Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/22—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Neisseriaceae (F)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
  • A61K39/095—Neisseria
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/70—Vectors or expression systems specially adapted for E. coli
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55505—Inorganic adjuvants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55544—Bacterial toxins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/70—Multivalent vaccine
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the present invention relates to field of vaccine formulations and methods for manufacturing thereof. Particularly, the present invention relates to Upstream, Downstream and formulation development of Neisseria meningitidis (meningococcal) serogroup B based recombinant/ chimeric protein antigens, methods of preparing such chimeric protein-based formulations and use of these formulations for prevention and/ or treatment of subjects with Neisseria meningitidis (meningococcal) serogroup B infections.
• Neisseria meningitidis is an important pathogen, particularly in children and young adults. Septicemia and meningitis are the most life-threatening forms of invasive meningococcal disease (IMD). Case fatality rates remain around 10% for disseminated disease, while a third of survivors of meningococcal disease suffer from significant debilitating, and long-term sequelae.
• Toxoid-based vaccines have almost eliminated diphtheria and tetanus in wealthy countries, while capsule-based vaccines have substantially reduced disease caused by Haemophilus influenzae, Streptococcus pneumoniae, and some strains of Neisseria meningitidis.
• challenges remain in developing vaccines against pathogens for which toxoid and capsule- based vaccines are not feasible.
• pathogens include non-typeable strains of H. influenzae and S. pneumoniae, un-encapsulated pathogens such as Neisseria gonorrhoeae and Moraxella catarrhalis and encapsulated serogroup B N.
• meningitidis is a Gram-negative bacterium which colonizes the human upper respiratory tract and is responsible for worldwide sporadic and cyclical epidemic outbreaks of, most notably, meningitis and sepsis.
• N. meningitidis typically possess a cytoplasmic membrane, a peptidoglycan layer, an outer membrane which together with the capsular polysaccharide constitute the bacterial wall, and pili, which project into the outside environment.
• Neisseria meningitidis (Nm) remains a leading cause of sepsis and bacterial meningitis in children and young adults. There are approximately 500,000 cases of meningococcal disease each year with around 50,000 deaths.
• N meningitidis In developed countries, the bacterium is a leading cause of mortality among children, has important public health impacts during outbreaks in schools and universities, and can cause profound disability in survivors. Encapsulated strains of N. meningitidis are a major cause of bacterial meningitis and septicemia in children and young adults. N meningitidis can be classified into at least 12 serogroups (including serogroups A, B, C, H, I, K, L, 29E, W135, X, Y and Z) based on chemically and antigenically distinctive polysaccharide capsules. The most common serogroups being A, B and C which are responsible for 90% of disease worldwide.
• Serogroup B is the most common cause of meningococcal disease in Europe, USA and several countries in Latin America and causes epidemics in sub-Saharan Africa every 5-10 years. Meningococcal is a devastating disease that can kill children and young adults within hours despite the availability of antibiotics. Therefore, prophylactic immunisation is the best way to protect individuals from meningococcal infection. Vaccines are available based on the bacterial polysaccharide capsule, but the polysaccharide capsule of N. meningitidis serogroup B is poorly immunogenic as it has structural identity with a human glycoprotein in neural tissue and could induce autoimmunity if used as a vaccine.
• serogroup B ‘humanization’ put on-hold early efforts towards the development of a safe and immunogenic conjugate polysaccharide vaccine against B group since the polysaccharide does not elicit serum bactericidal antibodies and in vitro anti-capsule B antibodies recognize neural cell adhesion molecules in fetal brain tissue.
• Two main approaches have been used to develop vaccines against serogroup B N. meningitidis; outer membrane vesicle vaccines (OMVV) and recombinant protein subunit vaccines.
• OMVVs were first developed in the 1980s.
• the immunodominant antigen in meningococcal OMVVs is PorA, an abundant outer-membrane porin with eight surface- exposed loops.
• Loops one and four are termed variable region 1 and 2 (VR1 and VR2), respectively, as they generate immune responses and are subject to antigenic variation.
• the VR2 loop dominates PorA-specific immunity elicited by OMVVs, which offer limited or no cross-protection against strains expressing PorA with a different VR2.
• OMVVs include an OMV vaccine studied over 20 months in Chile, the efficacy decreased to 50%, indicating poor longevity of response (A.L. Wilkins, M.D. Snape et al 2017).
• OMVVs containing multiple PorAs have been developed and selected for the prevalence of PorA sequences in circulating strains.
• OMVVs present complex manufacturing and regulatory issues.
• blebs which represent vesicles shed from the cell surface of the particular organism of interest.
• a crude product carries many problems. For example, there is wide variation in the composition of these blebs. There is no reliable way of controlling which proteins are included or excluded from these blebs. These blebs may or may not include polysaccharide- coating elements of the organism of interest. The proportions of the various components of the blebs in relation to one another cannot be reliably determined. The composition of these blebs cannot be easily determined or controlled.
• Bexsero® provides uncertain coverage as 1) antigens are derived from a single meningococcal strain (Tan, L., et al. 2010) 2) most studies of immunogenicity were done with a 3+1 schedule and not the 2+1 schedule planned for the UK (doses at 2, 4 and 12 months) 3) indirect not direct correlates of protection have been mostly measured in 13 month olds, and not in infants who are at the greatest risk. N.
• OMVs Outer membrane vesicle
• PorA an outer membrane porin
• LPS toxic lipopolysaccharide
• PorA is the most abundant meningococcal outer membrane protein (OMP), which also elicits SBA, and is the main target of immune responses elicited by OMV vaccines which have been used successfully in outbreaks.
• PorA variants differ in their variable regions (VRs), surface exposed loops which are the target of immune responses.
• VR2 is responsible for most SBA elicited by OMV vaccines.
• PorA is an integral OMP with multiple hydrophobic domains. This makes PorA difficult to produce as a recombinant protein in its native conformation, limiting its use as an antigen in subunit vaccines.
• Factor H binding protein (fHbp) is also an antigen that elicits serum bactericidal antibody responses in immunised individuals and is a key component of investigational vaccines for the prevention of meningococcal, in particular serogroup B, disease that are currently being evaluated in clinical trials.
• fHbp Factor H Binding Protein
• lipoprotein 2086 Fletcher et al (2004) Infect Immun 72:2088-2100
• GAA Genome-derived Neisserial antigen 1870
• fHbps from different Neisseria meningitidis strains have been categorised using several schemes. These include two subfamilies (A and B) (Murphy E, et al.
