range is 15 to 20 hours.

[0295] In one embodiment, TEV protease digestion is carried out in the presence of 10-50 mM sodium phosphate buffer pH 7.4, after which dithiothreitol (DTT) is added to a final concentration of 0.5 mM-5 mM.

[0296] In one embodiment, the TEV protease used for tag removal / digestion is as follows: Proliferating host cells containing an expression vector in a nutrient medium; Inducing the expression of TEV protease in the host cells; To collect and isolate the host cells in question; Lysing the recovered cells and separating the host cells to obtain TEV protease; and Purify TEV protease; It is generated using a method that includes [something].

[0297] In one embodiment, the host cell may include Escherichia coli, and the Escherichia coli strain may be selected from the group consisting of 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)*, T7 Express, m15 pREP4*, C41(DE3), C43(DE3), or B834(DE3).

[0298] In one embodiment, the host cell for TEV protease expression is Escherichia coli Rosetta®(DE3)pLysS.

[0299] In one embodiment, the TEV protease enzyme produced by the above method includes a TEV protease that has SEQ ID NO: 12 or is encoded by SEQ ID NO: 11.

[0300] In one embodiment, the TEV protease is as follows: Lysizing the harvested cells; The process of separating host cell fragments and collecting the supernatant; Wash the supernatant at least once with a washing buffer; Elute the purified TEV protease with elution buffer; Concentrating purified TEV protease; It is purified using a non-specific process.

[0301] In one embodiment, the washing buffer comprises sodium phosphate, sodium chloride, and imidazole.

[0302] In one embodiment, the supernatant is subjected to at least four washing steps using a washing buffer with a pH of 7.4.

[0303] In other embodiments, the supernatant is subjected to at least two washing steps using a wash buffer with a pH of 8.5.

[0304] The methods and parameters for the production and purification of TEV protease are the same as those for the tagged recombinant protein / modified fHbp fusion protein described above and will be described in detail in the following examples.

[0305] In one embodiment, after tag removal, the recombinant protein / modified fHbp fusion protein is subjected to purification including chromatographic purification methods, ultrafiltration, dialysis, or a combination thereof.

[0306] In the 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 bonding chromatography, reversed-phase chromatography, and combinations thereof.

[0307] In one embodiment, the chromatography-based purification method includes ion exchange chromatography, affinity chromatography, or a combination of both.

[0308] In other embodiments, the chromatography-based purification method includes ion exchange followed by purification based on affinity chromatography.

[0309] In yet another embodiment, ion exchange chromatography includes anion exchange chromatography.

[0310] In yet another embodiment, the anion exchange chromatography includes strong anion exchange chromatography.

[0311] In one embodiment, the affinity column may contain an immobilized metal affinity column resin.

[0312] Immobilized metal affinity chromatography (IMAC) resins are highly binding resins for purifying His-tagged proteins, and their effectiveness depends on the affinity of His to the immobilized transition metal.

[0313] In one embodiment, the immobilized metal affinity column resin includes uncharged or pre-charged Ni2+ resin.

[0314] In one embodiment, the uncharged form is selectively charged with metal ions that are more flexible for further purification.

[0315] In one embodiment, the immobilized metal affinity column resin includes a Ni Sepharose 6 Fast Flow column.

[0316] The chromatography may be a combination of multimodal anion exchange resins Capto™ Adhere, Capto adhere ImpRes, CaptomMC ImpRes, or any further mixed modes of chromatography including ion exchange, electrostatic interaction, hydrogen bonding, and hydrophobic interaction.

[0317] In one embodiment, concentration and dialysis filtration are performed using tangential flow filtration (TFF) through a filter typically having a molecular weight cutoff (MWCO) in the range of 5 kDa to 50 kDa.

[0318] In one embodiment, the eluate containing the recombinant protein / modified fHbp fusion protein is sterilized by direct flow filtration (DFF) through at least one sterile-grade filter to obtain a filtrate containing the sterile recombinant protein / modified fHbp fusion protein. Here, the sterile-grade filter may be selected from the group including 0.8 μm, 0.45 μm, and 0.2 μm. Furthermore, commercially available filters and filtration devices are well known in the art and can be selected by those skilled in the art. Exemplary filtration devices may be made from polypropylene or cellulose acetate or polyethersulfone or polyvinylidene fluoride, and commercially available filters may be Millipak (Millipore), Kleenpak (Pall), and Sartobran (trademark) P filtration devices.

[0319] In one embodiment, the recombinant protein / modified fHbp fusion protein further comprises an inclusion body (IB).

[0320] Inclusion bodies (IB): Protein aggregates / accumulation of insoluble proteins are known as IBs. IB formation arises from an unbalanced equilibrium between protein aggregation and solubilization.

[0321] In one embodiment, the recombinant protein / modified fHbp fusion protein containing the inclusion body (IB) is (a) Washing and recovery of the inclusion body; (b) Solubilization and refolding of inclusion bodies (IB); That is the case.

[0322] In one embodiment, a recombinant protein / modified fHbp fusion protein containing inclusion bodies (IB) is subjected to urea denaturation, the inclusion bodies solubilized in urea are bound to an affinity column, and the column is further washed with a refolding buffer that reduces the urea concentration and promotes accurate refolding. The target protein is then eluted with an elution buffer.

[0323] In one embodiment, denaturation is carried out using a denaturation buffer composition containing 20-50 mM sodium phosphate buffer, 50-120 mM sodium chloride (NaCl) pH 7.4, and 5-10 M urea.

[0324] In other embodiments, washing with decreasing urea concentration includes washing with 20–50 mM sodium phosphate buffer and 50–120 mM sodium chloride (NaCl) pH 7.4. In other embodiments, the urea reduction concentrations include 8 M, 6 M, 4 M, 2 M, and 1 M (5–6 CV each).

[0325] In one embodiment, target protein elution is performed using an elution buffer containing 40 mM to 400 mM imidazole.

[0326] In one embodiment, the affinity column includes an immobilized metal affinity column resin.

[0327] Immobilized metal affinity chromatography (IMAC) resins are highly binding resins for purifying His-tagged proteins, and their effectiveness depends on the affinity of His to the immobilized transition metal.

[0328] In one embodiment, the immobilized metal affinity column resin includes uncharged or pre-charged Ni2+ resin.

[0329] In other embodiments, the uncharged form is selectively charged with metal ions that are more flexible for further purification.

[0330] In yet another embodiment, the immobilized metal affinity column resin includes a Ni Sepharose 6 Fast Flow column.

[0331] In one embodiment, the supernatant containing the inclusion bodies is subjected to protein precipitation before urea denaturation.

[0332] In one embodiment, protein precipitation is performed using ammonium sulfate.

[0333] In one embodiment, after urea denaturation, the supernatant is filtered using a 0.22 μM filter.

[0334] In one embodiment, after elution, the eluate containing the target recombinant protein / modified fHbp fusion protein is further subjected to concentration and dialysis.

[0335] In one embodiment, concentration and dialysis filtration are performed using tangential flow filtration (TFF) through a filter typically having a molecular weight cutoff (MWCO) in the range of 5 kDa to 50 kDa.

[0336] In one embodiment, the concentration of recombinant protein / modified fHbp fusion protein is higher than 0.95 mg / ml.

[0337] In other embodiments, when the OD at 590 / 600 nm is 20-100, the culture is induced by stopping the glucose supply, starting the glycerol supply, and adding and / or maintaining lactose at 1-50 g / L in a fed-batch mode.

[0338] In one embodiment, the purified chimeric / recombinant protein / modified fHbp fusion protein is stored at 2°C to 8°C until further use. In other embodiments, the purified chimeric / recombinant protein / modified fHbp fusion protein is stored at 2°C to 8°C in the presence of a stabilizer. In one embodiment, the stabilizer is selected from TRIS, Tween / polysorbate, nonionic surfactants such as polyethylene glycol lauryl ether (BRIJ® 35), sucrose (up to 5%), etc.

[0339] In another aspect of the present invention, recombinant protein / modified fHbp fusion protein formulations are optimized to improve the immunogenicity of recombinant protein / modified fHbp fusion protein antigens, improve stability, and maintain stability over long-term storage.

[0340] In one embodiment, the optimized vaccine formulation has low viscosity, does not aggregate, and is stable over a wide temperature range for extended periods. In one embodiment, the optimized vaccine formulation comprises a solid or liquid carrier.

[0341] In one embodiment, the vaccine formulation is entirely liquid. Suitable forms of the liquid formulation include buffered solutions, suspensions, emulsions, syrups, isotonic aqueous solutions, viscous compositions, and elixirs at a selected pH.

[0342] In one embodiment, the vaccine formulation comprises a polymer or further agent for controlling the viscosity of the composition and / or controlling the release of antigen / secreted proteins from the composition.

[0343] In one embodiment, the vaccine formulation is in the form of a transdermal formulation, including a lotion, gel, spray, ointment, or further suitable technology. If nasal or respiratory (mucosal) administration is desired (e.g., aerosol inhalation or inhalation), the composition may be in a certain form and may be dispensed by a press spray dispenser, pump dispenser, or aerosol dispenser. Aerosols are usually under pressure due to hydrocarbons. Pump dispensers may preferably dispense a measured dose or a dose with a specific particle size. If in the form of a solution, suspension, or gel, in one embodiment the immunogenic composition contains a large amount of water (preferably purified water) in addition to the active ingredient.

[0344] In one embodiment, the vaccine formulation is stable for 12 to 36 months at 2 to 8°C; 2 to 6 months at 25°C; 1 to 4 weeks at 37°C; 2 to 7 days at 42°C; and 2 to 7 days at 55°C.

[0345] In one embodiment, the vaccine formulation is a lyophilized / freeze-dried formulation.

[0346] In one embodiment, the final pH of the formulation may be in the range of pH 6.0 to pH 8.0.

[0347] In a further aspect of the present invention, a modified fHbp, nucleic acid, or formulation according to the present invention is provided for use in the treatment or prevention of pathogenic infection or colonization in a subject. In a further aspect of the present invention, a method for treating or preventing pathogenic infection or colonization in a subject is provided, comprising administering a modified fHbp, nucleic acid, or composition according to the present invention to a subject. In another aspect of the present invention, a method of vaccination is provided, comprising administering a modified fHbp, nucleic acid, or composition according to the present invention to a subject.

[0348] In one embodiment, the modified fHbp is immunogenic and includes parenteral, subcutaneous, intradermal, intramuscular, intraperitoneal, or intravenous administration, or injectable administration, or sustained release from an implant, or administration via eye drops, nasal, rectal, buccal, or vaginal, oral, or gastric, mucosal, alveolar, gingival, olfactory, or respiratory mucosa, or any further immune pathway to administer an immunologically effective amount of the immunogenic preparation to a human subject.

[0349] As used herein, the term "concurrently administered" means that different immunogenic compositions / vaccines may be administered separately or in combination.

[0350] When vaccines are administered separately, they are usually administered to different sites; for example, one vaccine is administered to the left upper arm and the second to the right upper arm. Thus, the two vaccines may be administered contralaterally (e.g., both arms or both legs, or contralateral arms and legs) or ipsilaterally (e.g., arms and legs on the same side of the body). Although the vaccines are administered separately, they are administered substantially simultaneously (e.g., during the same medical consultation or visit to a medical professional or vaccination center), for example, within one hour of each other.

[0351] However, co-immunization may be performed in combination rather than separately. Therefore, co-immunization may be performed using a mixed vaccine, i.e., a single composition containing a mixture of different immunogens. Mixed vaccines offer the subject the advantage of fewer injections, which can lead to the clinical benefit of increased compliance.

[0352] The compositions of the present invention are generally administered directly to the patient. Direct delivery can be achieved by parenteral injection (e.g., subcutaneous, intraperitoneal, intravenous, intramuscular, or into the interstitial space of tissue) or by rectal, oral, vaginal, topical, transdermal, intranasal, ocular, ear, lung, or further mucosal administration. Intramuscular administration into the thigh or upper arm is preferred. The injection may be administered via a needle (e.g., a subcutaneous injection needle), but a needleless injection may be used instead. The usual intramuscular dose is about 0.5 ml.

[0353] Furthermore, the composition may be provided in a “multi-dose” kit, i.e., a single container containing enough composition for multiple immunizations. The multi-dose kit may include a preservative, or the multi-dose container may have a sterile adapter for dispensing individual doses of the composition.

[0354] The subjects to be immunized may be humans of any age, for example, 0-12 months, 1-5 years, 5-18 years, 18-55 years, or over 55 years. Preferably, the subjects to be immunized are adolescents (e.g., 12-18 years) or adults (18 years or older).

[0355] In some cases, the subjects are adolescents or adults who were immunized with Neisseria meningitidis in childhood (e.g., before the age of 12) and are receiving an additional immunization dose of the immunogenic composition according to the present invention.

[0356] In one embodiment, the vaccine formulation of the present invention elucidates cross-protection against Neisseria gonorrhea strain and Neisseria meningitidis serogroup ACWYX.

[0357] The administration may be provided in a therapeutically effective dose. Those skilled in the art can determine the appropriate dose and repetition for administration.

[0358] The vaccine formulation can be formulated as a single-dose vial or a multi-dose vial (2-dose, 5-dose, or 10-dose vials), a multi-dose kit, or a pre-filled syringe. The vaccine formulation can be administered on a single-dose schedule or, preferably, on a multi-dose schedule, with 1 to 3 separate doses administered at subsequent time intervals, as needed, 1 to 3 years after the initial course of vaccination. The administration schedule is also determined, at least in part, based on the need for a booster dose necessary to confer protective immunity.

[0359] In one embodiment, the vaccine formulation is prepared for administration to human subjects under 2 years of age or over 2 years of age, including elderly, adolescent, adult, or child subjects, according to a one-dose, two-dose, or three-dose regimen consisting of a first dose and / or a second dose administered 3 months to 2 years after the first dose and / or a third dose administered 3 months to 2 years after the second dose.

[0360] In one embodiment, the subject is a mammal such as a human.

[0361] In one embodiment, the infection is a bacterial infection. In another embodiment, the infection is meningitis, for example, caused by Neisseria meningitidis or Neisseria gonorrhoeae.

[0362] In a further aspect of the present invention, the modified fHbp according to the present invention is used in combination with at least one further prophylactic or therapeutically active molecule.

[0363] In one embodiment, at least one further prophylactic or therapeutically active molecule comprises a vaccine or antigen different from the modified fHbp according to the present invention as herein. In embodiments of the present invention, the antigen is diphtheria toxin (D), tetanus toxin (T), whole-cell pertussis (wP), hepatitis B virus surface antigen (HbsAg), Haemophilus influenzae type b PRP-carrier protein complex (Hib), Haemophilus influenzae (a, c, d, e, f serotypes and unencapsulated strains), meningococcal A antigen, meningococcal C antigen, meningococcal W-135 antigen, meningococcal Y antigen, meningococcal X antigen, pneumococcal antigen, meningococcal B bleb or purified antigen, or grape aureus. Cocci antigens, anthrax bacteria, BCG, hepatitis (A, C, D, E, F, and G strains) antigens, human papillomavirus, HIV, typhi antigen, acellular pertussis, modified adenylyl cyclase, malaria antigen (RTS,S), measles, mumps, rubella, dengue, Zika, Ebola, chikungunya, Japanese encephalitis, rotavirus, diarrhea antigen, flavivirus, smallpox, yellow fever, herpes zoster, varicella virus antigen, and combinations thereof are selected, but are not limited to these.

[0364] In other embodiments, the present invention envisions a composition comprising an fHbp-PorA chimeric antigen and an antigen for immunization against further diseases or infections. According to embodiments, the composition comprises the following further antigens: —Protein antigens derived from PorB, Fet A, OmpC, NHBA, NadA, meningococcal antigen 287, NspA, HmbR, NhhA, App, and 936 — Glycoantigens derived from meningococcal serotypes A, C, W, Y and / or X — Streptococcus pneumoniae-derived sugar antigens —Diphtheria antigen, e.g., diphtheria toxin, e.g., CRM197 variant ―Tetanus antigens such as tetanus toxin — Antigen derived from Bordetella pertussis, cell-free pertussis antigen, or whole-cell pertussis antigen —Sugar antigen derived from Haemophilus influenzae type B —Polio antigen, e.g., IPV - Measles, mumps, and / or rubella antigens —Influenza antigens, e.g., hemagglutinin and / or neuraminidase surface proteins —Antigen (protein or sugar) derived from Streptococcus agalactie (Streptococcus group B) — Antigens (proteins or sugars) derived from Streptococcus pyogenes (Group A Streptococcus) —Antigens (proteins or sugars) derived from Staphylococcus aureus — Antigens (proteins or sugars) derived from Salmonella species Includes.