• fHbp is antigenically variable; databases of genome sequences contain more than 900 different fHbp peptides, which fall into three variant groups or two subfamilies: V1 (subfamily B), V2 and V3 (both subfamily A).
• V1 subfamily B
• V2 subfamily A
• V3 both subfamily A
• immunisation with a particular fHbp induces cross-protection against strains that express fHbp belonging to the same, but not a different, variant group, although there can be cross- protection between fHbp variant groups 2 and 3 (subfamily A).
• Bexsero® contains a single fHbp peptide (V1.1), with two other recombinant antigens as well as an OMV, while Trumenba®is composed solely of two fHbp peptides (V1.55 and V3.45).
• V1.1 fHbp peptide
• Trumenba® is composed solely of two fHbp peptides (V1.55 and V3.45).
• V2 fHbp even though strains expressing this variant account for around 20-30% of all isolates (38% of UK cases).
• fHbp on Neisseria meningitidis is a 27 KDa lipoprotein that consists of two beta barrels (an N terminal barrel and a C terminal barrel) joined by a short amino acid linker.
• V2 fHbp The reason for the lack of V2 fHbp in current licensed vaccines is the instability of its N terminal ⁇ -barrel (Prymula, R. 2014; Johnson, S 2012). Further, protein stability is important during vaccine manufacturing as it affects yield and is a significant issue for quality control. Also, antigens in Bexsero® and Trumenba® have exact sequence matches to 36 and 4.8%, respectively of serogroup B N. meningitidis disease isolates currently circulating in the UK, leading to concerns about their ability to provide broad coverage against an antigenically diverse pathogen.
• modified V2 fHbp having increased stability over wild type V2 fHbp has been disclosed wherein modified V2 fHbp comprising atleast six mutations at Ser35, Aspl07, Vall l2, Leul l4, Serl37 and Glyl38. Since, a single fHbp does not provide universal protection against meningococcal disease, therefore, immunisation with a vaccine comprising representatives from each of the three variants, VI, V2 and V3, is necessary for a broad-N meningitidis serogroup B protection.
• Upstream process development includes scale-up of a fermentation process to ensure a similar product yield with quality at large scale as is produced at small scale.
• Various cultivation parameters such as media composition, pH, agitation, aeration, temperature, cell density, the concentration of inducers, induction time, and feeding strategies affect the protein expression level depending upon expression systems. Thus, it is essential to evaluate each of the cultivation conditions for the expression of every recombinant protein and the development of effective bioprocesses.
• Escherichia coli is the most widely used bacterial host for the production of recombinant proteins due to: (1) its fast growth rate with a generation time spanning 20 min under optimized conditions (Clark and Maal ⁇ e, 1967), (2) well-developed tools of molecular manipulations along with in-depth knowledge of its biology, and (3) the ability to achieve high cell density using inexpensive culture reagents. But in practice, a number of obstacles encountered along the pipeline must be overcome. These include poor growth of the host strain, protein instability or toxicity, aggregation and inclusion body formation, unsuitability of environmental conditions (temperature, pH, salt concentration, etc) and even no amplified expression at all.
• E. coli cannot perform post-translational modifications, limiting the product range that can be produced in a soluble and active form in this host organism. Furthermore, E. coli cannot secrete recombinant proteins. Consequently, recombinant E. coli cells need to be disrupted to access the intracellular product, which is then usually purified by several steps of filtration. In addition, high level expression of recombinant proteins in E. coli results in aggregation of expressed proteins into inclusion bodies (IB's).
• IB's inclusion bodies
• HCPs Host cell proteins
• DNA DNA are the main source of impurities, and the HCPs of each process vary significantly from each other in their molecular mass, charge, hydrophobicity, and structure. Therefore, they present a challenge for chromatographic purification.
• a purified soluble active recombinant protein is needed, it is invaluable to have means to (i) detect it along the expression and purification scheme, (ii) attain maximal solubility, and (iii) easily purify it from the E. coli cellular milieu.
• the expression of a stretch of amino acids (peptide tag) or a large polypeptide (fusion partner) in tandem with the desired protein to form a chimeric protein may allow these three goals to be straightforwardly reached. Being small, peptide tags are less likely to interfere when fused to the protein. However, in some cases they may provoke negative effects on the tertiary structure or biological activity of the fused chimeric protein.
• peptide tags should be removed too because it can affect protein conformation, hamper the interaction with a partner molecule or decrease the biological activity. Indeed, when these tags are removed, the final solubility of the desired product is unpredictable.
• expression vectors possess sequences that encode for protease cleavage sites downstream of the gene coding for the tag. Choosing among the different proteases is based on specificity, cost, number of amino acids left in the protein after cleavage and ease of removal after digestion C-type cysteine protease from Tobacco Etch Virus (TEV) is among the most widely used.
• TEV protease in Escherichia coli faced difficulties regarding protein yield (product yield is reduced) or low solubility of the protein at the industrial scales which means that large volumes and often long incubation times are required for efficient cleavage.
• Vaccines based on recombinant protein antigens generally require an adjuvant to achieve protection from the associated disease.
• Aluminum salts are the most prevalent adjuvants in vaccines approved for human use by the U.S. Food and Drug Administration.
• the point of zero charge (PZC) of the adjuvant is the point at which the net surface charge is zero; the PZC for aluminum oxyhydroxide is approximately 11, whereas the PZC for aluminum phosphate is approximately 4–5.5.
• Protein adsorption to the adjuvant surfaces is generally maximized when the sign of the net charge of the protein is opposite that of the adjuvant surface, allowing for an electrostatic attraction. Therefore, the protein vaccine formulation is formulated with adsorption buffers to improve adsorption to the surface.
• protein conformation can change when proteins bind to liquid-solid interfaces.
• conformational changes induced by binding to adjuvant could alter protein stability during long-term storage. For example, if adsorption is essentially complete, aggregation via pathways that occur in bulk solution are not likely to occur. Conversely, unfolding upon binding may expose normally buried residues to solvent, promoting degradation processes such as oxidation. (J Pharm Sci.2009 September; 98(9): 2970–2993).
• zeta potential can reduce the time needed to produce trial formulations. It can also be used as an aid in predicting long-term stability. In certain circumstances, the particles in dispersion may adhere to one another and form aggregates of successively increasing size, which may settle out under the influence of gravity. Further it can be seen that the zeta potential depends on the nature of the buffer used. There are various factors affecting zeta potential which include excipient used in formulation, excipient compatibilities, concentration of a formulation components, pH and conductivity. The variation in pH influences the electrostatic force, whereby the van der Waals forces remain constant for a given system.
• ChAs Chimeric antigens
• McAs serogroup B N. meningitidis
• MenB meningitidis
• ChAs exploit fHbp (non-lipidated) as a molecular scaffold to present the surface exposed PorA VR2 loop, which is achieved by inserting the VR2 loop (“10-20 amino acid” PorA VR2 loops instead of a “whole PorA protein) into a ⁇ -turn region in fHbp.
• ChAs retain epitopes from both fHbp and PorA and are found to elicit functional immune responses against both antigens.
• Yield of tagged fHbp protein could be at least 700mg of purified tagged protein from 100 grams of wet cell mass (Harvest) due to use of optimal concentration of inducer (IPTG (1 mM to 10 mM) or Lactose (1 g/L to 50 g/L)); use of Modified M9 salt media (Chemically defined media)/ Fed Batch Mode supplemented with L-Methionine (maintaining the L-Methionine concentration between 1-10 mM, preferably 2-5 mM, as compared to Luria Broth (LB) Media (Complex media)/ Batch Mode; stopping glucose feed and starting Glycerol feed when OD at 590/ 600 nm is ⁇ 20-100 and inducing the culture by adding and/or maintaining Lactose at 1-50 g/L in Fed batch mode.10) Using two step chromatography (Ion Exchange followed by Affinity) instead of previously known 3 step chromatography (multistep Affinity chromatography, Ion Exchange,
• Figures 1-5 illustrate the vector map for the recombinant proteins/ modified fHbp fusion proteins in accordance with the embodiments of the present invention
• Figure-6 illustrates the vector map for TEV protease in accordance with an embodiment of the present invention
• Figure-7 illustrates the flow chart for seed development for 10 L scale fermentation batch for the production of tagged fHbp protein
• Figure-8 illustrates the flow chart for the production of tagged fHbp protein at 10 L scale fermenter
• Figures 9a-9h illustrate the growth profile for the tagged fHbp proteins at 10 L scale fermentation batch along with the SDS PAGE gel images
• Figure-10 illustrates the flow chart for seed development for 10 L scale fermentation batch for the production of TEV protease
• Figure-11 illustrates the production of TEV protease at 10 L scale fermenter
• Figures 12a-12b illustrate the growth profile for the TEV protease at 10 L scale
• An object of the present disclosure is to provide an efficient platform process for manufacturing an effective vaccine formulation against Neisseria meningitidis that meets multiple criteria including improved immunogenicity, safety and affordability.
• Another object of the present disclosure is to provide an efficient platform process for manufacturing an effective vaccine formulation comprising of one or more recombinant protein/ modified fHbp fusion protein derived from Neisseria meningitidis serogroup B and pharmaceutically acceptable carrier or excipient.
• Another object of the present disclosure is to develop and optimize upstream bioprocess to increase cell density and recombinant protein/ modified fHbp fusion protein productivity for the lead cell lines.
• ChAs chimeric antigens
• ChAs exploit fHbp (non- lipidated) as a molecular scaffold to present the surface exposed PorA VR2 loop, which is achieved by inserting the VR2 loop (instead of a “whole PorA protein) into a ⁇ -turn region in fHbp. ChAs retain epitopes from both fHbp and PorA and are found to elicit functional immune responses against both antigens wherein the integration of a VR2 loop does not alter the overall architecture of fHbp and that the VR2 loop folds into a conformation recognised by a bactericidal mAb.
• fHbp non- lipidated
• Another object of the present disclosure is to develop and optimize formulation comprising chimeric proteins that are soluble and high yielding, are stable, wherein fHbp and the PorA VR2 loop both are immunogenic.
• Another object of the present disclosure is to develop and optimize formulation comprising Chimeras composed of the most prevalent fHbp and PorA antigens to maximize vaccine coverage.
• Another object of the present disclosure is to develop and optimize formulation comprising chimeric proteins having Molecular weight in the range of 20 kDa to 40 kDa while preserving immunogenic epitopes of both fHbp and PorA: Table-2: fHbp-PorA chimeras Sr. No.
• Another object of the present disclosure is to develop and optimize downstream comprising inserting TEV cleavage site in between His-MBP tags and chimera, to ease removal of tag during downstream processing and optimized Substrate to enzyme ratio/
• Another object of the present disclosure is using a combination of chemical lysis and homogeniser (with optimized conditions instead of sonication or only chemical method) for large scale lysis of cells.
• Another object of the present disclosure is developing high yield of chimeric fHbp-PorA proteins due to use of optimal concentration of inducer; use of Modified M9 salt media (Chemically defined media)/ Fed Batch Mode supplemented with L-Methionine; stopping glucose feed and starting Glycerol feed when reaching a specific OD at 590/600 nm and inducing the culture by adding Lactose or IPTG.
• Another object of the present disclosure is developing two step chromatography instead of previously known methods using at least 3 step chromatography.
• a method comprising steps a), b) and c) encompasses, in its narrowest sense, a method which consists of steps a), b) and c).
• the phrase "consisting of” means that the composition (or device, or method) has the recited elements (or steps) and no more.
• the term “comprises” can encompass also a method including further steps, e.g., steps d) and e), in addition to steps a), b) and c).
• the terms first, second, third, etc. should not be construed to limit the scope of the present disclosure as the aforementioned terms may be only used to distinguish one element, component, region, layer or section from another component, region, layer or section.
• any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.
• Reference throughout this specification to "one embodiment” or “an embodiment” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention.
• appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment but may do.
• the features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments.
• each of these types of embodiments is a non- limiting example of a feature that is intended to be combined with any other feature, or combination of features, described herein without having to list every possible combination.
• Such features or combinations of features apply to any of the aspects of the invention.
• the ranges defined throughout the specification include the end values as well, i.e. a range of 1 to 10, between 1 to 10 imply that both 1 and 10 are included in the range. For the avoidance of doubt, the applicant shall be entitled to any equivalents according to applicable law.
• the term “about” when qualifying a value of a stated item, number, percentage, or term refers to a range of plus or minus 10 percent, 9 percent, 8 percent, 7 percent, 6 percent, 5 percent, 4 percent, 3 percent, 2 percent or 1 percent of the value of the stated item, number, percentage, or term. Preferred is a range of plus or minus 10 percent. In case numerical ranges are used herein such as “in a concentration between 1 and 5 micromolar”, the range includes not only 1 and 5 micromolar, but also any numerical value in between 1 and 5 micromolar, for example, 2, 3 and 4 micromolar.
• in vitro as used herein denotes outside, or external to, the animal or human body.
• in vitro should be understood to include “ex vivo”.
• ex vivo typically refers to tissues or cells removed from an animal or human body and maintained or propagated outside the body, e.g., in a culture vessel.