[0365] In one embodiment, at least one further prophylactic or therapeutically active compound comprises a monovalent capsule-capsule polysaccharide protein complex vaccine. The monovalent protein-capsule polysaccharide vaccine may comprise a meningococcal serotype C or A capsule containing a bacterial toxin, a bivalent vaccine (containing serotype C and A capsule polysaccharides complexed with the bacterial toxin), a tetravalent (serotype A, C, Y, W) or pentavalent (A, C, Y, W, X) complex vaccine. Alternatively, at least one further prophylactic or therapeutically active molecule may comprise a complex vaccine in which an antigen containing an fHbp scaffold with an exogenous peptide loop (such as a PorA loop) can be incorporated into the complex vaccine as a protein carrier molecule. The complex vaccine may comprise any of the serotype capsule polysaccharides derived from strains A, C, Y, W, or X individually or in combination.

[0366] Combination vaccines can be selected from hexavalent (ACWYX-B), tetravalent (AC-Hib-B), trivalent (AC-B), and bivalent (A-B, X-B, C-B).

[0367] In one embodiment, the liquid meningococcal serotype B vaccine is reconstituted with a lyophilized ACWYX complex vaccine for bedside administration.

[0368] In embodiments, the present invention relates to a method for inducing an immune response against Neisseria meningitidis in mammals. The method comprises (i) at least one fusion protein comprising a stable non-functional / non-lipidized fHbp and a PorA VR2 loop, and at least one complex selected from (a)(i) a capsular sugar of Neisseria meningitidis serotype A and (ii) tetanus toxin; (b)(i) a capsular sugar of Neisseria meningitidis serotype C and (ii) CRM197; (c)(i) a capsular sugar of Neisseria meningitidis serotype Y and (ii) CRM197; (d)(i) a capsular sugar of Neisseria meningitidis serotype W135 and (ii) CRM197; and (d)(i) a capsular sugar of Neisseria meningitidis serotype X and (ii) tetanus toxin.

[0369] One aspect of the present invention relates to a method for inducing an immune response to Neisseria meningitidis in mammals. The method comprises (i) at least two fusion proteins, each consisting of one fHbp mutant bound to one PorA VR2 loop, and (a) a complex of (i) Neisseria meningitidis serotype A and (ii) tetanus toxin; (b) a complex of (i) Neisseria meningitidis serotype C and (ii) CRM197; (c) a complex of (i) Neisseria meningitidis serotype Y and (ii) CRM197; (d) a complex of (i) Neisseria meningitidis serotype W135 and (ii) CRM197; and (d) a complex of (i) Neisseria meningitidis serotype X and (ii) tetanus toxin.

[0370] One aspect of the present invention relates to a method for inducing an immune response to Neisseria meningitidis in mammals. The method comprises (i) at least three fusion proteins, each consisting of one fHbp mutant bound to one PorA VR2 loop, and (a) a complex of (i) Neisseria meningitidis serotype A and (ii) tetanus toxin; (b) a complex of (i) Neisseria meningitidis serotype C and (ii) CRM197; (c) a complex of (i) Neisseria meningitidis serotype Y and (ii) CRM197; (d) a complex of (i) Neisseria meningitidis serotype W135 and (ii) CRM197; and (d) a complex of (i) Neisseria meningitidis serotype X and (ii) tetanus toxin.

[0371] One aspect of the present invention relates to a method for inducing an immune response to Neisseria meningitidis in mammals. The method comprises (i) at least four fusion proteins, each consisting of one fHbp mutant bound to one PorA VR2 loop, and (a) a complex of (i) Neisseria meningitidis serotype A and (ii) tetanus toxin; (b) a complex of (i) Neisseria meningitidis serotype C and (ii) CRM197; (c) a complex of (i) Neisseria meningitidis serotype Y and (ii) CRM197; (d) a complex of (i) Neisseria meningitidis serotype W135 and (ii) CRM197; and (d) a complex of (i) Neisseria meningitidis serotype X and (ii) tetanus toxin.

[0372] The fHbp-PorA chimeric antigens used in the present invention include amino acid sequences that have 50% or more identity (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with the sequences listed in sequence listings. The sequence listings are attached in standard ST.26 format and are incorporated herein by reference.

[0373] List of Men B strains used in trials of fHbp-PorA chimeric vaccines fHbp isogenic strains expressing different fHbp strains (V1.1, V1.14, V2.19, and V3.45 in the H44 / 76ΔfHbpΔPorA construct) were used in the testing of fHbp-PorA chimeric vaccines.

[0374] A. fHbp expressing strain 1.H44 / 76ΔfHbpΔPorA:fHbp V1.1 2.H44 / 76ΔfHbpΔPorA:fHbp V1.14 3.H44 / 76ΔfHbpΔPorA:fHbp V2.19 4.H44 / 76ΔfHbpΔPorA:fHbp V3.45 B.PorA-expressing strain: 1.H44 / 76ΔfHbpΔPorA:PorA 1.4 2.H44 / 76ΔfHbpΔPorA:PorA 1.9 C. Clinical isolates: M11.240413 (expresses fHbp V1.13 and PorA 1.9) For the trial of the fHbp-PorA chimeric vaccine, the following clinical strains of Neisseria meningitidis will be used as SBA at Oxford / UKHSA. TIFF2026511179000013.tif39170

[0375] Furthermore, meningococcal strains M15 240912, M16 240272, M15 240460, or any other suitable strain may be used for SBA.

[0376] In another aspect of the present invention, factor H-binding protein (fHbp) is used as an epitope display scaffold. For use as an epitope display scaffold, factor H-binding protein (fHbp) containing any of the modifications described herein may be used. They may be used as vaccines, and the compositions or modified fHbp according to the present invention may be useful as diagnostic reagents and as measures of the immunoassay of vaccines.

[0377] The immune response induced by the modified fHbp of the present invention may affect the function of Neisseria meningitidis (Nm) infecting subjects immunized with the modified fHbp of the present invention. Preferably, the function of Nm infecting subjects immunized with the modified fHbp of the present invention is interfered with or prevented. The induced immune response may recognize and destroy Nm.

[0378] Alternatively, the induced immune response may interfere with or prevent the replication of Nm. Alternatively, the induced immune response may interfere with or prevent diseases that can induce Nm in humans or non-human animals.

[0379] The above description of specific embodiments fully illustrates the general nature of the embodiments herein, and others can readily modify and / or adapt such specific embodiments to various uses without departing from the general concept by applying their current knowledge. Therefore, such adaptations and modifications should and are intended to fall within the meaning and scope of the equivalents of the disclosed embodiments. It should be understood that the terminology and expressions used herein are descriptive and not limiting. Thus, while the embodiments herein describe preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modifications within the spirit and scope of the embodiments described herein.

[0380] Throughout this specification, variations of the term "comprise," "comprises," or "comprising" will be understood to mean that they include the elements, integers, or processes or groups of elements, integers, or processes described, but not that they exclude any further elements, integers, or processes or groups of elements, integers, or processes.

[0381] The terms “one or more” or “at least one” imply the use of one or more elements, components, or quantities, which may be used in embodiments of the present invention to achieve one or more desired objectives or results.

[0382] Any consideration of documents, actions, materials, apparatus, articles, etc., included herein is solely for the purpose of providing the context of this disclosure. It should not be construed as acknowledging that any or all of such matters constituted a fundamental part of the prior art or existed somewhere prior to the priority date of this application, and therefore were common general knowledge in the art relevant to this disclosure.

[0383] The numerical values ​​given for various physical parameters, dimensions, and quantities are merely approximations, and values ​​higher than those assigned to physical parameters, dimensions, and quantities are assumed to fall within the scope of the present invention unless otherwise stated herein.

[0384] While this specification has given considerable emphasis to certain features of preferred embodiments, it will be understood that many further features can be added and many modifications can be made in preferred embodiments without departing from the principles of the disclosure. These modifications and further modifications in preferred embodiments of the disclosure will be obvious to those skilled in the art from the disclosure herein, and it should be clearly understood that the foregoing descriptions should be construed as merely illustrative and not as limiting of the disclosure.

[0385] Technical advantages: 1. The present invention provides an efficient platform process for producing an effective vaccine against Neisseria meningitidis that meets multiple criteria, with improved immunogenicity, safety, and cost.

[0386] 2. The present invention provides a method for developing and optimizing upstream bioprocesses to increase the cell density of lead cell lines and the productivity of recombinant protein / modified fHbp fusion protein.

[0387] 3. The present invention provides a method for developing and optimizing downstream bioprocesses to produce recombinant protein / modified fHbp fusion protein in high yield and high purity.

[0388] 4. The improved formulation overcomes the limitations of prior art, exhibiting low viscosity, no aggregation, long-term stability over a wide temperature range, high stability, and indirectly preserving the desired characteristics of the recombinant protein / modified fHbp fusion protein, including immunogenicity.

[0389] 5. The novel formulation / composition (i) is free from aggregate and particle formation, has a higher osmotic pressure, optimized zeta potential, and low viscosity, and (iii) possesses the desired physicochemical and immunogenic properties of the recombinant protein / modified fHbp fusion protein, and can be stored at 2-8°C for 12 months, 25°C for 6 months, and 40°C for 30 days.

[0390] 6. Chimeric antigen (CHA) against meningococcal serotype B. CHA utilizes fHbp (non-lipidized) as a molecular scaffold for presenting a surface-exposed PorA VR2 loop, achieved by inserting a VR2 loop (a "10-20 amino acid" PorA VR2 loop instead of the "entire PorA protein") into the β-turn region of fHbp. CHA has been found to carry epitopes derived from fHbp and PorA and to induce a functional immune response to both antigens. The overall structure of fHbp remains unchanged even with the incorporation of the VR2 loop, and the VR2 loop folds into a three-dimensional structure recognized by bactericidal mAbs.

[0391] 7. The soluble, high-yield chimeric protein is stable, and both the fHbp and PorA VR2 loops are immunogenic.

[0392] 8. Since the chimeric composition reflects the prevalent fHbp and PorA antigens circulating within a given geographical area, the generated chimera is composed of the most prevalent fHbp and PorA antigens to maximize vaccine coverage.

[0393] 9. Inserting a PorA loop at a specific position in fHbp reduces H factor binding to the desired extent (at least 10%, at least 50%, preferably >70% compared to the wild type) while preserving both the immunogenic epitopes of fHbp and PorA, and maintaining a molecular weight range of 20kDa to 40kDa (Table 12).

[0394] [Table 12]

[0395] The present invention is described in more detail by the following embodiments and combinations of embodiments arising from corresponding dependent references and links.

[0396] I. A modified factor H binding protein (fHbp) comprising a wild-type fHbp mutant and at least one exogenous peptide loop, wherein, a. A modified factor H binding protein (fHbp) is selected from an amino acid sequence that is at least 75% identical to one of the sequences of sequence numbers 6-10; b. At least one exogenous peptide loop is immunogenic; c. At least one exogenous peptide loop originates from a bacterial membrane protein; d. The modified fHbp is a fusion protein. e.fHbp mutants are selected from v1, v2, and v3, which are modified with at least one PorA loop containing at least 10 amino acids inserted into the β-turn region of fHbp; and The f.PorA loop is selected from VR1 and VR2.

[0397] II. The modified H factor-binding protein according to Embodiment I, wherein the modified fHbp comprises a wild-type fHbp mutant and at least one exogenous bacterial membrane protein peptide loop, or the modified fHbp is a fusion protein.

[0398] III. A modified H factor-binding protein according to any one of the above embodiments, wherein the modified fHbp is modified to reduce H factor-binding activity.

[0399] IV. A nucleic acid sequence encoding a modified fHbp that is at least 75% identical to any one of the sequences of sequence numbers 1-5.

[0400] V. An immunogenic composition comprising a nucleic acid sequence encoding at least one modified fHbp described in any one of the above embodiments or a modified fHbp described in Embodiment IV.

[0401] VI. The immunogenic composition according to Embodiment V, comprising two or more different modified fHbp.

[0402] VII. An immunogenic composition according to any one of the above embodiments, comprising a pharmaceutically acceptable carrier.

[0403] VIII. An immunogenic composition according to any one of the above embodiments, further comprising an adjuvant.

[0404] IX. The immunogenic composition according to any one of the above embodiments, further comprising at least one further prophylactic or therapeutically active molecule, including a monovalent protein:capsular polysaccharide vaccine.

[0405] X. The immunogenic composition according to any one of the above embodiments, wherein a scaffold of fHbp having an exogenous peptide loop is incorporated into the complex vaccine as a protein carrier molecule.

[0406] XI. The following: —Protein antigens derived from PorB, Fet A, OmpC, NHBA, NadA, meningococcal antigen 287, NspA, HmbR, NhhA, App, and 936 — Glycoantigens derived from meningococcal serotypes A, C, W, Y and / or X — Streptococcus pneumoniae-derived sugar antigens —Diphtheria antigen, e.g., diphtheria toxin, e.g., CRM197 variant ―Tetanus antigens such as tetanus toxin — Antigen derived from Bordetella pertussis, cell-free pertussis antigen, or whole-cell pertussis antigen —Sugar antigen derived from Haemophilus influenzae type B —Polio antigen, e.g., IPV - Measles, mumps, and / or rubella antigens —Influenza antigens, e.g., hemagglutinin and / or neuraminidase surface proteins —Antigen (protein or sugar) derived from Streptococcus agalactie (Streptococcus group B) — Antigens (proteins or sugars) derived from Streptococcus pyogenes (Group A Streptococcus) —Antigens (proteins or sugars) derived from Staphylococcus aureus — Antigens (proteins or sugars) derived from Salmonella species An immunogenic composition according to any one of the above embodiments, comprising a recombinant protein / modified fHbp fusion protein used in combination with at least one further antigen selected from the above.

[0407] XII. An immunogenic composition according to any one of the above embodiments, wherein a scaffold of fHbp having an exogenous peptide loop is incorporated into a polysaccharide complex 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).

[0408] XIII. A modified fHbp according to any one of Embodiments I to III, a nucleic acid according to Embodiment IV, or an immunogenic composition according to any one of Embodiments V to IX, for use as a pharmaceutical or for the treatment or prevention of pathogenic infection or colonization in a subject.

[0409] XIV. Combination of a modified fHbp according to any one of Embodiments I to III, a nucleic acid according to Embodiment IV, or an immunogenic composition according to any one of Embodiments V to IX with at least one further prophylactic or therapeutically active molecule.

[0410] XV. The combination according to Embodiment XIII or IX, comprising a conjugate vaccine containing at least one further prophylactic or therapeutically active molecule, which is one of the serotype capsular polysaccharides selected from strains A, C, Y, W, or X, or combinations thereof.

[0411] XVI. Protein: Capsule polysaccharide vaccine comprising any of the following: serotype C or A capsule containing a bacterial toxin, bivalent vaccine (containing serotype C and A capsule polysaccharides combined with the bacterial toxin), tetravalent (serotype A, C, Y, W polysaccharides combined with the bacterial toxin), or pentavalent (serotype A, C, Y, W, X polysaccharides combined with the bacterial toxin) complex vaccine, as disclosed in Embodiment XV or as disclosed in Embodiment IX.

[0412] XVII. H factor-binding protein (fHbp) as described in Embodiments I-III, used as an epitope display scaffold.

[0413] 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 the group consisting of transferrin-binding protein, Neisseria heparin-binding protein, Neisseria surface protein A, PorA, meningococcal enterobactin receptor FetA, Neisseria adhesin A, factor H-binding protein (fHbp) disclosed in any one of the preceding embodiments, or a combination thereof.

[0414] XIX. The recombinant protein is the fHbp described in any one of the above embodiments, and the fHbp is derived from meningococcal serotypes A, B, C, H, I, K, L, 29E, W135, X, Y, and Z, according to Embodiment XVIII.

[0415] XX. The vaccine formulation according to any one of the above embodiments, wherein the recombinant protein is the fHbp described in any one of the above embodiments, and the fHbp is derived from meningococcal serotype B.

[0416] XXI. The vaccine formulation according to any one of the above embodiments, wherein the recombinant protein is fHbp according to any one of the above embodiments, and the molecular weight of the fHbp is in the range of 10 kDa to 200 kDa, preferably up to 50 kDa.