• in vivo denotes inside, or internal to, the animal or human body. Definitions: In order for the present disclosure to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms may be set forth throughout the specification.
• protein or “polypeptide” or “(poly)peptide” or “peptide” (all terms are used interchangeably, if not indicated otherwise) as used herein encompasses isolated and/or purified and/or recombinant (poly)peptides being essentially free of other host cell polypeptides.
• peptide as referred to herein comprises at least two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300 or even more amino acid residues where the alpha carboxyl group of one is bound to the alpha amino group of another.
• a post-translational modification of the protein or peptide as used and envisaged herein is the modification of a newly formed protein or peptide and may involve deletion, substitution or addition of amino acids, chemical modification of certain amino acids, for example, amidation, acetylation, phosphorylation, glycosylation, formation of pyroglutamate, oxidation/reduction of sulfa group on a methionine, or addition of similar small molecules, to certain amino acids.
• Homologues refers to bacterial, fungal, plant or animal homologues of the oxidase enzyme or rubredoxin or rubredoxin reductase useful in the invention, preferably plant homologues, but also includes truncated sequences, single- stranded DNA or RNA of the coding and non-coding DNA sequence. Sequence identity, homology or similarity is defined herein as a relationship between two or more amino acid sequences or two or more nucleic acid sequences, as determined by comparing those sequences. Usually, sequence identities or similarities are compared over the whole length of the sequences but may also be compared only for a part of the sequences aligning with each other.
• sequence identities or similarities are compared over the whole length of the sequences, herein.
• identity or “similarity” also means the degree of sequence relatedness between polypeptide sequences or nucleic acid sequences, as the case may be, as determined by the match between such sequences.
• Sequence alignments can be generated with a number of software tools, such as: 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, for example, implemented into the “NEEDLE” program, which performs a global alignment of two sequences.
• the NEEDLE program is contained within, for example, 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
• BLOSUM62 One out of the many BLOSUMs is “BLOSUM62”, which is often the “default” setting for many programs, when aligning protein sequences.
• BLAST Basic Local Alignment Search Tool
• BlastP Basic Local Alignment Search Tool
• BlastN BLAST program
• BLAST2 The “original” BLAST: Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) "Basic local alignment search tool.” J. Mol. Biol. 215:403-410; BLAST2: Altschul, Stephen F., Thomas L. Madden, Alejandro A.
• Sequence identity usually is provided as “% sequence identity” or “% identity”.
• % sequence identity or “% identity”.
• a pairwise sequence alignment is generated between those two sequences, wherein the two sequences are aligned over their complete, entire or full length (i.e., a pairwise global alignment).
• the alignment is generated with a program or software described herein.
• the preferred alignment for the purpose of this invention is that alignment, from which the highest sequence identity can be determined.
• sequence “identity” used herein refers to the percentage identity between two aligned sequences using standard NCBI BLAST parameters (http://blast.ncbi.nlm.nih.gov).
• the nucleic acids (or polynucleotides) of the invention comprises nucleic acid sequences which encode the fusion proteins of the invention.
• the nucleic acid sequences encoding the fusion proteins of the invention are preferably recombinant and/or isolated and/or purified nucleic acid sequences.
• the nucleic acid sequences which encode the fusion proteins of the invention can be produced and isolated using known molecular-biological standard techniques, the sequence information and organisms provided herein.
• nucleic acid includes reference to a deoxyribonucleotide or ribonucleotide polymer, i.e. a polynucleotide, in either single-or double-stranded form, and unless otherwise limited, encompasses known analogues having the essential nature 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).
• a polynucleotide can be full- length or a sub-sequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to the specified sequence as well as the complementary sequence thereof.
• DNAs or RNAs with backbones modified for stability or for other reasons are “polynucleotides” as that term is intended herein.
• DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples are “polynucleotides” as the term is used herein. Lt will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art.
• polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including among other things, simple and complex cells. Every nucleic acid sequence herein that encodes a polypeptide such as the oxidase enzyme or rubredoxin or rubredoxin reductase also, by reference to the genetic code, describes every possible silent variation of the nucleic acid.
• the term “conservatively modified variants” applies to both amino acid and nucleic acid sequences.
• the term “conservatively modified variants” if used, may refer to those nucleic acids which encode identical or conservatively modified variants of the amino acid sequences due to the degeneracy of the genetic code.
• the term “degeneracy of the genetic code” refers to the fact that a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
• Such nucleic acid variations are “silent variations” and represent one species of conservatively modified variation.
• polypeptide “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues.
• polypeptide “peptide” and “protein” apply also to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
• the essential nature of such analogues of naturally occurring amino acids is that, when incorporated into a protein, that protein is specifically reactive to antibodies elicited to the same protein but consisting entirely of naturally occurring amino acids.
• polypeptide polypeptide
• peptide protein
• oligomers are considered a species of the group of polymers. Oligomers have a relatively low number of monomeric units, in general 2-100, in particular 6-100, including, e.g., primer sequences, such as used for cloning of the oxidase enzyme or rubredoxin or rubredoxin reductase useful in the invention, in the Examples.
• heterologous when used with respect to a nucleic acid (DNA or RNA) or protein of the invention refers to a nucleic acid or protein that does not occur naturally as part of the organism, cell, genome or DNA or RNA sequence in which it is present, or that is found in a cell or location or locations in the genome or DNA or RNA sequence that differ from that in which it is found in nature.
• Heterologous nucleic acids or proteins of the invention are not endogenous to the cell into which they are introduced but have been obtained from another cell or synthetically or recombinantly produced. Generally, though not necessarily, such nucleic acids encode proteins that are not normally produced by the cell in which the DNA is expressed.
• heterologous A gene that is endogenous to a particular host cell but has been modified from its natural form, though, for example, the use of DNA shuffling, is also called heterologous.
• heterologous also includes non-naturally occurring multiple copies of a naturally occurring DNA sequence.
• the term “heterologous” may refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position and/or a number within the host cell nucleic acid in which the segment is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides.
• a “homologous” DNA sequence of the invention is a DNA sequence that is naturally associated with a host cell into which it is introduced.
• heterologous nucleic acid or protein Any nucleic acid or protein that one of skill in the art would recognize as heterologous or foreign to the cell in which it is expressed is herein encompassed by the term heterologous nucleic acid or protein.
• modified modified, “modification”, “mutated”, or “mutation”, as used herein regarding proteins or polypeptides compared to another protein or polypeptide apply mutatis mutandis to nucleotide or nucleic acid sequences.
• the mentioned terms are used to indicate that the modified nucleotide or nucleic acid sequences encoding the protein or polypeptide has at least one difference in the nucleotide or nucleic acid sequence compared to the nucleotide or nucleic acid sequence of the protein or polypeptide with which it is compared.
• Mutagenesis is a well-known method in the art, and includes, for example, site-directed mutagenesis by means of PCR or via oligonucleotide- mediated mutagenesis, as described in Sambrook, J., and Russell, D.W. Molecular Cloning: A Laboratory Manual.3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (2001).
• modified is used to indicate that at least one nucleotide in the nucleotide sequence of that gene or a regulatory sequence thereof, is different from the nucleotide sequence that it is compared.
• a modification or mutation may in a particular be a replacement of a nucleotide by a different one, a deletion of a nucleotide or an insertion of a nucleotide.
• modified fHbp/ modified fHbp fusion protein/ recombinant protein/ chimera/ chimeric protein/ chimeric molecule/ chimeric antigen/ fusion protein/ clone are interchangeably used throughout the specification and refers to proteins created through the joining of two or more genes which originally coded for separate or same proteins resulting in a polypeptide comprising a combination of sequences from different gene products or sources. Translation of this recombinant/ chimeric/ fusion gene results in a single polypeptide with functional properties derived from each of the original proteins along with few additional characteristics.
• tagged protein used herein refers to proteins having specific peptide sequence(s) (also known as tags) grafted genetically.
• the tags serve various purposes and can be added to either end of the target protein are either C-terminus or N-terminus specific or are both C- terminus and N-terminus specific. Some tags are also inserted at sites within the protein of interest; they are known as internal tags. Affinity tags are appended to proteins so that they can be purified from their crude biological source using an affinity technique. Affinity tags include chitin binding protein (CBP), maltose binding protein (MBP), Strep-tag and glutathione-S-transferase (GST).
• CBP chitin binding protein
• MBP maltose binding protein
• Strep-tag and glutathione-S-transferase
• GST glutathione-S-transferase
• the poly(His) tag is a widely used protein tag, which binds to matrices bearing immobilized metal ions.
• peptide loop used herein is intended to refer to a single chain polypeptide sequence anchored at both ends (e.g. anchored to a scaffold such as fHbp).
• the term “loop” does not infer or require any particular secondary structure adopted by the polypeptide.
• exogenous used herein in the context of “exogenous peptide loop” is understood to mean that the peptide loop is derived from a different source relative to the fHbp protein (i.e. it is not fHbp or a fragment thereof). However, it may be from the same organism as the fHbp.
• a modified fHbp may include an N.
• meningitidis fHbp modified with (exogenous) peptide loop(s) derived from N. meningitidis PorA.
• isolated when applied to the modified fHbp of the present invention means a protein: (i) encoded by nucleic acids using recombinant DNA methods or a viral vector; or (ii) synthesized by, for example, chemical synthetic methods; or (iii) separated from biological materials, and then purified.
• An isolated polypeptide of the invention includes a protein expressed from a nucleotide sequence encoding the protein, or from a recombinant vector containing a nucleotide sequence encoding the protein.
• immunological refers to a molecule that is capable of eliciting an immune response in a human or animal body.
• the immune response may be protective.
• protecting used herein means prevention of a disease, a reduced risk of disease infection, transmission and/or progression, reduced severity of disease, a cure of a condition or disease, an alleviation of symptoms, or a reduction in severity of a disease or disease symptoms.
• prophylaxis used herein means prevention of or protective treatment for a disease. The prophylaxis may include a reduced risk of infection, transmission and/or progression, or reduced severity of disease.
• treatment means a cure of a condition or disease, an alleviation of symptoms, or a reduction in severity of a disease or disease symptoms.
• freeze-drying/ lyophilize/ lyophilization refers to the process by which a suspension/solution is frozen, after which the water is removed by sublimation at low pressure.
• sublimation refers to a change in the physical properties of a composition, wherein the composition changes directly from a solid state to a gaseous state without becoming a liquid.
• a modified factor H binding protein (fHbp) is provided.
• the modified factor H binding protein (fHbp) is a fusion protein and comprises a wild type fHbp variant and at least one exogenous peptide loop(s).
• the fHbp is meningococcal fHbp.
• the fHbp is gonococcal fHbp.
• the modified factor H binding protein (fHbp) has an amino acid sequence with at least 75% identity with any one of sequences of SEQ ID NOs 6 to 10. In an embodiment, the modified factor H binding protein (fHbp) includes an amino acid sequence with at least 75% identity with any one of sequences of SEQ ID NOs 6 to 10.
• substitutions may be to similar amino acid residues, for example having similar MW, charge, hydrophobicity or moieties, or synthetic analogues. Such modifications are envisaged as part of the invention.
• 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 sequence described herein.
• the modified factor H binding protein (fHbp) is selected from amino acid sequence with 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 sequences of SEQ ID Nos 6 to 10.
• the modified factor H binding protein (fHbp) is selected from amino acid sequence with at least 75.0% identity with sequence of SEQ ID No 6.
• the modified factor H binding protein (fHbp) is selected from amino acid sequence with 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 sequence of SEQ ID No 6. In one embodiment, the modified factor H binding protein (fHbp) is selected from amino acid sequence with at least 75.0% identity with sequence of SEQ ID No 7. In a preferred embodiment, the modified factor H binding protein (fHbp) is selected from amino acid sequence with 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 sequence of SEQ ID No 7.
• the modified factor H binding protein (fHbp) is selected from amino acid sequence with at least 75.0% identity with sequence of SEQ ID No 8. In a preferred embodiment, the modified factor H binding protein (fHbp) is selected from amino acid sequence with 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 sequence of SEQ ID No 8. In one embodiment, the modified factor H binding protein (fHbp) is selected from amino acid sequence with at least 75.0% identity with sequence of SEQ ID No 9.
• the modified factor H binding protein (fHbp) is selected from amino acid sequence with 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 sequence of SEQ ID No 9. In one embodiment, the modified factor H binding protein (fHbp) is selected from amino acid sequence with at least 75.0% identity with sequence of SEQ ID No 10. In a preferred embodiment, the modified factor H binding protein (fHbp) is selected from amino acid sequence with 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 sequence of SEQ ID No 10.
• the at least one exogenous peptide loop(s) is immunogenic and is derived from a bacterial membrane protein.
• the fHbp variant is selected from v1, v2 and v3, and is modified with at least one PorA loop inserted into a ⁇ -turn region in fHbp.
• the fHbp may comprise fHbp v1.
• the fHbp may comprise fHbp v2.