[0417] XXII. Adjuvants include aluminum hydroxide, aluminum phosphate, aluminum hydroxyphosphate, potassium aluminum sulfate, MF-59, liposomes, lipopolysaccharides, saponins, lipid A, lipid A derivatives, monophosphoryl lipid A, GLA, 3-deacylated monophosphoryl lipid A, AS01, AS03, AF3, IL-2, RANTES, GMCSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L and 41 BBL, oligonucleotides, oligonucleotides and / or liposomes containing at least one unmethylated CpG, Freund's adjuvants, complete Freund's adjuvants, incomplete Freund's adjuvants, polymers, copolymers such as polyoxyethylene-polyoxypropylene copolymers containing block copolymers, polymer p 1005, CRL-8300 adjuvant, muramyl dipeptide, e.g., TLR1 / 2 agonist, TLR2, TLR3, TLR-4 agonist, TLR5, TLR7, TLR7 / 8, TLR8, TLR9, ODN 2216 (Type A), TLR11 / 12, TLR-4 agonist, flagellin, flagellin derived from Gram-negative bacteria, TLR-5 agonist, flagellin fragments capable of binding to the TLR-5 receptor, alpha-C galactosylceramide, chitosan, interleukin-2, QS-21, squalene, Quil A, cholera toxin B subunit, polyphosphazene and derivatives, mycobacterial cell wall preparations, mycolic acid derivatives, nonionic block copolymer surfactants, OMV, fHbp, sterols and saponins in combination with lipids, dmLT, 1, A vaccine formulation according to any one of Embodiments 1 to 50, selected from the group consisting of 25-dihydroxyvitamin D3, CAF01, poly[di(carboxylatofenoxy)-phosphazene] (PCPP), and Venezuelan encephalitis virus (VEE) replicon particles or combinations thereof.

[0418] XXIII. A vaccine formulation according to any one of the above embodiments, wherein the adjuvant is aluminum hydroxide having a particle size greater than 500 nm.

[0419] XXIV. One or more pharmaceutically acceptable excipients, as follows: (a) Buffers selected from carbonates, phosphates, acetates, HEPES, succinates, TRIS, borates, citrates, lactates, glucons, tartrates, or combinations thereof; (b) Sugars selected from trehalose, mannose, raffinose, lactobionic acid, glucose, maltulose, isomaltulose, maltose, lactose, dextrose, fructose, or combinations thereof; (c) Sugar alcohols or polyols selected from mannitol, lactitol, sorbitol, glycerol, xylitol, maltitol, lactitol, erythritol, isomalt and hydrolyzed starch products or combinations thereof; (d) Surfactants selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, oxytoxynol 40, nonoxynol 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene 660 hydroxystearate, polyoxyethylene ricinoleic acid 35, soy lecithin, poloxamer, copolymers of ethylene oxide (EO), propylene oxide (PO) and / or butylene oxide (BO), octoxynol, phospholipids, nonylphenol ethoxylate, polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols, sorbitan esters or combinations thereof; (e) polymers selected from dextran, carboxymethylcellulose, hyaluronic acid, cyclodextrin, or combinations thereof; (f) Salts selected from NaCl, KCl, KH2PO4, Na2HPO4.2H2O, CaCl2, MgCl2, or combinations thereof; (g) Amino acids selected from tricine, 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 proteins selected from gelatin, lactalbumin hydrolysate, monosodium glutamate, collagen hydrolysate, keratin hydrolysate, peptides, casein hydrolysate, whey protein hydrolysate, serum albumin, or combinations thereof; (i) Preservatives selected from phenoxyethanol, benzethonium chloride (Phemerol), phenol, m-cresol, thiomersal, formaldehyde, paraben esters, 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 physiological saline; A vaccine preparation according to any one of the embodiments described above.

[0420] XXV. At least one recombinant protein / at least one modified fHbp as described in any one of the embodiments above; Aluminum hydroxide; Mannitol; Phosphate; and A vaccine formulation according to any one of the above embodiments, comprising polysorbate.

[0421] XXVI. At least one modified fHbp represented by an amino acid sequence that is at least 75% identical to any one of the amino acid sequences represented by SEQ ID NOs: 6-10; or At least one modified fHbp encoding a nucleic acid sequence that is at least 75% identical to any one of the nucleic acid sequences represented by sequence numbers 1 to 5; and Aluminum hydroxide; Mannitol; Phosphate; and A vaccine formulation according to any one of the above embodiments, comprising polysorbate.

[0422] XXVII. A vaccine formulation according to any one of the above embodiments, comprising (i) at least one recombinant protein / modified fHbp fusion protein; (ii) an amount of aluminum hydroxide in the range of 0.5 mg / ml to 4.5 mg / ml; (iii) an amount of mannitol in the range of 5 mg / ml to 100 mg / ml; (iv) an amount of phosphate buffer in the range of 1 mM to 10 mM; and (v) an amount of polysorbate 20 in the range of 0.01 mg / ml to 2 mg / ml, wherein each recombinant protein / modified fHbp fusion protein is present in an amount in the range of 15 μg / ml to 150 μg / ml.

[0423] XXVIII. A vaccine formulation according to any one of the above embodiments, 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.

[0424] XXIX. A vaccine formulation according to any one of the above 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 μg / ml to 150 μg / ml.

[0425] XXX.A vaccine formulation according to any one of the above embodiments, comprising (i) at least four recombinant protein / modified fHbp fusion proteins; (ii) an amount of aluminum hydroxide in the range of 0.5 mg / ml to 4.5 mg / ml; (iii) an amount of mannitol in the range of 5 mg / ml to 100 mg / ml; (iv) an amount of phosphate buffer in the range of 1 mM to 10 mM; and (v) an amount of polysorbate 20 in the range of 0.01 mg / ml to 2 mg / ml, wherein each recombinant protein / modified fHbp fusion protein is present in an amount in the range of 15 μg / ml to 150 μg / ml.

[0426] XXXI. At least one modified fHbp represented by an amino acid sequence that is at least 75% identical to any one of the amino acid sequences represented by SEQ ID NOs: 6-10; or At least one modified fHbp encoded by a nucleic acid sequence that has at least 75% identity with any one of the nucleic acid sequences represented by sequence numbers 1 to 5; and Aluminum hydroxide in amounts ranging from 0.5 mg / ml to 4.5 mg / ml; Mannitol in amounts ranging from 5 mg / ml to 100 mg / ml; A phosphate buffer solution in the range of 1 mM to 10 mM; and A vaccine formulation according to any one of the above embodiments, comprising polysorbate 20 in an amount in the range of 0.01 mg / ml to 2 mg / ml.

[0427] XXXII.FHbpV3.45M5:PorA316―320 / exP1.14 (represented by sequence number 6 or coded by sequence number 1) in quantities in the range of 15 μg / ml to 150 μg / ml; or fHbpV2.19 M6:PorA316―320 / exP1.4 (represented by sequence number 7 or coded by sequence number 2) in quantities in the range of 15 μg / ml to 150 μg / ml; or fHbpV1.14:PorA307―311 / exP1.9 (represented by sequence number 8 or coded by sequence number 3) in quantities in the range of 15 μg / ml to 150 μg / ml; or fHbpV1.1:PorA307―311 / exP1.4 (represented by sequence number 9 or coded by sequence number 4) in quantities in the range of 15 μg / ml to 150 μg / ml; and Aluminum hydroxide in amounts ranging from 0.5 mg / ml to 4.5 mg / ml; Mannitol in amounts ranging from 5 mg / ml to 100 mg / ml; A phosphate buffer solution in the range of 1 mM to 10 mM; and A vaccine formulation according to any one of the above embodiments, comprising polysorbate 20 in an amount in the range of 0.01 mg / ml to 2 mg / ml.

[0428] XXXIII.fHbpV3.45M 5:PorA316―320 / ExP 1.14 (represented by sequence number 6 or coded by sequence number 1) in quantities ranging from 15 μg / ml to 150 μg / ml; fHbpV2.19 M 6:PorA 316-320 / ExP1.4 (represented by sequence number 7 or coded by sequence number 2) in quantities ranging from 15 μg / ml to 150 μg / ml; fHbpV1.14:PorA307―311 / ExP1.9 (represented by sequence number 8 or coded by sequence number 3) in quantities ranging from 15 μg / ml to 150 μg / ml; fHbpV1.1:PorA307―311 / ExP1.4 (represented by sequence number 9 or coded by sequence number 4) in quantities in the range of 15 μg / ml to 150 μg / ml; and Aluminum hydroxide in amounts ranging from 0.5 mg / ml to 4.5 mg / ml; Mannitol in amounts ranging from 5 mg / ml to 100 mg / ml; A phosphate buffer solution in the range of 1 mM to 10 mM; and A vaccine formulation according to any one of the above embodiments, comprising polysorbate 20 in an amount in the range of 0.01 mg / ml to 2 mg / ml.

[0429] XXXIV.FHbpV3.45M5:PorA316―320 / exP1.14 (represented by Sequence ID 6 or coded by Sequence ID 1) in quantities ranging from 15 μg / mL to 150 μg / mL; or fHbpV2.19 M6:PorA316―320 / exP1.4 (represented by Sequence ID 7 or coded by Sequence ID 2) in quantities in the range of 15 μg / mL to 150 μg / mL; or fHbpV1.1:PorA307―311 / exP1.9 (represented by sequence number 10 or coded by sequence number 5) in the range of 15 μg / mL to 150 μg / mL; or fHbpV1.14:PorA307―311 / exP1.9 (represented by sequence number 8 or coded by sequence number 3) in quantities in the range of 15 μg / mL to 150 μg / mL; and Aluminum hydroxide in amounts ranging from 0.5 mg / mL to 4.5 mg / mL; Mannitol in amounts ranging from 5 mg / mL to 100 mg / mL; A phosphate buffer solution in the range of 1 mM to 10 mM; and A vaccine formulation according to any one of the above embodiments, comprising polysorbate 20 in an amount in the range of 0.01 mg / mL to 2 mg / mL.

[0430] XXXV.fHbpV3.45M5: Amounts in the range of 15 μg / mL to 150 μg / mL, represented by PorA 316-320 / ExP1.14 (indicated by SEQ ID NO: 6 or coded by SEQ ID NO: 1); fHbpV2.19M6: Amounts in the range of 15 μg / mL to 150 μg / mL, represented by PorA316-320 / ExP1.4 (indicated by SEQ ID NO: 7 or coded by SEQ ID NO: 2); HbpV1.1: Amounts in the range of 15 μg / mL to 150 μg / mL represented by PorA307-311 / ExP1.9 (represented by SEQ ID NO: 10 or coded by SEQ ID NO: 5); fHbpV1.14: Amounts in the range of 15 μg / mL to 150 μg / mL represented by PorA307-311 / ExP1.9 (represented by SEQ ID NO: 8 or coded by SEQ ID NO: 3); Aluminum hydroxide in amounts ranging from 0.5 mg / mL to 4.5 mg / mL; Mannitol in amounts ranging from 5 mg / mL to 100 mg / mL; A phosphate buffer solution in the range of 1 mM to 10 mM; and A vaccine formulation according to any one of the above embodiments, comprising polysorbate 20 in an amount in the range of 0.01 mg / mL to 2 mg / mL.

[0431] XXXVI. A vaccine formulation according to any one of the above embodiments, comprising 2-phenoxyethanol in an amount ranging from 1 mg / mL to 10 mg / mL.

[0432] XXXVII. A vaccine formulation according to any one of the above embodiments, wherein the vaccine composition is stable for 6 months at 2-8°C, 25°C, and 40°C.

[0433] XXXVIII. A vaccine formulation according to any one of the above embodiments, wherein the zeta potential is in the range of -16mV to -30mV; and the osmotic pressure is in the range of 200mOsmol / kg to 500mOsmol / kg.

[0434] XXXIX. Diphtheria toxin (D), tetanus toxin (T), whole-cell pertussis (wP), hepatitis B virus surface antigen (HbsAg), Haemophilus influenzae type b PRP-carrier protein complex (Hib), Haemophilus influenzae (a, c, d, e, f serotypes and unencapsulated strains), meningococcal A antigen, meningococcal C antigen, meningococcal W-135 antigen, meningococcal Y antigen, meningococcal X antigen, Streptococcus pneumoniae antigen, meningococcal B bleb or purified antigen, Staphylococcus aureus antigen, Bacillus anthracis, BCG, hepatitis (A A vaccine formulation according to any one of the above embodiments, further comprising one or more antigens selected from (C, D, E, F, and G strain) antigens, human papillomavirus, HIV, Salmonella typhi antigen, acellular pertussis, modified adenylyl cyclase, malaria antigen (RTS,S), measles, mumps, rubella, dengue, Zika, Ebola, chikungunya, Japanese encephalitis, rotavirus, diarrhea antigen, flavivirus, smallpox, yellow fever, herpes zoster, varicella virus antigen, and combinations thereof.

[0435] XL. A vaccine formulation according to any one of the above embodiments, comprising (i) at least one fusion protein comprising a stable non-functional / non-lipidized fHbp and a PorA VR2 loop, and (ii) at least one polysaccharide protein complex.

[0436] XLI. The vaccine formulation according to any one of the above embodiments, wherein the recombinant protein / modified fHbp fusion protein is administered concurrently with one or more vaccines selected from Bexcelo®, MENVEO, MENACTRA, NIMENRIX, MenQuadFi, MENFIVE, MenAfriVac, Men AC, and Men ACHib.

[0437] XLII. Recombinant protein / modified fHbp fusion protein, administered concurrently with MENFIVE, in the vaccine formulation according to any one of the above embodiments.

[0438] XLIII.i) A fusion protein comprising a stable, non-functional / non-lipidized fHbp and at least one PorA VR2 loop, and the following: (a)(i) a complex of meningococcal serotype A capsule sugar and (ii) tetanus toxin; (b)(i) Capsule sugar of Neisseria meningitidis serotype C and (ii) CRM197 complex; (c)(i) Capsular sugar of Neisseria meningitidis serotype Y and (ii) Complex of CRM197; (d)(i) Capsule sugar of Neisseria meningitidis serotype W135 and (ii) CRM197 complex; and (e)(i) a complex of meningococcal serotype X capsule sugars and (ii) a complex of tetanus toxin; A vaccine formulation according to any one of the above embodiments, comprising at least one complex selected from, and

[0439] XLIV.(i) A fusion protein consisting of at least two fHbp mutants, each bound to one PorA VR2 loop, and the following: (a)(i) a complex of meningococcal serotype A capsule sugar and (ii) tetanus toxin; (b)(i) Capsule sugar of Neisseria meningitidis serotype C and (ii) CRM197 complex; (c)(i) Capsular sugar of Neisseria meningitidis serotype Y and (ii) Complex of CRM197; (d)(i) Capsule sugar of Neisseria meningitidis serotype W135 and (ii) CRM197 complex; and (e)(i) a complex of meningococcal serotype X capsule sugars and (ii) a complex of tetanus toxin; A vaccine formulation according to any one of the above embodiments, including the above.

[0440] XLV.(i) consists of at least three fusion proteins, each comprising one fHbp mutant bound to two PorA VR2 loops, and the following: (a)(i) a complex of meningococcal serotype A capsule sugar and (ii) tetanus toxin; (b)(i) Capsule sugar of Neisseria meningitidis serotype C and (ii) CRM197 complex; (c)(i) Capsular sugar of Neisseria meningitidis serotype Y and (ii) Complex of CRM197; (d)(i) Capsule sugar of Neisseria meningitidis serotype W135 and (ii) CRM197 complex; and (e)(i) a complex of meningococcal serotype X capsule sugars and (ii) a complex of tetanus toxin; A vaccine formulation according to any one of the above embodiments, including the above.

[0441] XLVI.(i) A fusion protein consisting of at least four fHbp mutants, each bound to three PorA VR2 loops, and the following: (a)(i) a complex of meningococcal serotype A capsule sugar and (ii) tetanus toxin; (b)(i) Capsule sugar of Neisseria meningitidis serotype C and (ii) CRM197 complex; (c)(i) Capsular sugar of Neisseria meningitidis serotype Y and (ii) Complex of CRM197; (d)(i) Capsule sugar of Neisseria meningitidis serotype W135 and (ii) CRM197 complex; and (e)(i) a complex of meningococcal serotype X capsule sugars and (ii) a complex of tetanus toxin; A vaccine preparation according to any one of the above embodiments, comprising...

[0442] XLVII. A vaccine preparation according to any one of the above embodiments for use in the treatment or prevention of infections and / or diseases caused by Neisseria meningitidis serogroup B.

[0443] XLIII. A vaccine preparation according to any one of the above embodiments for elucidating cross - protection against Neisseria gonorrhea strains and Neisseria meningitidis serogroups ACWYX.

[0444] XLIX. A vaccine preparation according to any one of the above embodiments, wherein the adsorption ratio of the recombinant protein / modified fHbp fusion protein onto the adjuvant is in the range of 70% - 100%.

[0445] L. A vaccine preparation according to any one of the above embodiments, wherein the adsorption ratio of fHbpV3.45M5 PorA onto the adjuvant is in the range of 80% - 100%. 316―320exP1.14 onto the adjuvant is in the range of 80% - 100%.

[0446] LI. A vaccine preparation according to any one of the above embodiments, wherein the adsorption ratio of fHbpV1.14PorA onto the adjuvant is in the range of 80% - 90%. 307―311 exP1.9 onto the adjuvant is in the range of 80% - 90%.