• the fHbp may comprise fHbp v3.
• the variant of the wild type fHbp comprises a wild type meningococcal orthologue of fHbp.
• a variant of fHbp may comprise Ghfp, the Gonococcal homologue of fHbp.
• Ghfp is non-functional and closely related to v3 fHbps (>95% aa identity, dissociation constant KD > 100 ⁇ with factor H).
• the PorA loop is selected from VR1, and VR2.
• the PorA loop is VR1.
• the PorA loop is VR2.
• the modified factor H binding protein is modified to reduced factor H binding activity.
• the modified fHbp has >80% decreased binding to human factor H (fH), as compared to wild type fHbp.
• nucleic acid encoding essentially or at least the modified fHbp according to the invention herein.
• the present invention is directed to a nucleic acid sequence encoding the modified fHbp, wherein the nucleic acid sequence is with 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 sequences of SEQ ID NO.1 to 5.
• the present invention is directed to a nucleic acid sequence encoding the modified fHbp, wherein the nucleic acid sequence is with at least 75.0% identity with sequence of SEQ ID No 1.
• the present invention is directed to a nucleic acid sequence encoding the modified fHbp, wherein the nucleic acid sequence is with 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 sequence of SEQ ID No 1.
• the present invention is directed to a nucleic acid sequence encoding the modified fHbp, wherein the nucleic acid sequence is with at least 75.0% identity with sequence of SEQ ID No 2.
• the present invention is directed to a nucleic acid sequence encoding the modified fHbp, wherein the nucleic acid sequence is with 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 sequence of SEQ ID No 2.
• the present invention is directed to a nucleic acid sequence encoding the modified fHbp, wherein the nucleic acid sequence is with at least 75.0% identity with sequence of SEQ ID No 3.
• the present invention is directed to a nucleic acid sequence encoding the modified fHbp, wherein the nucleic acid sequence is with 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 sequence of SEQ ID No 3.
• the present invention is directed to a nucleic acid sequence encoding the modified fHbp, wherein the nucleic acid sequence is with at least 75.0% identity with sequence of SEQ ID No 4.
• the present invention is directed to a nucleic acid sequence encoding the modified fHbp, wherein the nucleic acid sequence is with 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 sequence of SEQ ID No 4.
• the present invention is directed to a nucleic acid sequence encoding the modified fHbp, wherein the nucleic acid sequence is with at least 75.0% identity with sequence of SEQ ID No 5.
• the present invention is directed to a nucleic acid sequence encoding the modified fHbp, wherein the nucleic acid sequence is with 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 sequence of SEQ ID No 5.
• a nucleic acid sequence encoding the modified fHbp of the present invention there is provided a nucleic acid sequence encoding the modified fHbp of the present invention.
• the nucleic acid is a vector, such as a viral vector.
• the present invention is directed to an immunogenic composition comprising at least one modified fHbp as described herein.
• the present invention is directed to an immunogenic composition comprising at least one modified fHbp as disclosed herein or encoded by the nucleic acid sequence encoding the modified fHbp as disclosed herein.
• an immunogenic composition comprising at least one modified fHbp of the present invention.
• the present invention is directed to an immunogenic composition
• an immunogenic composition comprising at least one modified fHbp of the present invention, wherein the modified fHbp includes - at least one modified fHbp of the amino acid sequence with at least 75% identity with from any one of amino acid sequences SEQ ID NO.6 to 10, or combinations thereof, or - at least one modified fHbp encoded the nucleic acid sequence is with at least 75% identity with by any one of nucleic acid sequence SEQ ID NO.1 to 5, or combinations thereof.
• the present invention is directed to an immunogenic composition comprising at least one modified fHbp of the present invention, wherein the modified fHbp includes - fHbpV3.45 M5:PorA316-320/exP1.14 (of SEQ ID NO.6 or encoded by SEQ ID NO.1), or - fHbpV2.19 M6:PorA316-320/exP1.4 (of SEQ ID NO.7 or encoded by SEQ ID NO.2), or - fHbpV1.14:PorA307-311/exP1.9 (of SEQ ID NO.8 or encoded by SEQ ID NO.3), or - fHbpV1.1:PorA307-311/exP1.4 (of SEQ ID NO.9 or encoded by SEQ ID NO.4), or - fHbpV1.1:PorA307-311/exP1.9 (of SEQ ID NO.
• the immunogenic composition comprises two or more different modified fHbp. In still another embodiment, the immunogenic composition comprises three or more different modified fHbp. In yet another embodiment, the immunogenic composition comprises four different modified fHbp. In an embodiment, the immunogenic composition comprises a pharmaceutically acceptable carrier. In a further embodiment, the immunogenic composition comprises an adjuvant. In another embodiment, the immunogenic composition further comprises at least one other prophylactically or therapeutically active molecule.
• the at least one other prophylactically or therapeutically active molecule comprises: - a monovalent protein: capsule polysaccharide vaccine; or - a conjugate vaccine, wherein antigen(s) comprising the fHbp scaffold bearing exogenous peptide loops is incorporated as the protein carrier molecule in the conjugate vaccine.
• the modified fHbp, the nucleic acid or the composition of the present invention may be used as a medicament, or in treatment or prevention of a pathogenic infection or colonization of a subject.
• the present invention further envisages an immunogenic composition comprising a combination of the modified fHbp, a nucleic acid, and at least one other prophylactically or therapeutically active molecule.
• the at least one other prophylactically or therapeutically active molecule comprises a protein: capsule polysaccharide conjugate vaccine.
• the protein: capsule polysaccharide vaccine comprises any of serogroup C or A capsule with bacterial toxoids, bi-valent vaccines (with serogroup C and A capsular polysaccharide conjugated to bacterial toxoids), quadrivalent- (serogroups A, C, Y, W polysaccharides conjugated to bacterial toxoids) or pentavalent- (serogroups A, C, Y, W, X polysaccharides conjugated to bacterial toxoid) conjugate vaccines.
• the at least one other prophylactically or therapeutically active molecule comprises a conjugate vaccine, wherein antigen(s) comprising the fHbp scaffold bearing exogenous peptide loops is incorporated as the protein carrier molecule in the conjugate vaccine, optionally wherein the conjugate vaccine comprises any of serogroup capsular polysaccharides selected from A, C, Y, W, or X strains, or combinations thereof.
• the factor H binding protein (fHbp) is used as an epitope display scaffold.
• fHbp found on the surface of Neisseria meningitidis is a 27 kDa lipoprotein that consists of two beta barrels (an N terminal barrel and a C terminal barrel) joined by a short amino acid linker. While charged carbohydrates on the surface of the vascular endothelium engage fH, charged amino acids in the fHbp bind to fH at nanomolar affinities at the same site of this complement regulator. Based on differences in the nucleotide and predicted amino acid sequences, fHbps from different Neisseria meningitidis strains have been categorised using several schemes.
• fHbp Factor H Binding Protein
• lipoprotein 2086 Fletcher et al (2004) Infect Immun 72:2088-2100
• GAA Genome-derived Neisserial antigen 1870
• fHbps from different Neisseria meningitidis strains have been categorised using several schemes. These include two subfamilies (A and B) (Murphy E, et al.
• fHbps belonging to the same variant group share over 85% amino acid similarity, and only 60-70% similarity between the three variant groups.
• fHbp is also an antigen that elicits serum bactericidal antibody responses in immunised individuals and is a key component of investigational vaccines for the prevention of meningococcal, in particular serogroup B, disease that are currently being evaluated in clinical trials.
• Meningococcal outer membrane vesicle is PorA, an integral outer membrane protein (OMP) in the meningococcus.
• OMV outer membrane vesicle
• Variants of PorA are identified by sequences in the variable-regions (VR) of the protein, which are located in the surface- exposed peptide loops of the protein and are the target of immune responses.
• PorA has seven extracellular peptide loops; the fourth loop is variable region 2 (VR2) and is the target of most serum bactericidal activity (SBA) generated by PorA following natural infection and after immunisation with OMVs.
• SBA serum bactericidal activity
• immunogenic peptides such as those from PorA
• fHbp factor H binding protein
• the peptides that are introduced into fHbp are presented to the immune system and are able to elicit protective responses such as SBA.
• the fHbp molecule provides an ideal molecular scaffold for stable inclusion of peptide loops for the display of epitopes, particularly for epitopes that are difficult to stabilise and display in their native conformation, for example loops from integral OMPs such as PorA.
• many OMPs, such as PorA are difficult to express because of the insolubility of their membrane spanning domains.
• PorA has a 16-beta stranded barrel structure with the surface-exposed loops between strands 1 and 2 (loop 1), strands 7 and 8 (loop 4), strands 9 and 10 (loop 5) and strands 11 and 12 (loop 7) demonstrated to be the most effective antigens.
• fHbp contains two beta barrels, therefore the peptide loop sequences from OMPs can be inserted into the tips of the loops between bet ⁇ - strands of fHbp to present the extra-cellular loop fragments from integral OMPs, in their native conformations for immunisation.
• the modified fHbp scaffold molecule of the invention may be used as a prophylactic or a therapeutic vaccine directed to Nm or the gonococcus in which a single protein presents key epitopes from two different antigens.
• the modified fHbp is a fusion protein, such as a recombinant fusion protein.
• the modified fHbp is an isolated modified fHbp molecule.
• the modified fHbp molecule of the invention is included as a single protein in multi-valent vaccine.
• the modified fHbp is included in an OMV vaccine.
• the exogenous peptide loops are same, e.g. the same sequence, or substantially similar.
• some epitopes such as PorA epitopes, may not elicit sufficient functional responses when displayed singly on fHbp.
• the present invention may be used to provide the same epitope at multiple sites on the same modified fHbp molecule, thereby enhancing the immunogenic recognition of the epitope.
• the exogenous peptide loops are different relative to each other.
• the different exogenous peptide loops are from distinct regions of the protein, such as PorA.
• the different exogenous peptide loops are derived from overlapping and distinct regions of the protein, such as PorA.
• the exogenous peptide loops are derived from different species or strains. For example, when a multivalent vaccine is desired for multiple different antigens including different organisms.
• an effective vaccine formulation comprising at least one recombinant protein/ modified fHbp fusion protein, an adjuvant and one or more pharmaceutically acceptable excipient.
• vaccine formulation comprising at least one recombinant protein/ modified fHbp fusion protein further additionally comprises an antigen selected from antigen peptide subvariants fHbp 3.45, fHbp 1.55, fHbp-fHbp-fHbp or any other fHbp fusion protein, fHbp-cholera toxin, multiple Por A fused to single fHbp, recombinant N. meningitidis group B NHBA fusion protein, recombinant N.
• the recombinant protein may comprise of a Transferrin Binding Protein, Neisserial Heparin Binding Protein, Neisserial Surface Protein A, PorA, meningococcal enterobactin receptor FetA, Neisserial Adhesin A, or factor H binding protein (fHbp).
• the recombinant protein/ modified fHbp fusion protein may comprise of a modified factor H binding protein (fHbp), comprising fHbp, or a variant thereof, to act as a molecular scaffold by modification with the addition of at least one exogenous peptide loop from a different antigen.
• the recombinant protein/ modified fHbp fusion protein may comprise of a modified factor H binding protein (fHbp), comprising fHbp, or a variant thereof, to act as a molecular scaffold by modification with the addition of at least two exogenous peptide loops from a different antigen.
• the vaccine formulation may comprise of fHbp variants selected from v1 or v2 or v3.
• the formulation may comprise of fHbp variants selected from v1 and v2.
• the formulation may comprise of fHbp variants selected from v2 and v3.
• the formulation may comprise of fHbp variants selected from v1, v2 and v3.
• the recombinant protein/ modified fHbp fusion protein is an fHbp variant selected from v1, v2 and v3, modified with at least one PorA loop comprising at least 10 amino acids inserted into a ⁇ -turn region in fHbp.
• the PorA loop is selected from VR1, and VR2, and has >80 % decreased binding to human factor H (fH), as compared to wild type fHbp. All the activities related to generation of recombinant modified factor H binding protein (fHbp) have been carried out at Oxford University Innovation Limited. Contents of PCT/GB2013/052215 and PCT/GB2017/052535 are incorporated herein in entirety.
• the vaccine formulation is developed against Neisseria meningitidis, wherein the vaccine formulation may comprise of one or more recombinant proteins/ modified fHbp fusion proteins derived from Neisseria meningitidis and pharmaceutically acceptable carrier or excipient.
• the recombinant protein/ modified fHbp fusion protein is derived from Neisseria meningitidis serogroup A, B, C, H, I, K, L, 29E, W135, X, Y and Z.
• the recombinant protein/ modified fHbp fusion protein is derived from Neisseria meningitidis serogroup B.
• the vaccine formulation may comprise of a one or more different variants of modified factor H binding protein (fHbp) as an antigen selected from the group comprising of: Table-3: Variants of modified factor H binding protein (fHbp) Sr. No.