[0447] LII. A vaccine preparation according to any one of the above embodiments, wherein the adsorption ratio of fHbpV2.19PorA onto the adjuvant is in the range of 80% - 90%. 316―320 exP1.4 onto the adjuvant is in the range of 80% - 90%.

[0448] LIII. A vaccine preparation according to any one of the above embodiments, wherein the adsorption ratio of fHbpV1.1PorA onto the adjuvant is in the range of 80% - 90%. 307―311 exP1.4 onto the adjuvant is in the range of 80% - 90%.

[0449] LIV. fHbpV1.1PorA on adjuvant 307―311 exP1.9 A vaccine formulation according to any one of the above embodiments, wherein the adsorption ratio is in the range of 70% to 80%.

[0450] LV. A method for producing a vaccine formulation according to any one of the embodiments described above, the following: (a) Growing host cells containing the expression vector in a nutrient medium; (b) Inducing the host cells to express the protein; (c) Recovering and separating the host cells; (d) Lyse the recovered cells to separate the host cell fragments and obtain tagged proteins; (e) Purifying the tagged protein; (f) Remove the tag from the tagged protein to obtain a recombinant protein / modified fHbp fusion protein; (g) Purifying the recombinant protein / modified fHbp fusion protein; and (h) Prepare a vaccine formulation containing the purified recombinant protein / modified fHbp fusion protein. Methods that include...

[0451] LVI. The method according to any one of the above embodiments, wherein the host cell is a bacterial expression host system.

[0452] LVII. The method according to any one of the above embodiments, wherein the bacterial expression host system is an Escherichia coli strain selected from the group consisting of 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)*, T7 Express, m15 pREP4*, C41(DE3), C43(DE3), or B834(DE3).

[0453] LVIII. The method according to any one of the above embodiments, wherein the nutrient medium is selected from undefined medium, terrific broth (TB) medium, lysogenia broth, luria broth (LB) medium or luria-bertani medium, chemically defined medium, M9 minimum medium, chemically defined M9 modified salt medium, 2xYT medium or catabolic metabolite suppression (SOC) medium, superoptimal broth, and combinations thereof.

[0454] LIX. The method according to any one of the above embodiments, wherein the concentration of L-methionine during the proliferation of host cells in (a) is maintained in the range of 1 mM to 10 mM, and the fermentation is in a fed-batch mode.

[0455] LX. The method according to any one of the above embodiments, 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 volumes of gas per unit volume of liquid (VVM).

[0456] LXI. The method according to any one of the above embodiments, wherein the host cells are induced using an inducer selected from lactose and its non-hydrolyzable analog isopropyl β-D-1-thiogalactopyranoside (IPTG).

[0457] LXII. The method according to any one of the above embodiments, wherein the lactose concentration is in the range of 1 g / L to 50 g / L and the IPTG concentration is in the range of 1 mM to 10 mM.

[0458] LXIII. The method according to any one of the above embodiments, wherein the host cells are lysed using a method selected from chemical, biological, physical, mechanical, and combinations thereof.

[0459] LXIV. The method according to any one of the above embodiments, wherein the host cells are lysed using a combination of chemical and mechanical methods.

[0460] LXV. The method according to any one of the above embodiments, wherein the host cells are lysed using a lysis buffer having a pH range of 7 to 9, and then mechanically lysed for 3 to 8 cycles at a pressure range of 1000 to 1500 bar.

[0461] LXVI. A method according to any one of the above embodiments, wherein mechanical dissolution is performed using a homogenizer.

[0462] LXVII. The method according to any one of the above embodiments, wherein, in (e) above, the tagged protein is purified by chromatography, followed by concentration and dialysis.

[0463] LXVIII. The method according to any one of the above embodiments, wherein the tag is removed using a TEV protease having a protein:TEV protease ratio in the range of 5:1 to 30:1.

[0464] LXIX. The method according to any one of the above embodiments, wherein the tagged protein is purified by chromatography, concentration, and dialysis in the above (g).

[0465] The method according to any one of the above embodiments, wherein LXX. 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 bonding chromatography, reversed-phase chromatography, and combinations thereof.

[0466] LXXI. The method according to any one of the above embodiments, wherein the tagged protein is expressed as inclusion bodies (IBs), and is purified by urea unfolding of the tagged protein and column refolding of the tagged protein.

[0467] LXXII. The method according to any one of the above embodiments, wherein the vaccine formulation is prepared by adsorbing individual recombinant protein / modified fHbp fusion proteins onto an adjuvant and then adding it to an excipient mixture comprising a sugar alcohol, a buffer, a stabilizer, and a liquid carrier.

[0468] LXXIII. The method according to any one of the above embodiments, wherein the excipient mixture contains a preservative.

[0469] A method for producing LXXIV.TEV protease, wherein the following: (a) Growing host cells containing the expression vector in a nutrient medium; (b) Inducing the host cells to express TEV protease; (c) Recovering and separating the host cells; (d) Lyse the recovered host cells and separate them to obtain the TEV protease; (e) Purifying the recovered TEV protease; and (f) Concentrate the purified TEV protease, dialysis filter it, and store it; A method according to any one of the above embodiments, including:

[0470] The method according to any one of the above embodiments, wherein the host cell for expressing the TEV protease is an Escherichia coli strain selected from the group consisting of 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)*, T7 Express, m15 pREP4*, C41(DE3), C43(DE3), Rosetta(trademark)(DE3)pLysS, or B834(DE3).

[0471] The method according to any one of the above embodiments, wherein when the OD at LXXVI.590 / 600nm is 20-100, glucose supply is stopped and glycerol supply is started, and the culture is induced by adding and / or maintaining lactose at 1-50 g / L in a fed-batch mode.

[0472] LXXVII. A method for inducing an immune response to meningococcal serotype B strain in an individual by administering a vaccine formulation described in any one of the above embodiments, wherein the administration is equivalent to inducing an immune response to meningococcal serotype B strain.

[0473] [Examples] The above description of embodiments is provided for illustrative purposes only and is not intended to limit the scope of the disclosure. Individual components of a particular embodiment are generally interchangeable and not limited to that specific embodiment. Such modifications should not be considered departures from the disclosure, and all such modifications should be considered within the scope of the disclosure. The present invention is further described with reference to the following embodiments, which are provided for illustrative purposes only and should not be construed as limiting the scope of the disclosure.

[0474] The method for manufacturing the vaccine formulation described herein comprises the following extensive steps: (a) Growing host cells containing the expression vector in a nutrient medium; (b) Inducing the host cells to express the protein; (c) Recovering and separating the host cells; (d) Lyse the recovered cells to separate the host cell fragments and obtain tagged proteins; (e) Purifying the tagged protein; (f) Remove the tag from the tagged protein to obtain a recombinant protein / modified fHbp fusion protein; (g) Purifying the recombinant protein / modified fHbp fusion protein; and (h) preparing a vaccine formulation comprising the purified recombinant protein / modified fHbp fusion protein, which will be described in further detail in the following paragraphs. [Examples]

[0475] Expression of tagged fHbp protein and TEV protease Sources of biological resources used in this invention: (1) Host cells: Escherichia coli B834 (DE3) was used for recombinant protein expression. Catalog number D48175, Sigma Aldrich (transferred from Oxford University to the Serum Institute Pvt. Ltd., India) (2) Plasmid*: pET-28a(+)DNA; catalog number D48556, EMD Millipore (transferred from Oxford University to the Serum Institute of India Pvt. Ltd.) (3) Host cells: Rosetta(DE3)pLysS competent cells were used for TEV protease expression. Catalog number D49062, Sigma Aldrich (4) Plasmid*: pET-28a(+)DNA; catalog number D48556, EMD Millipore (transferred from Oxford University to the Serum Institute of India Pvt. Ltd.) (*The target gene is cloned / inserted into the pET-28a(+) plasmid backbone.) Example 1A: Expression of tagged fHbp protein We received 33 clone constructs expressing the fHbp-PorA chimeric protein from the University of Oxford. Of the 33 clones received, 24 were screened for protein expression, and the details are shown in Table 3. Of the 24 screened clones, 5 were finally selected for the vaccine formulations listed in Table 13 based on molecular and biochemical characterization. The sequence listings for the recombinant protein / modified fHbp fusion protein and TEV protease are provided in Table 13a.

[0476] [Table 13]

[0477] [Table 14]

[0478] The expression vector used for expressing fHbp-tagged proteins is pET28a-His-MBP-TEV-fHbp-PorA. Detailed vector maps and nucleotide sequences are provided in Table 14 and Figures 1-5 below.

[0479] [Table 15]

[0480] fHbp-tagged protein production was carried out in 10 L-scale fermentation batches. Details of the culture media used are shown in Tables 15-19.

[0481] Stock solutions for culture medium and feed preparation: (1) Base solution: 14% ammonia solution (2) Acid solution: 6% orthophosphate (3) Antifoaming agent solution: 10% antifoaming agent (STRUKTOL® J 673 A)

[0482] [Table 16]

[0483] [Table 17]

[0484] [Table 18]

[0485] Details of the supplied solution: L-methionine stock solution: 45 mg / mL L-methionine solution

[0486] [Table 19]

[0487] Inducer: Lactose monohydrate solution Figure 7 provides a seed development process for a 10 L scale fermentation batch for the production of tagged fHbp protein. Figure 8 provides a process for the production of tagged fHbp protein in a 0 L scale fermenter.

[0488] Each tagged fHbp protein was grown individually according to the process shown in Figures 7-8.

[0489] During the purification of tagged proteins derived from cell lysates, protein precipitation occurred in clones fHbp V3.45 M5:PorA 316–320 / exP1.14 and fHbp V2.19 M6:PorA 316–320 / exP1.4. To address this issue, the temperature setpoint was shifted from 37°C to 33°C during induction in the fermentation process.

[0490] The effects of the temperature shift were shown as follows:

[0491] Clone fHbp V2.19 M6:PorA 316-320 / exP1.4: Low-temperature induction (33°C) resulted in the expression of the major portion of the tagged fHbp protein in a soluble form.

[0492] By lowering the temperature by 4°C, the problem of protein precipitation during downstream processing was resolved.

[0493] Clone fHbp V3.45 M5:PorA 316―320 / exP1.14: Low-temperature induction (33°C) resulted in the majority of the expressed tagged fHbp protein being in a soluble form.

[0494] This problem is resolved to some extent, but issues still remain during downstream processing.

[0495] Figures 9a–9h provide growth profiles of tagged fHbp proteins in 10 L scale fermentation batches, along with SDS-PAGE gel images. Figures 9a and 9b show the growth profiles and SDS-PAGE gel images for fHbpV3.45 M5:PorA316–320 / exP1.14 (SEQ ID NOs. 1 and 6) in 10 L scale fermentation batches, respectively. Figures 9c and 9d show the growth profiles for fHbpV2.19 M6:PorA316–320 / exP1.4 (SEQ ID NOs. 2 and 7) in 10 L scale fermentation batches and SDS-PAGE gel images, respectively. Figures 9e and 9f show the growth profiles of 10 L scale fermentation batches and SDS-PAGE gel images for fHbpV1.14:PorA307-311 / exP1.9 (SEQ ID NOs. 3 and 8), respectively; Figures 9g and 9h show the growth profiles of 10 L scale fermentation batches and SDS-PAGE gel images for fHbpV1.1:PorA307-311 / exP1.4 (SEQ ID NOs. 4 and 9), respectively.

[0496] SDS-PAGE analysis (Figures 9b, 9d, 9f, and 9h) revealed that the majority (60kDa–80kDa) of all recombinant tagged fHbp proteins were expressed in a soluble form.

[0497] Example 1B: Expression of TEV protease The expression vector used for TEV protease expression is His-Gst-TEV protease (pET28a) (Kan / chloramphenicol). Detailed vector maps and nucleotide sequences are provided in Table 20 and Figure below.

[0498] [Table 20]

[0499] TEV protease production was performed in 10L scale fermentation batches. Details of the culture media used are shown in Tables 21-24.

[0500] Stock solutions for culture medium and feed preparation: (1) Base solution: 14% ammonia solution (2) Acid solution: 6% orthophosphate (3) Antifoaming agent solution: 10% antifoaming agent (STRUKTOL® J 673 A)

[0501] [Table 21] TIFF2026511179000024.tif109170

[0502] Details of the supplied solution: L-methionine stock solution: 45 mg / mL L-methionine solution

[0503] [Table 22]

[0504] Inducer: IPTG solution Figure 10 shows the process for developing seeds for a 10 L scale fermentation batch for TEV protease production. Figure 11 shows the production of TEV protease in a 10 L scale fermenter.

[0505] Protein precipitation occurred during the purification of TEV protease from cell lysates. To address this issue, the temperature setpoint was shifted from 37°C to 20°C during induction in the fermentation process, i.e., the cultures were induced at 20°C for 12–16 hours using IPTG.

[0506] The effects of the temperature shift were shown as follows: • The majority of the TEV protein expressed by low-temperature induction (20°C) was obtained in a soluble form. • Low-temperature induction (20°C) resolved the problem of protein precipitation during downstream processing. Since the culture was induced at 20°C, the induction time was extended to 12-16 hours.

[0507] Along with SDS-PAGE gel images, the growth profiles of TEV protease in a 10 L scale fermentation batch are provided in Figures 12a and 12b, respectively. SDS-PAGE analysis (Figure 12b) shows that the major portion of the TEV protease (50 kDa) was expressed in a soluble form. [Examples]

[0508] Purification of tagged fHbp protein and TEV protease The cells harvested in Example 1 are processed using the following general procedure: Cell lysis by chemical and mechanical means; Chromatography separation; One or more washing steps; Protein elution; Protein concentration and dialysis Protein storage until further use In what order are the materials used for purification?

[0509] When tagged proteins were expressed in inclusion bodies (IBs), further steps were performed, including ammonium sulfate precipitation, urea unfolding of the protein, and column refolding of the protein.

[0510] The purification process is described in detail in the following sections. Studies were conducted to optimize various parameters such as the concentration of the chimeric protein and excipients, and these are described in detail below.

[0511] Optimization research: We optimized parameters such as mechanical cell lysis and TEV protease cleavage.

[0512] (i) Homogenizer optimization: The recovered E. coli cell pellet (for expression of tagged fHbp protein and TEV protease) was dissolved in lysis buffer (25 mM sodium phosphate buffer + 100 mM NaCl + 20 mM imidazole, pH 7.4), and the cells were lysed for 6 cycles at 1000–1200 Bar using a Panda Homogenizer. The first cycle was performed without pressure, and the next 5 cycles were performed at a pressure of 1000–1200 Bar.

[0513] After each cycle, the dissolved material was collected and its optical density was determined at 600 nm using a spectrophotometer. The results are shown in Table 25.

[0514] [Table 23]

[0515] Conclusion: Engineering absorbance (OD) decreased only after pressurization in the first cycle. Further decreases in OD were observed up to cycle 3. Cycles 4 and 5 did not show significant differences in OD.

[0516] Considering resuspended samples with 100% viable cells and 0% lysis, the lysis ratio was calculated for all homogenization cycles, and the results are shown in Table 26 and Figure 13.

[0517] [Table 24]

[0518] Conclusion: From Table 26 and Figure 13, it can be seen that approximately 80% dissolution was achieved in Cycle 1, and this further decreased to approximately 98% by the end of Cycle 3.

[0519] Therefore, homogenization at a pressure of 1000-1200 bar for at least three cycles is necessary for the efficient lysis of E. coli cells.

[0520] It should be noted that this method uses both mechanical and chemical lysis to destroy cells. A lysis buffer with a higher osmotic pressure was used for chemical lysis, and a cell homogenizer was used for mechanical lysis.

[0521] Homogenizers are used to provide uniformity during large-scale (industrial / production scale; model-wise capacity 10 L / hour to several thousand L / hour) cell disruption and lysis, while sonication is used for small-scale (less than 100 ml) cell disruption.

[0522] (ii) TEV protease cleavage process: Enzyme: Optimization of substrate concentration: The TEV protease enzyme:substrate concentration was optimized using His-MBP tagged protein (fHbpV1.1:PorA 307-311 / exP1.4) (where the substrate is the tagged fHbp protein). Different ratios of 5, 10, 20, 30, and 40 parts substrate to 1 part TEV protease enzyme were studied. Samples were collected after incubation at 30°C for 1 hour with gentle mixing. The same reaction was then incubated at 30°C for 18 hours with gentle mixing, as in Example 2C. The results obtained by loading the TEV protease cleavage reaction product onto SDS-PAGE are shown in Figures 14a and 14b.

[0523] Furthermore, densitometry analysis (Figure 14b) was performed using the Biorad gel documentation system, measuring the intensity of the bands and calculating the ratio of the formed products, considering 100% cleavage at a 1:5 ratio. The results are shown in Table 27 and Figure 15.

[0524] [Table 25]

[0525] Conclusion: Table 27 and Figure 15 show that a higher degree of cleavage was observed after 1 hour of incubation using enzyme:substrate ratios of 1:5 and 1:10. However, after 18 hours of incubation, saturation was observed at 1:5, 1:10, and 1:20 ratios, i.e., approximately 95% and above of product formation was observed. Uncleaved substrate was observed at higher enzyme:substrate ratios of 1:30 and 1:40, with product formation of approximately 81% and 76%, respectively, suggesting that longer incubation times may be required for complete cleavage at these higher ratios. Therefore, it is found that for TEV cleavage, an enzyme:substrate ratio of 1:20, or 18 hours of incubation at lower enzyme:substrate ratios, is optimal.

[0526] Similar findings were observed for the remaining tagged fHbp proteins.

[0527] Temperature optimization and results for TEV protease cleavage experiments TEV protease cleavage at various temperatures from 5°C to 50°C was optimized using a His-MBP-tagged protein (FHBP V1.1:PorA 307-311 / exP1.4). Twenty parts of substrate were used for every one part of TEV protease enzyme. Samples were collected after 1 hour of incubation at various temperatures from 5°C to 50°C while gently mixing. As shown in Figure 16, the TEV protease cleavage reactions at various temperatures were loaded onto SDS-PAGE. Furthermore, densitometry analysis of Figure 16 was performed using the Biorad gel documentation system. Band intensity was measured, and the magnification change of the formed product was calculated, taking into account that the intensity of the formed product at the initial temperature of 5°C was a value of 1. The results are shown in Table 28 and Figure 17.

[0528] [Table 26]

[0529] Conclusion: From Table 28 and Figure 17, it can be seen that no cleavage was observed at 0 hours. After 1 hour, the formed product / chimera was higher at 30°C, which was 2.5 times higher than at 5°C. Therefore, the optimal temperature for TEV protease cleavage was found to be approximately 30°C.

[0530] Example 2 A: Purification of TEV protease The TEV protease recovered in Example 1B was purified using the process shown in Figure 18, and the lysis buffer had a pH of 7.4.

[0531] The isoelectric point (pI) of a protein is defined as the pH at which the net charge of the protein molecule is zero. At solution pH levels higher than pI, the protein surface is predominantly negatively charged, causing repulsion between similarly charged molecules. Similarly, at solution pH levels lower than pI, the protein surface is predominantly positively charged, leading to repulsion between proteins. However, at pI, the negative and positive charges balance out, reducing electrostatic repulsion, and attraction becomes dominant, resulting in aggregation and precipitation.

[0532] Since the pI of HIS-GST-TEV protease is 7.8, the desired purity could not be obtained at a working pH of 7.4. Therefore, the working pH was increased to 8.5, as shown in the process in Figure 19, to improve the purity of HIS-GST-TEV protease.

[0533] Figures 20a and 20b show the purification profiles of TEV protease at pH 8.5 and pH 7.4, respectively. Lanes marked with an asterisk (★) represent the final elution fraction of TEV protease. The purity of the TEV protease produced using the pH 8.5 process is higher than that of the TEV protease produced using the pH 7.4 process.

[0534] Example 2B: Purification of tagged fHbp protein The tagged fHbp proteins recovered in Example 1A were individually purified according to Figure 21.

[0535] Tagged fHbp proteins are soluble at the working pH used for purification, and the process described above (Figure 21) yields tagged fHbp proteins of the desired purity. The purification process shown in Figure 21 was used for the purification of fHbpV3.45 M5:PorA316-320 / exP1.14, fHbpV2.19 M6:PorA316-320 / exP1.4, fHbpV1.14:PorA307-311 / exP1.9, fHbpV1.1:PorA307-311 / exP1.9, and fHbpV1.1:PorA307-311 / exP1.4.

[0536] However, during scale-up, rapidly expressed tagged fHbp proteins may have solubility issues due to protein aggregation into inclusion bodies (IBs). IBs are formed as a result of aggregation of partially folded and misfolded protein molecules.

[0537] To avoid the solubility problem and recover the protein expressed from IB, an alternative process involving urea (Figure 22) can be used.

[0538] Inclusion bodies are known to contain protein molecules with natural-like conformations, and some inclusion bodies exhibit significant biological activity.

[0539] In the alternative method (Figure 22), the tagged protein expressed as IB was solubilized with 8M urea and then conjugated to a Ni-Sepharose 6 FF column for on-column refolding and purification.

[0540] Urea was removed from the column by reducing its concentration from 8 M to 1 M in the first step. Subsequently, urea was completely removed from the column and from the recombinant protein / modified fHbp fusion protein bound to the column by washing over 5-6 column volumes with 25 mM sodium phosphate buffer + 100 mM NaCl, pH 7.4.

[0541] Example 2 C: Process for tag removal from purified tagged fHbp protein The His-MBP tag was removed from purified protein (tagged fHbp protein) using the TEV protease enzyme (encoded by SEQ ID NO: 12 or SEQ ID NO: 11) according to the process shown in Figure 23.

[0542] After removing the tag from the fHbp protein, the chimeric protein obtained in the digestion mixture was further purified by two-step chromatography using ion exchange chromatography (Figure 24) followed by affinity chromatography (Figure 25).

[0543] Table 29 provides characterizations of chimeric proteins produced using the method disclosed herein.

[0544] [Table 27]

[0545] fHbp specifically binds to human H factor (fH), which downregulates complement activation and enhances resistance to bactericidal activity. Modifications (multiple modifications) introduced into the fHbp antigen can reduce fH binding and enhance the protective antibody response.

[0546] Table 29 shows that recombinant fHbp has over 85% less fH binding compared to wild-type fHbp, which improves the immunogenicity of vaccine formulations containing recombinant fHbp.

[0547] Previously, multiple chromatography steps, including metal affinity chromatography, cation exchange, anion exchange, and gel filtration chromatography (GFC), were used to purify digested mixtures. In contrast, the present method uses two chromatography steps (anion exchange and metal affinity chromatography) after TEV protease-mediated tag removal. Chimeric proteins produced using the two chromatography steps of this disclosure exhibited similar characteristics and purity profiles to those produced using multi-step chromatography. [Examples]

[0548] Preparations containing recombinant proteins / modified fHbp fusion proteins / chimeric proteins Different formulations of recombinant protein / modified fHbp fusion protein were prepared and evaluated to obtain formulations with optimal physicochemical properties, stability, and immunogenicity.

[0549] (i) Adjuvant optimization: Formulation development began with adsorbing proteins onto different adjuvants, such as aluminum hydroxide (alkyl hydrogel), aluminum phosphate (adjuphos), and double mutagenic thermotonic toxin (dmLT), to enhance immunogenic response and physicochemical properties. These formulations were evaluated based on different physicochemical parameters. The summary of the tests, results, and conclusions are presented below.

[0550] [Table 28]

[0551] Conclusion: Three adjuvants, dmLT, alpha-hydrogel, and adjuphos, were evaluated. Table 30 shows that the zeta potentials of the alpha-hydrogel and adjuphos adsorbents were more satisfactory than those of the dmLT adsorbent.

[0552] The isoelectric point (pI) of the alpha-hydrogel is approximately 11.4, while the pI value of adjuphos is between 4.5 and 6.0. The pI of all fHbp proteins is in the range of 5.5 to 6.5. Therefore, the alpha-hydrogel was used in further studies to achieve optimal adsorption of all proteins on the adjuvant in the vaccine formulation.

[0553] (ii) Optimization of buffer and sugar:

[0554] [Table 29]

[0555] Table 31 shows that when sodium chloride and sucrose were used in the formulation, the desired zeta potential could not be obtained in batches 051021-A and 051021-B. Sucrose was used in the initial stages of composition formulation. However, since optimal physicochemical properties could not be obtained in compositions containing sucrose, it was not included in subsequent compositions.

[0556] When phosphate buffer was added to the formulation (batch number 061021), an improvement in zeta potential was observed, but the pH value was not within the expected range. Further studies were conducted using mannitol and phosphate buffer. Details of the formulation and the results obtained are summarized in Table 32.

[0557] [Table 30]

[0558] Table 32 shows that when mannitol and phosphate buffer were used in the formulation, the pH, osmotic pressure, and zeta potential were within the expected range. It was also observed that increasing the mannitol concentration from 4% to 5% resulted in the optimal osmotic pressure of the formulation.

[0559] (iii) Individual chimeric protein preparations: Similar studies were conducted on the remaining proteins. Details of the formulations and the results obtained are summarized in Table 33.

[0560] [Table 31]

[0561] Table 33 shows that by using the formulations provided, which include mannitol (5% v / v) and polysorbate 20 (0.05 mg / mL), optimal physicochemical parameters were obtained for all individual proteins.

[0562] (iv) Formulations containing multiple chimeric proteins: The physicochemical properties of formulations including those with combined protein formulations were determined, as with individual protein formulations. Details of the formulations and the results obtained are summarized in Table 34. Formulations were obtained by adsorbing individual proteins onto adjuvants and then adding them to an excipient mixture.

[0563] [Table 32]

[0564] Table 34 shows that the formulation remained stable when stored at 2-8°C for approximately 7 days. Furthermore, the pH, zeta potential, and adsorption percentage remained stable. Therefore, this combination of excipients in the formulation was adopted for further scale-up.

[0565] (v) Optimization of chimeric protein concentration: To determine the effects on pH, osmotic pressure, and zeta potential, studies were conducted using higher concentrations of chimeric protein. Details of the formulation and the results obtained are summarized in Tables 35a and 35b.

[0566] [Table 33]

[0567] Tables 35a and 35b show that the high-protein formulation using phosphate buffer, polysorbate 20, and 5% mannitol provided optimal pH, osmotic pressure, and zeta potential.

[0568] (vi) Optimization of excipient concentration: Studies were conducted to optimize the concentration of excipients. The details of the formulation and the results obtained are summarized in Tables 36-39.

[0569] [Table 34] TIFF2026511179000038.tif164170 TIFF2026511179000039.tif253170

[0570] Tables 36-39 show that the optimal physicochemical properties can be obtained by using a 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 in combination with each chimeric protein at a concentration of 120 μg / mL.

[0571] (vii) pH optimization: A study was conducted to investigate the effect of pH on adsorption. Details of the formulation and the results obtained are summarized in Tables 40 and 41.

[0572] [Table 35] TIFF2026511179000041.tif155170

[0573] Tables 40-41 show that increasing the pH of the phosphate buffer to 7.4 resulted in an increase in the pH of the final formulation (7.83-7.86). Furthermore, as the pH of the phosphate buffer increased, protein adsorption from 94% to 85% decreased.

[0574] [Table 36] TIFF2026511179000043.tif191170

[0575] Tables 42-43 show that when a phosphate buffer with a pH of 7.0 was used in the formulation, the final pH ranged from 7.3 to 7.5, indicating improved protein adsorption. [Examples]

[0576] Stability testing of vaccine formulations containing recombinant protein / modified fHbp fusion protein Stability tests were conducted on two batches at 2–8°C for 6 months and 10 months. Details of the formulation and the results obtained are summarized in Table 44.

[0577] [Table 37]

[0578] Conclusion: Table 44 shows that the protein adsorption %, pH, zeta potential, and PSD of vaccine formulations containing recombinant protein / modified fHbp fusion protein remain within the desired range for up to 6 months at 2–8°C. [Examples]

[0579] Immunogenicity testing of recombinant / chimeric protein formulations Details of the biological materials used are shown below.

[0580] Meningococcal serotype B strain: The glycerol stock of Neisseria meningitidis was received from Professor Chris Tang (Sir William Dunn School of Pathology, UK). We received the Neisseria meningitidis serotype B4 strain. Strain details are provided below and further summarized in Table 45.

[0581] 1. Neisseria meningitidis M08.240157 (fHbp V1.1:PorA VR2 16) 2. Neisseria meningitidis M17, Sequence ID No. 240832 (fHbp 1.4:PorA VR2 4) 3. Neisseria meningitidis M17, Sequence ID No. 240156 (fHbp 3.45:PorA VR2 14) 4. N. meningitidis M18 240043 (fHbp 1.15:PorA VR2 15-11)

[0582] [Table 38]

[0583] Table 46 summarizes the details of the formulations used in the immunogenicity tests.

[0584] [Table 39]

[0585] Serum sterilization assay hSBA procedures for Men-B shares Preparation using working assay stocks for SBA The master stock vial was removed from the freezer, and the culture was rapidly streaked onto a pre-dried blood agar plate (Columbia blood agar containing 5% horse blood). The plate was incubated overnight at 37°C in 5% CO2. Approximately 50 colonies were picked up using a sterile loop and resuspended in a sterile 50 ml centrifuge tube containing 15 ml of BHI broth with glycerol (thus creating a heavy suspension). The suspension was divided equally into 1.8 ml cryovials of 0.25 ml each. The labeled vials were stored in a freezer at -70°C.

[0586] Assay procedure for serum bactericidal assay for titration of antibodies against Men B strain: Meningococcal strain B (working stock) was streaked onto a single blood agar plate and incubated overnight at 37°C with 5% CO2. The culture was streaked again onto a fresh blood agar plate in a square shape in the center of the plate for confluent growth. The plates were incubated for 4 hours (+15 minutes) in a 37°C CO2 incubator with 5% CO2 for all Men B strains. The growth swept from the center of the plate was treated with Ca +2 and Mg +2A suspension was prepared by suspending the sample in 5 ml of Hanks equilibrium salt solution / buffer (Invitrogen) containing the sample. Absorbance at 650 nm was read using 1 ml of the suspension. The suspension was adjusted by diluting it to 650 nm = 0.1 OD, then by diluting it 1 / 10 and then 1 / 250 with buffer. 20 μl of buffer was added to all wells up to row 11 of a 96-well microtiter plate. 20 μl of (diluted or undiluted) test serum sample was added to the first row of the plate; the last two rows were reserved for quality control serum. 20 μl was serially diluted from row 1 to row 2, up to row 9, and 20 μl from row 9 onwards was discarded. Columns 10 and 11 were reserved for complement control. 10 μl of the already prepared microbial suspension was added to all wells up to row 11. 10 μl of human complement was added to all wells up to row 10. 10 μl of heat-inactivated (56°C for 30 minutes) human complement was added to all wells of the 11th column. The contents of the wells were thoroughly mixed, and the plate was incubated at 37°C for 1 hour. After incubation, 10 μl of the contents of each well was spotted onto a suitably labeled blood agar plate. The blood agar plate was incubated in a 37°C CO2 incubator containing 5% CO2. The following day, bacterial colonies were counted using an automated colony counter (Synbiosis-ProtoCOL3).

[0587] Research details: Immunogenicity studies were conducted in rabbits (New Zealand White) using three formulations / groups: G1-Tolmenba® (Pfizer), G2-Formulation 1, and G3-Formulation 2. Each formulation was injected into four male and four female rabbits. Injections were performed on days 0, 14, and 28, and blood samples were collected on days D0 and D37. Serum samples were analyzed for immunogenicity using hSBA (human complement serum bactericidal assay). All rabbits were given the full human dose.

[0588] Drug product: Meningococcal B vaccine (MenB) (250423 - Formulations 1 and 2) Research Design: The research design is summarized in Table 47.

[0589] [Table 40]

[0590] Criteria for statistical significance between groups: A twofold change in titer is considered assay variation, and a difference of >4 times in titer between comparison groups is considered statistically significant.

[0591] Purpose and scope: The purpose of this study was to compare the immunogenicity response of Tormenba® to individual MenB formulations—Formulation 1 and Formulation 2—and MenB strains—M 08240157 (fhbp 1.1), M 17240832 (fhbp 1.4), M 17240156 (fhbp 3.45), and M 18240043 (fhbp 1.15) hSBA. The results are shown in Table 48.

[0592] [Table 41]

[0593] Figure 26 provides a graph displaying individual data points and GMT for the blood collection date, with each point highlighted.

[0594] hSBA data show that the formulations containing the recombinant protein / modified fHbp fusion protein of this disclosure (Formulation 1 / Quad 1 and Formulation 2 / Quad 2) are highly immunogenic and can induce protective SBA titers against different strains of Neisseria meningitidis (M 08240157, M 17240832, M 17240156 and M 18240043).

[0595] (i) ELISA to evaluate the binding of human complement factor H to the fHbp-PorA chimeric protein compared to recombinant wild-type Men B samples. the purpose: The purpose of this study is to describe a method for evaluating the binding of human complement factor H to fHbp-PorA chimeric protein compared to recombinant wild-type Men B samples at the Monoclonal Antibody IPQC Laboratory (MAb IPQC).

[0596] procedure: The necessary chemical substances and reagents are shown in Table 49.

[0597] [Table 42]

[0598] For all of the above chemical substances, equivalent products from other brands can also be used.

[0599] Required equipment: An ELISA plate reader capable of measuring wavelengths of 450 nm and 630 nm. Microplate washer An incubator that can achieve and maintain a temperature of 37°C. refrigerator Required consumables: NUNC-Immunoplate, manufactured by Thermo Scientific; catalog number: 442404 or equivalent.

[0600] Plate sealing tape Reagent reservoir 50 mL Applicable glassware Micropipettes (single-channel and multi-channel) and their respective tips Vortex mixture Microcentrifuge tubes - 15 mL and 50 mL Preparation of reagents and solutions: Coating buffer: 1×PBS pH 7.4 Dissolve two PBS tablets in 800 μL of WFI, then increase the volume to 1 L. Store at room temperature (RT) and use one month before the preparation date.

[0601] Wash buffer: 1X PBST (0.05% Tween20 in 1X PBS, pH 7.4) Prepare 1 L of PBS as described above. Add 0.5 mL of Tween 20 solution to 999.5 mL of 1×PBS and mix using a magnetic stirrer. Store at room temperature (RT) and use one month before the preparation date. This 1×PBST can also be used as a blocking solution and a diluent for antibody preparation.

[0602] Blocking: 4% BSA in PBST Accurately weigh 4.0 g of BSA and dissolve it in 100 mL of 1 × PBST.

[0603] TMB solution: binary mixture Accurately measure equal volumes of solutions A and B (provided as colorimetric reagents). Equilibrate to RT (store in the dark). Prepare a fresh solution and gently mix before use.

[0604] Note: The above procedures for preparing reagents and solutions are for illustrative purposes only. The actual volume / amount of chemicals / reagents should be calculated according to the number of assay plates.

[0605] method: Antigen coating: The side of the 96-well plate displays antigen details, the analyst's initials, and the date the plate was coated.

[0606] Determine the volume of each antigen and coating buffer required to coat the desired number of plates.

[0607] Each well of an ELISA plate is coated with 5.0 μg of antigen (fHbp-PorA chimeric) diluted to a final concentration of 0.1 μg / μL in phosphate-buffered saline (1×PBS). Using a multichannel pipette, 50 μL of the diluted antigen is added to each well of a 96-well Nunc flat-bottom plate.

[0608] As a control, only 50.0 μL of PBS is added to the wells.

[0609] Incubate the sealed plate overnight at 2-8°C.

[0610] Cleaning: Remove the coated 96-well microtiter plate, place the plate in a microplate washer, and perform the washing procedure by selecting a prefeed program for three washes.

[0611] For manual washing, empty all wells by discarding the contents into the sink, add 300 μL / well of washing buffer / 1 X PBST, immerse for a few seconds, and empty all wells into the sink. Gently tap an unbound tissue paper to ensure all wells are completely empty. Repeat the washing process twice.

[0612] blocking: The following day, unbound antigens are removed by washing the plate three times with 300 μL / well of 1×PBST.

[0613] Block the wells with 200 μL of 4% BSA in PBST. Incubate the plate at 37°C for 1 hour.

[0614] Addition of human complement factor H: Unbound BSA is removed by washing the plate three times with 300 μL / well of 1×PBST.

[0615] The human complement factor H dilution scheme is summarized in Table 50.

[0616] [Table 43]

[0617] Add 50.0 μL of factor H diluted to an appropriate final concentration (according to the dilution scheme shown in Table 50, the following are the final concentrations of factor H in 50.0 μL: 0.0 μg, 0.001 μg, 0.005 μg, 0.01 μg, 0.05 μg, 0.1 μg, 0.2 μg, 0.3 μg, 0.4 μg, 0.5 μg, and 1.0 μg of protein), and incubate the plate at 37°C for 1 hour.

[0618] Addition of primary antibody: Unbound factor H is removed by washing the plate three times with 300 μL / well of 1×PBST.

[0619] Add 50.0 μL of OX24 mAb diluted to 1 / 10,000 in PBST, and incubate the plate at 37°C for 1 hour.

[0620] Addition of secondary antibody: Unbound primary antibody (OX24 mAb) is removed by washing the plate three times with 300 μL / well of 1×PBST.

[0621] Add 50.0 μL of secondary antibody diluted to a final concentration of 1 / 10,000 in PBST, and incubate the plate at 37°C for 1 hour.

[0622] Addition of TMB substrate: Unbound secondary antibodies are removed by washing the plate three times with 300 μL / well of 1×PBST.

[0623] The plate is colored by adding 100.0 μL of chromogenic reagent (the chromogenic reagent in a 1:1 mixture of solution A and solution B). The plate is incubated at 25°C / RT for 20 minutes, and then stopped by adding 50.0 μL of stop solution, which causes the reactants to turn yellow.

[0624] The absorbance of the colored wells is read on the plate reader at 450 nm and 630 nm. Normalized absorbance (A450 nm–A630 nm) is used to plot graphs in Excel and Graph-pad PRISM.

[0625] Figure 27 and Table 51 show representative data on the binding of human complement factor H to wild-type fHbp compared to Men B chimeric protein.

[0626] [Table 44]

[0627] Conclusion: Table 51 and Figure 27 show that the recombinant protein / modified fHbp fusion protein of the present invention exhibits a reduction of more than 80% in human complement factor H binding compared to the wild-type fHbp protein. [Examples]

[0628] Combination formulations including recombinant protein / modified fHbp fusion protein preparations Physicochemical characterization of combination formulations: The vaccine formulation of the present invention, containing a recombinant protein / modified fHbp fusion protein derived from Neisseria meningitidis serotype B, was found to be stable and immunogenic. Furthermore, combination formulations including the vaccine formulation were studied with additional Neisseria meningitidis serotypes to determine their effects on physicochemical characteristics, stability, and immunogenicity.

[0629] The applicant's MenFive formulation, containing a polysaccharide-protein complex against meningococcal serotype ACYWX, was used in the study. Lyophilized MenFive vaccine was reconstituted with the MenB formulation and stored at room temperature for 2 hours to study the effect on the physicochemical parameters of the entire reconstituted solution. This study evaluated the stability of the reconstituted solution during injection into animals to simulate routine clinical practice.

[0630] MenB and MenFive formulations from batch number 090823 (batch number 2352M001: 5-dose lyophilized vials were used in this study, with each antigen (A, C, Y, W, X) = 32 μg; sucrose = 15 mg; trisodium citrate = 2.5 mg; Tris buffer = 0.61 mg) being used in the tests. The test parameters and results obtained are summarized in Table 52.

[0631] [Table 45]

[0632] Conclusion: Table 52 shows that the reconstituted solution was physicochemically stable for 2 hours, and no particle aggregation / sedimentation was visually observed.

[0633] Therefore, further studies were planned to determine the immunogenicity and non-interference of the MenFive + MenB combination formulation.

[0634] Immunogenicity and non-interference studies of combination formulations: Details of the biological materials used are shown below.

[0635] Meningococcal serotype B strain: The meningococcal serotype B strain used was the same as the one used in Example 5.

[0636] Meningococcal serotypes A, C, W, Y, and X) strains: Glycerol stocks of meningococcal strains were received in July 2012 from Professor Ray Borrow (Health Protection Agency, Manchester Laboratory, UK). The five serotypes of meningococcal strains were obtained as follows:

[0637] 1. Meningococcal serotype A, F8238 2. Meningococcal serotype C11, M05 240852 3. Meningococcal serotype W135, M01 0240070 4. Meningococcal serotype X, BF2 / 97 5. Meningococcal serotype Y, M03 0241125 hSBA procedures for Men-B shares Preparation using working assay stocks for SBA The master stock vial was removed from the freezer, and the culture was rapidly streaked onto a pre-dried blood agar plate (Columbia blood agar containing 5% horse blood). The plate was incubated overnight at 37°C in 5% CO2. Approximately 50 colonies were picked up using a sterile loop and resuspended in a sterile 50 ml centrifuge tube containing 15 ml of BHI broth with glycerol (thus creating a heavy suspension). The suspension was divided equally into 1.8 ml cryovials of 0.25 ml each. The labeled vials were stored in a freezer at -70°C.

[0638] Assay procedure for serum bactericidal assay for titration of antibodies against Men B strain: Meningococcal strain B (working stock) was streaked onto a single blood agar plate and incubated overnight at 37°C with 5% CO2. The culture was streaked again onto a fresh blood agar plate in a square shape in the center of the plate for confluent growth. The plates were incubated for 4 hours (+15 minutes) in a 37°C CO2 incubator with 5% CO2 for all Men B strains. The growth swept from the center of the plate was treated with Ca +2 and Mg +2A suspension was prepared by suspending the sample in 5 ml of Hanks equilibrium salt solution / buffer (Invitrogen) containing the sample. Absorbance at 650 nm was read using 1 ml of the suspension. The suspension was diluted to 650 nm = 0.1 OD, then adjusted by diluting 1 / 10 and then 1 / 250 with buffer. 20 μl of buffer was added to all wells up to row 11 of a 96-well microtiter plate. 20 μl of (diluted or undiluted) test serum sample was added to the first row of the plate; the last two rows were reserved for quality control serum. 20 μl was serially diluted from row 1 to row 2, up to row 9, and 20 μl from row 9 onwards was discarded. Columns 10 and 11 were reserved for complement control. 10 μl of human complement was added to all wells up to row 10. 10 μl of human complement was added to all wells up to row 10. 10 μl of heat-inactivated (56°C for 30 minutes) human complement was added to all wells of the 11th column. The contents of the wells were thoroughly mixed, and the plate was incubated at 37°C for 1 hour. After incubation, 10 μl of the contents of each well was spotted onto a suitably labeled blood agar plate. The blood agar plate was incubated in a 37°C CO2 incubator containing 5% CO2. The following day, bacterial colonies were counted using an automated colony counter (Synbiosis-ProtoCOL3).

[0639] hSBA procedure for MenFive serotypes (A, C, W, Y, and X): hSBA procedures for Men-B shares Preparation using working assay stocks for SBA The master stock vial was removed from the freezer, and the culture was rapidly streaked onto a pre-dried blood agar plate (Columbia blood agar containing 5% horse blood). The plate was incubated overnight at 37°C in 5% CO2. Approximately 50 colonies were picked up using a sterile loop and resuspended in a sterile 50 ml centrifuge tube containing 15 ml of BHI broth with glycerol (thus creating a heavy suspension). The suspension was divided equally into 1.8 ml cryovials of 0.25 ml each. The labeled vials were stored in a freezer at -70°C.

[0640] Assay procedure for serum bactericidal assay for titration of antibodies against MenFive serotypes (A, C, W, Y, and X): Meningococcal serotypes A, C, W, Y, and X, working stocks, were streaked onto single blood agar plates and incubated overnight at 37°C with 5% CO2. The cultures were streaked again onto fresh blood agar plates in a square shape in the center of the plate for confluent growth. The plates were incubated for 4 hours (+15 mins) in a CO2 incubator at 37°C with 5% CO2 for serotypes C, W, Y, and X, and for 3 hours (+15 mins) for serotype A. The cultures swept from the center of the plate were streaked with Ca23 for serotypes A, Y, and X. +2 and Mg +2 A suspension was prepared by suspending the serum in 5 ml of Hanks equilibrium buffer (Hanks equilibrium Mgwith solution, Invitrogen), and for serotypes C and W, in Ca and salt BSA. Absorbance at 650 nm was read using 1 ml of the suspension. The suspension was diluted to 650 nm = 0.1 OD, then adjusted by diluting 1 / 10 and then 1 / 250 with buffer. 20 μl (diluted or undiluted) test serum sample was added to the first column of the plate; the last two columns were reserved for quality control serum. 20 μl was serially diluted from column 1 to column 2, up to column 9, and 20 μl from column 9 onwards was discarded. Columns 10 and 11 were reserved for complement control. 10 μl of thermo-inactivated human complement was added to all wells up to column 11. 10 μl of thermo-inactivated human complement was added to all wells in column 10. 10 μl of heat-inactivated human complement was added to all wells of the 11th column. The contents of the wells were thoroughly mixed, and the plate was incubated at 37°C for 1 hour. After incubation, 10 μl of the contents of each well was spotted onto a suitably labeled blood agar plate. The blood agar plate was incubated in a 37°C CO2 incubator containing 5% CO2. The following day, bacterial colonies were counted using an automated colony counter (Synbiosis-ProtoCOL3).

[0641] Research details: Immunogenicity studies were conducted in rabbits (New Zealand White) using five formulations / groups (G1 - placebo, G2 - MenFive, G3 - MenFive + MeNB - formulation 1, G4 - MenFive + MeNB - formulation 2, and G5 - Menfive + Tormenba). Each formulation was injected into four male and four female rabbits. Injections were given on days 0, 14, and 28, and blood samples were collected on days D0, D28, and D41. Serum samples were analyzed for immunogenicity using hSBA (human complement serum bactericidal assay). All rabbits were given the full human dose.

[0642] Preparation: Meningococcal (A-TT, C-CRM, Y-CRM, W-CRM, X-TT) polysaccharide complex vaccine - reconstituted with meningococcal B vaccine (MenB) in 5 dose vials (Men5) (2352M001) (090823 - Preparations 1 and 2) Research design: The research design is shown in Table 53.

[0643] [Table 46]

[0644] Criteria for statistical significance between groups: A twofold change in titer is considered assay variation, and a difference of >4 times in titer between comparison groups is considered statistically significant.

[0645] Purpose and scope: The objective of this study was to compare the immunogenicity responses of individual MenFive+MenB and mixed formulations against MenFive serotypes (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 M 18240043 (fhbp 1.15)—using hSBA. This scope also included checking for any inhibition / cross-protection in the response between the two individual vaccine formulations (MenFive and MenB). The results obtained are summarized in Tables 54-55 and Figures 28-29.

[0646] [Table 47]

[0647] Conclusion: Based on the results obtained in Tables 54-55 and Figures 28-29, the following observations were made.

[0648] Inferences regarding MenFive serotypes: The titers obtained for all five serotypes (A, C, W, Y, and X) in the combination formulations (MenFive+ formulation 1 and MenFive+ formulation 2) were comparable (within 2 times) to those of the individual MenFive mono-formulations, suggesting no loss of immunogenic response when mixed with the MenB component. SBA titers increased slightly in number, but no decrease was observed for any of the serotypes.

[0649] Inference regarding MenB serotypes: The titer of the MenFive+MenB preparation against MenB strains showed a more than fourfold increase in titer when comparing pre-(D0) versus post-(D41) titers, thus suggesting improved and acceptable immune response generation against four different strains of MenB.

[0650] The results suggest that the combination of MenFive and Men-B formulations does not result in any loss of immunogenic response, and therefore indicate no interference with either vaccine component of the combination formulation. [Examples]

[0651] Quantification of recombinant protein / modified fHbp fusion protein using ELISA The bicinchoninic acid (BCA) method is a standard technique for estimating the total recombinant protein / modified fHbp fusion protein content in the final vaccine formulation.

[0652] The vaccine formulation of the present invention contains more than one recombinant protein / modified fHbp fusion protein. BCA provides quantification of the total recombinant protein / modified fHbp fusion protein, but cannot provide the individual content of each protein present in the vaccine formulation.

[0653] As an alternative method, ELISA was used to quantify the content of individual recombinant proteins / modified fHbp fusion proteins in the vaccine formulation. This test can also be used as a batch release test.

[0654] Table 56 summarizes the materials used in ELISA for quantifying individual recombinant proteins / modified fHbp fusion proteins.

[0655] [Table 48]

[0656] Assay procedure: STEP-1 (Capture Antibody Coating): Dilute JAR-41 or JAR-5 antibody in PBS with 0.1% BSA, and use 0.5 μg to 10 μg per well for coating. Load 100 μL per well onto a 96-well plate. Coating incubation is 2.5 to 18 hours at room temperature or 2 to 8°C.

[0657] Step 2 (Blocking): Add 300-320 μL of 1% BSA in PBS to a 96-well plate and incubate at room temperature for 1-2.5 hours.

[0658] STEP 3 (Adding Standard / Test Sample): Dilute the reference standard chimera sequentially with 0.1% BSA in PBS from 0.5 mg / mL to 1 ng / mL, and add 50 μL to 150 μL to each well. Dilute the test sample with 0.1% BSA in PBS until 50 μL to 150 μL is loaded and falls within the quantification range. After adding both the standard and test sample, incubate at room temperature for 0.5 to 2.0 hours.

[0659] STEP 4 (Adding Detection Antibody): Dilute the detection antibody (anti-POR-A P1.14 mAb / anti-POR-A P1.4 mAb / anti-POR-A P1.9 mAb / JAR-11 mAb) to a dilution ratio between 100-fold and 100,000-fold, and load 100 μL per well into a 96-well plate. Incubation of the detection antibody is 0.5 to 2.0 hours at room temperature.

[0660] STEP 5 (Addition of secondary detection antibody): Dilute the secondary detection antibody (goat anti-mouse HRP antibody) to a concentration between 100-1 and 100,000-1, and load 100 μL per well onto a 96-well plate. The secondary detection antibody incubation is 0.5 to 2.0 hours at room temperature.

[0661] STEP-6 (Substrate Addition & Stop): Add 100 μL of TMB substrate to each well and incubate at room temperature for 10-45 minutes. Then, add 100 μL of 1N HCl to each well to stop the process. Immediately read the absorbance of the plate at 450 / 630°C. [Examples]

[0662] Protein prediction in recombinant protein / modified fHbp fusion proteins using BCA the purpose: The objective of this study is to provide a procedure for the estimation of proteins in Men B samples using the microbicinchoninic acid (BCA) method in 96-well plates.

[0663] BCA is a method for quantifying the concentration of protein in a sample. Under alkaline conditions, Cu 2+ It reacts with proteins to form Cu + This generates Cu + It reacts with bicinconic acid to form a purple complex. After incubation at 37°C, the color develops. The colored complex is detected at 562 nm using a spectrophotometer.

[0664] Materials and equipment: Micro BCA Protein Assay Kit (Thermoscientific, 23235) (a) Micro BCA reagent A (MA) (b) Micro BCA reagent B (MB) (c) Micro BCA Reagent C (MC) (d) Bovine serum albumin standard ampoule (2 mg / ml) Water for injection (WFI) Microtube (1.5 mL Eppendorf tube) Pipettes and tips 96-hole plate 96-hole plate leader (TECAN, Infinite M200) Plate incubator (Biosan Thermoshaker) Test procedure: Preparation of standards and working reagents: Set the incubator to 37°C.

[0665] Prepare a 200 μg / ml stock solution from the BSA standard (2 mg / ml).

[0666] Using a 200 μg / ml stock solution, create a calibration curve as shown in Table 57 and Figure 30. Create a standard curve each time the assay is performed.

[0667] Prepare the working reagent by mixing 25 parts of microBCA reagent MA and 24 parts of reagent MB with 1 part of reagent MC (25:24:1).

[0668] [Table 49]

[0669] Sample preparation: The MEN-B formulation samples were analyzed as follows: (a) the sample without centrifugation, (b) the supernatant after centrifugation, and (c) the pellet after centrifugation.

[0670] (a) Sample without centrifugation: The formulation sample is collected either as is or diluted to fit the standard curve.

[0671] (b) Supernatant: Take 500 μl of each sample and centrifuge at 8000 rpm for 5 minutes. After centrifugation, carefully transfer the supernatant to a new tube. Take the collected supernatant as a neat sample or dilute it to fit the curve.

[0672] (c) Pellet: The pellet formed after centrifugation is resuspended by adding 500 μl of WFI. These samples are diluted in the same manner as the sample itself (a).

[0673] For each sample and standard, prepare dilutions in two batches.

[0674] Transfer 150 μl of the standard substance and sample to the plate well.

[0675] Add 150 μl of working reagent to each well and mix the plate thoroughly on a plate shaker for 30 seconds.

[0676] Cover the plate and incubate at 37°C for 2 hours.

[0677] The absorbance was measured at 562 nm, and the results are shown in Table 58.

[0678] [Table 50]

[0679] The adsorption percentage was calculated as a percentage obtained by subtracting the concentration of the supernatant sample from 100 relative to the concentration of the same sample without centrifugation. The adsorption rates are shown in Table 59.

[0680] [Table 51]

Claims

1. A modified H factor-binding protein (fHbp) comprising a wild-type fHbp mutant and at least one exogenous loop, wherein: - The modified H factor-binding protein (fHbp) is selected from an amino acid sequence that has at least 75% identity with one of the sequences of SEQ ID NOs: 6 to 10; - The at least one exogenous peptide loop is immunogenic; - The at least one exogenous peptide loop is derived from a bacterial membrane protein; - The modified fHbp is a fusion protein; - The fHbp mutant is selected from v1, v2, and v3, which are modified with at least one PorA loop containing at least 10 amino acids inserted into the β-turn region of fHbp; and - The PorA loop is selected from VR1 and VR2. Modified H factor binding protein.

2. The modified H factor binding protein according to claim 1, wherein the modified fHbp is modified to reduce H factor binding activity.

3. A nucleic acid sequence encoding a modified fHbp that has at least 75% identity with one of the sequences of sequence numbers 1 to 5.

4. An immunogenic composition comprising a nucleic acid sequence encoding at least one modified fHbp according to claim 1 or 2, or a modified fHbp according to claim 3.

5. The immunogenic composition according to claim 4, comprising two or more different modified fHbp.

6. The immunogenic composition according to claim 4 or 5, comprising a pharmaceutically acceptable carrier.

7. Furthermore, the immunogenic composition according to any one of claims 4 to 6, further comprising an adjuvant.

8. Furthermore, the immunogenic composition according to any one of claims 4 to 7 comprises at least one further prophylactic or therapeutically active molecule, including a monovalent carrier protein:capsular polysaccharide conjugate vaccine.

9. The immunogenic composition according to any one of claims 4 to 8, wherein the fHbp scaffold having the exogenous peptide loop is incorporated into the complex vaccine as a protein carrier molecule.

10. below: —Protein antigens derived from PorB, FeA, OmpC, NHBA, NadA, meningococcal antigen 287, NspA, HmbR, NhhA, App, and 936; —Sugar or complex antigens derived from serotypes A, C, W, Y and / or X of Neisseria meningitidis; —Sugar or complex antigen derived from Streptococcus pneumoniae; —Diphtheria antigens such as diphtheria toxin, e.g., CRM197 variants; —Tetanus antigens such as tetanus toxin; —Antigen derived from Bordetella pertussis, cell-free pertussis antigen, or whole-cell pertussis antigen; —Sugar or complex antigen derived from Haemophilus influenzae B; —Polio antigens such as IPV; —Antigens of measles, mumps, and / or rubella; —Influenza antigens such as hemagglutinin and / or neuraminidase surface proteins; —Antigens (proteins, sugars, or complexes) derived from Agalactia bacteria (Streptococcus agalactiae) (Group B streptococcus); —Antigens (proteins, sugars, or complexes) derived from Streptococcus pyogenes (Group A streptococcus); —Antigens (proteins, sugars, or complexes) derived from Staphylococcus aureus; —Antigens (proteins, sugars, or complexes) derived from Salmonella bacteria (Salmonella Spp.); An immunogenic composition according to any one of claims 4 to 9, comprising a recombinant protein / modified fHbp fusion protein in combination with at least one further antigen selected from the above.

11. The immunogenic composition according to any one of claims 4 to 10, wherein a scaffold of fHbp having an exogenous peptide loop is incorporated into a polysaccharide complex vaccine selected from monovalent (A, X, C, W, Y), bivalent (A-C, A-B, X-B, C-B), trivalent (AC-B, AC-Hib), tetravalent (AC-Hib-B), pentavalent (ACWYX), or hexavalent (ACWYX-B).

12. A modified H factor-binding protein according to claim 1 or 2, a nucleic acid sequence according to claim 3, or an immunogenic composition according to any one of claims 4 to 8, for use as a pharmaceutical, or for use in the treatment or prevention of pathogenic infection or colonization of a subject.

13. A combination of the modified factor H binding protein according to claim 1 or 2, the nucleic acid sequence according to claim 3, or the immunogenic composition according to any one of claims 4 to 8, and at least one further prophylactic or therapeutically active molecule.

14. The combination according to claim 13 or the immunogenic composition according to claim 8, comprising a complex vaccine containing at least one further prophylactic or therapeutically active molecule which is one of the serotype capsular polysaccharides selected from strain A, strain C, strain Y, strain W, or strain X, or a combination thereof.

15. Protein: The combination according to claim 14 or the immunogenic composition according to claim 8, wherein the protein:capsular polysaccharide vaccine comprises any of the following: a capsule of serotype C or A containing a bacterial toxin, a bivalent vaccine (containing capsular polysaccharides of serotypes C and A combined with the bacterial toxin), a tetravalent vaccine (containing polysaccharides of serotypes A, C, Y, and W combined with the bacterial toxin), or a pentavalent vaccine (containing polysaccharides of serotypes A, C, Y, W, and X combined with the bacterial toxin).

16. The H factor-binding protein according to claim 1 or 2, 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 the at least one recombinant protein / modified fHbp fusion protein comprises one or more exogenous loops selected from, but not limited to, transferrin-binding protein, Neisseria heparin-binding protein, Neisseria surface protein A, PorA, meningococcal enterobactin receptor FeA, Neisseria adhesin A, the fHbp-fHbp fusion protein described in any one of claims 1 to 16, or a combination thereof.

18. The vaccine formulation according to claim 17, wherein the at least one recombinant protein / modified fHbp fusion protein is the H factor binding protein described in claim 1 or 2 or the nucleic acid sequence described in claim 3, and the fHbp is derived from Neisseria meningitidis serotypes A, B, C, H, I, K, L, 29E, W135, X, Y, and Z.

19. The vaccine formulation according to claim 17 or 18, wherein the at least one recombinant protein / modified fHbp fusion protein is derived from meningococcal serotype B.

20. The vaccine formulation according to any one of claims 17 to 19, wherein the molecular weight of the fHbp of the at least one recombinant protein / modified fHbp fusion protein is in the range of 10 kDa to 200 kDa, preferably up to 50 kDa.

21. The adjuvants include aluminum hydroxide, aluminum phosphate, aluminum hydroxyphosphate and potassium aluminum sulfate, MF-59, liposomes, lipopolysaccharides, saponins, lipid A, lipid A derivatives, monophosphoryl lipid A, GLA, 3-deacylated monophosphoryl lipid A, AS01, AS03, AF3, IL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L and 41. BBL, oligonucleotides, oligonucleotides and / or liposomes containing at least one unmethylated CpG, Freund's adjuvants, complete Freund's adjuvants, incomplete Freund's adjuvants, polymers, copolymers such as polyoxyethylene-polyoxypropylene copolymers containing block copolymers, polymer p1005, CRL-8300 adjuvants, muramyl dipeptides, e.g., TLR1 / 2 agonis TLR2, TLR3, TLR-4 agonists, TLR5, TLR7, TLR7 / 8, TLR8, TLR9, ODN2216 (Type A), TLR11 / 12, TLR-4 agonists, flagellin, flagellin derived from Gram-negative bacteria, TLR-5 agonist, flagellin fragments capable of binding to the TLR-5 receptor, alpha-C-galactosylceramide, chitosan, interleukin-2, QS-21, squalene, Quil A vaccine formulation according to any one of claims 17 to 20, selected from the group consisting of A, cholera toxin B subunit, polyphosphazene and its derivatives, mycobacterial cell wall preparations, mycolic acid derivatives, nonionic block copolymer surfactants, OMV, fHbp, sterols and saponin combinations with lipids, dmLT, 1,25-dihydroxyvitamin D3, CAF01, poly[di(carboxylatofenoxy)-phosphazene] (PCPP), and Venezuelan equine encephalitis (VEE) replicon particles or combinations thereof.

22. The vaccine formulation according to any one of claims 17 to 21, wherein the adjuvant is aluminum hydroxide having a particle size greater than 500 nm.

23. One or more pharmaceutically acceptable excipients are: (a) Buffers selected from carbonates, phosphates, acetates, HEPES, succinates, TRIS, borates, citrates, lactates, glucons, tartrates, or combinations thereof; (b) Sugars selected from trehalose, mannose, raffinose, lactobionic acid, glucose, maltulose, isomaltulose, maltose, lactose, dextrose, fructose, or combinations thereof; (c) Sugar alcohols or polyols selected from mannitol, lactitol, sorbitol, glycerol, xylitol, maltitol, lactitol, erythritol, isomalt and hydrolyzed starch products or combinations thereof; (d) Surfactants selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, oxytoxynol 40, nonoxynol 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene 660 hydroxystearate, polyoxyethylene ricinoleic acid 35, soy lecithin, poloxamer, copolymers of ethylene oxide (EO), propylene oxide (PO) and / or butylene oxide (BO), octoxynol, phospholipids, nonylphenol ethoxylate, polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols, sorbitan esters or combinations thereof; (e) Polymers selected from dextran, carboxymethylcellulose, hyaluronic acid, cyclodextrin, or combinations thereof; (f) NaCl, KCl, KH ２ PO ４ Na ２ HPO ４ 2H ２ O, CaCl ２ , MgCl ２ Or a salt selected from these combinations; (g) Amino acids selected from tricine, 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 proteins selected from gelatin, lactalbumin hydrolysate, monosodium glutamate, collagen hydrolysate, keratin hydrolysate, peptides, casein hydrolysate, whey protein hydrolysate, serum albumin, or combinations thereof; (i) Preservatives selected from phenoxyethanol, benzethonium chloride (Phemerol), phenol, m-cresol, thiomersal, formaldehyde, paraben esters, 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 physiological saline, The vaccine preparation according to any one of claims 17 to 20.

24. below: At least one recombinant protein / at least one modified fHbp according to claims 1 to 23; Aluminum hydroxide; Mannitol; Phosphate; and Polysorbate; A vaccine formulation according to any one of claims 17 to 23, including the vaccine formulation according to any one of claims 17 to 23.

25. below: At least one modified fHbp represented by an amino acid sequence that is at least 75% identical to any one of the amino acid sequences represented by SEQ ID NOs: 6-10; or At least one modified fHbp encoded by a nucleic acid sequence that has at least 75% identity with any one of the nucleic acid sequences represented by sequence numbers 1 to 5; and Aluminum hydroxide; Mannitol; Phosphate; and Polysorbate; A vaccine formulation according to any one of claims 17 to 24, including the vaccine formulation according to any one of claims 17 to 24.

26. A vaccine formulation according to any one of claims 17 to 25, comprising: (i) at least one recombinant protein / modified fHbp fusion protein; (ii) an amount of aluminum hydroxide in the range of 0.5 mg / ml to 4.5 mg / ml; (iii) an amount of mannitol in the range of 5 mg / ml to 100 mg / ml; (iv) an amount of phosphate buffer in the range of 1 mM to 10 mM; and (v) an amount of polysorbate 20 in the range of 0.01 mg / ml to 2 mg / ml, wherein the at least one recombinant protein / modified fHbp fusion protein is present in an amount in the range of 15 μg / ml to 150 μg / ml.

27. A vaccine formulation according to any one of claims 17 to 25, comprising: (i) at least two recombinant protein / modified fHbp fusion proteins; (ii) an amount of aluminum hydroxide in the range of 0.5 mg / ml to 4.5 mg / ml; (iii) an amount of mannitol in the range of 5 mg / ml to 100 mg / ml; (iv) an amount of phosphate buffer in the range of 1 mM to 10 mM; and (v) an amount of polysorbate 20 in the range of 0.01 mg / ml to 2 mg / ml, wherein each of the at least two recombinant protein / modified fHbp fusion proteins is present in an amount in the range of 15 μg / ml to 150 μg / ml.

28. A vaccine formulation according to any one of claims 17 to 25, comprising: (i) at least three recombinant protein / modified fHbp fusion proteins; (ii) an amount of aluminum hydroxide in the range of 0.5 mg / ml to 4.5 mg / ml; (iii) an amount of mannitol in the range of 5 mg / ml to 100 mg / ml; (iv) an amount of phosphate buffer in the range of 1 mM to 10 mM; and (v) an amount of polysorbate 20 in the range of 0.01 mg / ml to 2 mg / ml, wherein each of the at least three recombinant protein / modified fHbp fusion proteins is present in an amount in the range of 15 μg / ml to 150 μg / ml.

29. A vaccine formulation according to any one of claims 17 to 25, comprising: (i) at least four recombinant protein / modified fHbp fusion proteins; (ii) an amount of aluminum hydroxide in the range of 0.5 mg / ml to 4.5 mg / ml; (iii) an amount of mannitol in the range of 5 mg / ml to 100 mg / ml; (iv) an amount of phosphate buffer in the range of 1 mM to 10 mM; and (v) an amount of polysorbate 20 in the range of 0.01 mg / ml to 2 mg / ml, wherein each of the at least four recombinant protein / modified fHbp fusion proteins is present in an amount in the range of 15 μg / ml to 150 μg / ml.

30. below: At least one modified fHbp represented by an amino acid sequence that is at least 75% identical to any one of the amino acid sequences represented by SEQ ID NOs: 6-10; or At least one modified fHbp encoded by a nucleic acid sequence that has at least 75% identity with any one of the nucleic acid sequences represented by sequence numbers 1 to 5; and Aluminum hydroxide in amounts ranging from 5 mg / ml to 4.5 mg / ml; Mannitol in amounts ranging from 5 mg / ml to 100 mg / ml; A phosphate buffer solution in the range of 1 mM to 10 mM; and Polysorbate 20 in amounts ranging from 0.01 mg / ml to 2 mg / ml; A vaccine formulation according to any one of claims 17 to 24, including the vaccine formulation according to any one of claims 17 to 24.

31. below: (a) an amount of fHbpV3.45 M5:PorA316-320 / exP1.14 (represented by SEQ ID NO: 6 or coded by SEQ ID NO: 1) in the range of 15 μg / ml to 150 μg / ml; or (b) an amount of fHbpV2.19 M6:PorA316-320 / exP1.4 (represented by SEQ ID NO: 7 or coded by SEQ ID NO: 2) in the range of 15 μg / ml to 150 μg / ml; or (c) fHbpV1.14:PorA307-311 / exP1.9 in an amount in the range of 15 μg / ml to 150 μg / ml (represented by SEQ ID NO: 8 or coded by SEQ ID NO: 3); or (d) amounts of fHbpV1.1:PorA307-311 / exP1.4 (represented by SEQ ID NO: 9 or coded by SEQ ID NO: 4) in the range of 15 μg / ml to 150 μg / ml; and (e) an amount of aluminum hydroxide ranging from 0.5 mg / ml to 4.5 mg / ml; (f) Mannitol in amounts ranging from 5 mg / ml to 100 mg / ml; (g) an amount of phosphate buffer in the range of 1 mM to 10 mM; and (h) Polysorbate 20 in an amount ranging from 0.01 mg / ml to 2 mg / ml; A vaccine formulation according to any one of claims 17 to 30, including the vaccine formulation according to any one of claims 17 to 30.

32. below: (a) fHbpV3.45 M5:PorA316-320 / exP1.14 in quantities ranging from 15 μg / ml to 150 μg / ml (represented by SEQ ID NO: 6 or coded by SEQ ID NO: 1); (b) fHbpV2.19 M6:PorA316-320 / exP1.4 in amounts ranging from 15 μg / ml to 150 μg / ml (represented by SEQ ID NO: 7 or coded by SEQ ID NO: 2); (c) fHbpV1.14:PorA307-311 / exP1.9 in amounts ranging from 15 μg / ml to 150 μg / ml (represented by SEQ ID NO: 8 or coded by SEQ ID NO: 3); (d) fHbpV1.1:PorA307-311 / exP1.4 in amounts ranging from 15 μg / ml to 150 μg / ml (represented by SEQ ID NO: 9 or coded by SEQ ID NO: 4); (e) an amount of aluminum hydroxide ranging from 0.5 mg / ml to 4.5 mg / ml; (f) Mannitol in amounts ranging from 5 mg / ml to 100 mg / ml; (g) an amount of phosphate buffer in the range of 1 mM to 10 mM; and (h) Polysorbate 20 in an amount ranging from 0.01 mg / ml to 2 mg / ml; A vaccine formulation according to any one of claims 17 to 30, including the vaccine formulation according to any one of claims 17 to 30.

33. below: (a) an amount of fHbpV3.45 M5:PorA316-320 / exP1.14 (represented by SEQ ID NO: 6 or coded by SEQ ID NO: 1) in the range of 15 μg / ml to 150 μg / ml; or (b) an amount of fHbpV2.19 M6:PorA316-320 / exP1.4 (represented by SEQ ID NO: 7 or coded by SEQ ID NO: 2) in the range of 15 μg / ml to 150 μg / ml; or (c) fHbpV1.1:PorA307-311 / exP1.9 in an amount in the range of 15 μg / ml to 150 μg / ml (represented by SEQ ID NO: 10 or coded by SEQ ID NO: 5); or (d) amounts of fHbpV1.14:PorA307-311 / exP1.9 (represented by SEQ ID NO: 8 or coded by SEQ ID NO: 3) in the range of 15 μg / ml to 150 μg / ml; and (e) an amount of aluminum hydroxide ranging from 0.5 mg / ml to 4.5 mg / ml; (f) Mannitol in amounts ranging from 5 mg / ml to 100 mg / ml; (g) an amount of phosphate buffer in the range of 1 mM to 10 mM; and (h) Polysorbate 20 in an amount ranging from 0.01 mg / ml to 2 mg / ml; A vaccine formulation according to any one of claims 17 to 30, including the vaccine formulation according to any one of claims 17 to 30.

34. below: (a) fHbpV3.45 M5:PorA316-320 / exP1.14 in quantities ranging from 15 μg / ml to 150 μg / ml (represented by SEQ ID NO: 6 or coded by SEQ ID NO: 1); (b) fHbpV2.19 M6:PorA316-320 / exP1.4 in amounts ranging from 15 μg / ml to 150 μg / ml (represented by SEQ ID NO: 7 or coded by SEQ ID NO: 2); (c) fHbpV1.1:PorA307-311 / exP1.9 in amounts ranging from 15 μg / ml to 150 μg / ml (represented by SEQ ID NO: 10 or coded by SEQ ID NO: 5); (d) amounts of fHbpV1.14:PorA307-311 / exP1.9 (represented by SEQ ID NO: 8 or coded by SEQ ID NO: 3) in the range of 15 μg / ml to 150 μg / ml; and (e) an amount of aluminum hydroxide ranging from 0.5 mg / ml to 4.5 mg / ml; (f) Mannitol in amounts ranging from 5 mg / ml to 100 mg / ml; (g) an amount of phosphate buffer in the range of 1 mM to 10 mM; and (h) Polysorbate 20 in an amount ranging from 0.01 mg / ml to 2 mg / ml; A vaccine formulation according to any one of claims 17 to 30, including the vaccine formulation according to any one of claims 17 to 30.

35. A vaccine formulation according to any one of claims 17 to 34, comprising 2-phenoxyethanol in an amount ranging from 1 mg / mL to 10 mg / mL.

36. The vaccine formulation according to any one of claims 17 to 35, wherein the vaccine composition is stable for six months at 2 to 8°C, 25°C, and 40°C.

37. A vaccine formulation according to any one of claims 17 to 35, wherein the zeta potential is in the range of -16 mV to -30 mV; and the osmotic pressure is in the range of 200 mOsmol / kg to 500 mOsmol / kg.

38. Diphtheria toxin (D), tetanus toxin (T), whole-cell pertussis (wP), hepatitis B virus surface antigen (HbsAg), Haemophilus influenzae type b PRP-carrier protein complex (Hib), Haemophilus influenzae (a, c, d, e, f serotypes and unencapsulated strains), meningococcal A antigen, meningococcal C antigen, meningococcal W-135 antigen, meningococcal Y antigen, meningococcal X antigen, Streptococcus pneumoniae antigen, meningococcal B bleb or purified antigen, Staphylococcus aureus antigen, Bacillus anthrax, BCG, hepatitis (A, C, D A vaccine formulation according to any one of claims 17 to 35, further comprising one or more antigens selected from (strains E, F, and G) antigens, human papillomavirus, HIV, Salmonella typhi antigen, acellular pertussis, modified adenylyl cyclase, malaria antigen (RTS, S), measles, mumps, rubella, dengue, Zika, Ebola, chikungunya, Japanese encephalitis, rotavirus, diarrhea antigen, flavivirus, smallpox, yellow fever, herpes zoster, varicella virus antigen, and combinations thereof.

39. (i) a vaccine formulation according to any one of claims 17 to 35, 38, comprising at least one fusion protein containing a stable non-functional / non-lipidized fHbp and a PorA VR2 loop, and (ii) at least one polysaccharide protein complex.

40. The vaccine formulation according to any one of claims 17 to 39, wherein the recombinant protein / modified fHbp fusion protein is administered concurrently with one or more vaccines selected from Bexcelo®, MENVEO, MENACTRA, NIMENRIX, MenQuadFi, MENFIVE, MenAfriVac, Men AC, and Men ACHIb.

41. The vaccine formulation according to any one of claims 17 to 40, wherein the recombinant protein / modified fHbp fusion protein is administered simultaneously with MENFIVE.

42. i) A fusion protein comprising a stable, non-functional / non-lipidized fHbp and at least one PorA VR2 loop, and the following: (a) (i) a complex of meningococcal serotype A capsule sugar and (ii) tetanus toxin; (b) (i) a complex of meningococcal serotype C capsule sugar and (ii) CRM197; (c) (i) Capsular sugar of Neisseria meningitidis serotype Y and (ii) Complex of CRM197; (d) (i) Capsular sugar of Neisseria meningitidis serotype W135 and (ii) CRM197 complex; and (e) (i) a complex of meningococcal serotype X capsule sugars and (ii) a complex of tetanus toxin; At least one complex selected from, A vaccine preparation according to any one of claims 17 to 41, comprising the above.

43. (i) at least two fusion proteins, each consisting of one fHbp mutant bound to one PorA VR2 loop, and the following: (a) (i) a complex of meningococcal serotype A capsule sugar and (ii) tetanus toxin; (b) (i) a complex of meningococcal serotype C capsule sugar and (ii) CRM197; (c) (i) Capsular sugar of Neisseria meningitidis serotype Y and (ii) Complex of CRM197; (d) (i) Capsular sugar of Neisseria meningitidis serotype W135 and (ii) CRM197 complex; and (e) (i) a complex of meningococcal serotype X capsule sugars and (ii) a complex of tetanus toxin; A vaccine preparation according to any one of claims 17 to 41, comprising the above.

44. (i) at least three fusion proteins, each consisting of one fHbp mutant bound to two PorA VR2 loops, and the following: (a) (i) a complex of meningococcal serotype A capsule sugar and (ii) tetanus toxin; (b) (i) a complex of meningococcal serotype C capsule sugar and (ii) CRM197; (c) (i) Capsular sugar of Neisseria meningitidis serotype Y and (ii) Complex of CRM197; (d) (i) Capsular sugar of Neisseria meningitidis serotype W135 and (ii) CRM197 complex; and (e) (i) a complex of meningococcal serotype X capsule sugars and (ii) a complex of tetanus toxin; A vaccine preparation according to any one of claims 17 to 41, comprising the above.

45. (i) at least four fusion proteins, each consisting of one fHbp mutant bound to three PorA VR2 loops, and the following: (a) (i) a complex of meningococcal serotype A capsule sugar and (ii) tetanus toxin; (b) (i) a complex of meningococcal serotype C capsule sugar and (ii) CRM197; (c) (i) Capsular sugar of Neisseria meningitidis serotype Y and (ii) Complex of CRM197; (d) (i) Capsular sugar of Neisseria meningitidis serotype W135 and (ii) CRM197 complex; and (e) (i) a complex of meningococcal serotype X capsule sugars and (ii) a complex of tetanus toxin; A vaccine preparation according to any one of claims 17 to 41, comprising the above.

46. A vaccine formulation according to any one of claims 17 to 45, for use in the treatment or prevention of infection and / or disease caused by meningococcal serotype B.

47. A vaccine formulation according to any one of claims 17 to 45 for elucidating cross-protection against Neisseria gonorrhea strain and Neisseria meningitidis serogroup ACWYX.

48. The vaccine formulation according to any one of claims 17 to 45, wherein the adsorption ratio of the recombinant protein / modified fHbp fusion protein onto the adjuvant is in the range of 70% to 100%.

49. fHbpV3.45M5PorA on adjuvant ３１６―３２０ｅｘＰ１．１４ The vaccine formulation according to claim 48, wherein the adsorption ratio is in the range of 80% to 100%.

50. fHbpV1.14PorA adsorption onto the adjuvant ３０７―３１１ ｅｘＰ１．９ The vaccine preparation according to claim 48, wherein the adsorption ratio is in the range of 80% to 90%.

51. fHbpV2.19PorA on adjuvant ３１６―３２０ ｅｘＰ１．４ The vaccine formulation according to claim 48, wherein the adsorption ratio is in the range of 80% to 90%.

52. fHbpV1.1PorA on adjuvant ３０７―３１１ ｅｘＰ１．４ The vaccine formulation according to claim 48, wherein the adsorption ratio is in the range of 80% to 90%.

53. fHbpV1.1PorA on adjuvant ３０７―３１１ ｅｘＰ１．９ The vaccine formulation according to claim 48, wherein the adsorption ratio is in the range of 70% to 80%.

54. A method for producing a vaccine preparation according to any one of claims 17 to 53, the following: (a) Growing host cells containing the expression vector in a nutrient medium; (b) Inducing the host cells to express the protein; (c) Recovering and separating the host cells; (d) Lysing the recovered cells to separate the host cell fragments and obtain tagged proteins; (e) Purifying the tagged protein; (f) Remove the tag from the tagged protein to obtain a recombinant protein / modified fHbp fusion protein; (g) Purifying the recombinant protein / modified fHbp fusion protein; and (h) Prepare a vaccine formulation containing the purified recombinant protein / modified fHbp fusion protein. Methods that include...

55. The method according to claim 54, wherein the host cell is a bacterial expression host system.

56. The method according to claim 55, wherein the bacterial expression host system is an Escherichia coli strain selected from the group consisting of 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)*, T7 Express, m15 pREP4*, C41(DE3), C43(DE3), or B834(DE3).

57. The method according to claim 54, wherein the nutrient medium is selected from undefined medium, terrific broth (TB) medium, lysogenia broth, luria broth (LB) medium or luria-bertani medium, chemically defined medium, M9 minimum medium, chemically defined M9 modified salt medium, 2x YT medium or catabolic metabolite suppression (SOC) medium, superoptimal broth, and combinations thereof.

58. The method according to claim 54, wherein the concentration of L-methionine during the proliferation of host cells in (a) is maintained in the range of 1 mM to 10 mM, and the fermentation is in a fed-batch mode.

59. The method according to 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 of 0 to 2 volumes of gas per unit volume of liquid (VVM).

60. The method according to claim 54, wherein the host cells are induced using an inducer selected from lactose and its non-hydrolyzable analog isopropyl β-D-1-thiogalactopyranoside (IPTG).

61. The method according to claim 60, wherein the lactose concentration is in the range of 1 g / L to 50 g / L, and the IPTG concentration is in the range of 1 mM to 10 mM.

62. The method according to claim 54, wherein the host cells are lysed using a method selected from chemical, biological, physical, mechanical, and combinations thereof.

63. The method according to claim 62, wherein the host cells are lysed using a combination of chemical and mechanical methods.

64. The method according to any one of claims 62 to 63, wherein the host cells are lysed using a lysis buffer having a pH range of 7 to 9, and then mechanically lysed for 3 to 8 cycles at a pressure range of 1000 to 1500 bar.

65. The method according to claim 64, wherein mechanical dissolution is performed using a homogenizer.

66. The method according to claim 54, wherein, in (e) above, the tagged protein is purified by chromatography, concentration, and dialysis.

67. The method according to claim 54, wherein the tag is removed using a TEV protease having a protein:TEV protease ratio in the range of 5:1 to 30:

1.

68. The method according to claim 54, wherein in (g) above, the tagged protein is purified by chromatography, concentration, and dialysis.

69. The method according to claim 68, wherein 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 bonding chromatography, reversed-phase chromatography, and combinations thereof.

70. The method according to any one of claims 54 to 69, wherein the tagged protein is expressed as inclusion bodies (IBs), and is purified by urea unfolding of the tagged protein and column refolding of the tagged protein.

71. The method according to claim 54, wherein the vaccine formulation is prepared by adsorbing individual recombinant protein / modified fHbp fusion proteins onto an adjuvant, and then adding it to an excipient mixture containing a sugar alcohol, a buffer, a stabilizer, and a liquid carrier.

72. The method according to claim 71, wherein the excipient mixture contains a preservative.

73. A method for producing TEV protease, the following: (a) Growing host cells containing the expression vector in a nutrient medium; (b) Inducing the host cells to express TEV protease; (c) Recovering and separating the host cells; (d) Lyse the recovered host cells and separate them to obtain the TEV protease; (e) Purifying the recovered TEV protease; and (f) Concentrate the purified TEV protease, dialysis filter it, and store it; The method according to claim 67, including the method described in claim 67.

74. The method according to claim 73, wherein the host cell for expressing the TEV protease is an Escherichia coli strain selected from the group consisting of 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)*, T7 Express, m15 pREP4*, C41(DE3), C43(DE3), Rosetta(trademark)(DE3)pLysS, or B834(DE3).

75. The method according to any one of claims 54 to 74, wherein when the OD at 590 / 600 nm is 20 to 100, the glucose supply is stopped and the glycerol supply is started, and the culture is induced by adding and / or maintaining lactose at 1 to 50 g / L in a fed-batch mode.

76. A method for inducing an immune response to meningococcal serotype B strain in an individual by administering a vaccine formulation according to any one of claims 1 to 10, wherein the administration is equivalent to inducing an immune response to meningococcal serotype B strain.

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