• fHbp-PorA chimeric protein variants SEQ ID 1 fHbpV3.45 M5:PorA316-320/exP1.14 1 (NA) 6 (AA) 2 fHbpV2.19 M6:PorA316-320/exP1.4 2 (NA) 7 (AA) 3 fHbpV1.14:PorA307-311/exP1.9 3 (NA) 8 (AA) 4 fHbpV1.1:PorA307-311/exP1.4 4 (NA) 9 (AA) 5 fHbpV1.1:PorA307-311/exP1.9 5 (NA) 10 (AA) Table-3a: Additional Variants of modified factor H binding protein (fHbp) that may be used Sr. No.
• the molecular weight of the recombinant protein/ modified fHbp fusion protein is in the range of 20 kDa to 40 kDa.
• the formulation may comprise of four different variants of modified factor H binding protein (fHbp) selected from the group summarized in Table-4. Table-4: Variants of modified factor H binding protein (fHbp) Sr. No.
• the formulation may comprise of four different variants of modified factor H binding protein (fHbp) as an antigen selected from the Table-1.
• the recombinant protein/ modified fHbp fusion protein is adsorbed onto an adjuvant to improve the immunogenicity of the antigen and maximise the protection from the associated disease.
• the adsorption of recombinant protein/ modified fHbp fusion protein onto an adjuvant is evaluated and optimized for percent adsorption of same onto an adjuvant.
• adsorption buffers are used to improve adsorption to the surface, reduce aggregation and unfolding upon binding, reduced- dose efficacy of the recombinant protein/ modified fHbp fusion protein as an antigen post adsorption.
• the recombinant protein/ modified fHbp fusion protein is adsorbed onto an adjuvant selected from the group comprising of alum adjuvant based salt such as aluminum hydroxide, aluminum phosphate, aluminum hydroxyphosphate, and potassium aluminum sulfate, or immunostimulatory component based adjuvant selected from the group consisting of an oil and water emulsion (MF-59, a liposome, a lipopolysaccharide, a saponin, lipid A, lipid A derivatives, Monophosphoryl lipid A, GLA, 3–deacylated monophosphoryl lipid A, AS01, AS03, AF3)( Include all 3 vendors of MPLA that we are exploring including synthetic etc; MPL from Salmonella enterica serotype Minnesota Re 595) (for example, Sigma Aldrich Catalog # L6895).), IL-2, RANTES, GM- CSF, TNF-a, IFN-g, G-CSF, LFA- 3, CD72, B7-1,
• the amount of the adjuvant is in the range of in the range of 0.5 mg/ml to 4.5 mg/ml.
• the adjuvant is aluminium hydroxide having particle size > 500 nm.
• the percent adsorption of the recombinant protein/ modified fHbp fusion protein on to an adjuvant is in the range of 70 % to 100 %.
• the percent adsorption of fHbp V3.45 M5 PorA 316-320 exP1.14 on to an adjuvant is in the range of 80 % to 100 %.
• the percent adsorption of fHbp V1.14 PorA 307-311 exP1.9 on to an adjuvant is in the range of 80 % to 90 %. In another embodiment, the percent adsorption of fHbp V2.19 PorA 316-320 exP1.4 on to an adjuvant is in the range of 80 % to 90 %. In another embodiment, the percent adsorption of fHbp V1.1 PorA 307-311 exP1.4 on to an adjuvant is in the range of 80 % to 90 %. In another embodiment, the percent adsorption of fHbp V1.1 PorA 307-311 exP1.9 on to an adjuvant is in the range of 70 % to 80 %.
• the pharmaceutically acceptable carrier or excipient may be selected from the group comprising of buffering agent, sugar, sugar alcohol or polyol, surfactants, polymers, salts, amino acids or pH modifiers, hydrolysed protein, preservative and liquid carriers.
• the buffering agent selected from the group consisting of carbonate, phosphate, acetate, HEPES, Succinate, TRIS, borate, citrate, lactate, gluconate and tartrate, as well as more complex organic buffering agents including a phosphate buffering agent that contains sodium phosphate and/or potassium phosphate in a ratio selected to achieve the desired pH.
• the buffering agent contains Tris (hydroxymethyl) aminomethane, or "Tris", formulated to achieve the desired pH.
• the buffering agent could be the minimum essential medium with Hanks salts.
• Other buffers such as HEPES, piperazine-N, N′-bis (PIPES), and 2-ethanesulfonic acid (MES) are also envisaged by the present disclosure.
• the buffer aids in stabilizing the recombinant protein/ modified fHbp fusion protein of the present disclosure.
• the amount of the buffer is in the range of 0.1 mM to 300 mM.
• sugars as excipient selected from the group of trehalose, mannose, raffinose, lactobionic acid, glucose, maltulose, iso- maltulose, maltose, lactose, dextrose, fructose or a combination thereof.
• the amount of the sugar is in the range of 5 mg/ml to 100 mg/ml.
• sugar alcohol or polyol as excipient selected from the group of mannitol, lactitol, sorbitol, glycerol, xylitol, maltitol, lactitol, erythritol, isomalt and hydrogenated starch hydrolysates or a combination thereof.
• the amount of the sugar alcohol or polyol is in the range of 5 mg/ml to 100 mg/ml.
• Surfactants can be classified by their 'HLB' (hydrophile/lipophile balance).
• Preferred surfactants of the invention have HLB of at least 10, preferably at least 15, and more preferably at least 16.
• Surfactants as excipient may include non-ionic surfactants such as polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 85, nonylphenoxypolyethoxethanol, octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer - 0.001%-0.05%.
• non-ionic surfactants such as polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 85, nonylphenoxypolyethoxethanol, octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxyste
• EO ethylene oxide
• PO propylene oxide
• BO butylene oxide
• DOWF AXTM tradename such as linear EO/PO block copolymers
• octoxynols which can vary in the number of repeating ethoxy (oxy-l,2-ethanediyl) groups, with octoxynol-9 (Triton X-1OO, or t-octylphenoxypolyethoxyethanol) being of particular interest
• phospholipids such as phosphatidylcholine (lecithin); nonylphenol ethoxylates, such as the TergitolTM NP series
• polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols known as Brij surfactants
• Non-ionic surfactants are preferred.
• Preferred surfactants for including in the emulsion are Tween 20.
• the amount of the surfactant is in the range of 0.01 mg/ml to 2 mg/ml.
• the polymers may include dextran, carboxymethylcellulose, hyaluronic acid, cyclodextrin, etc.
• the salts may include NaCl, KCl, KH2PO4, Na2HPO4.2H2O, CaC12, MgC12, etc.
• aminoacids as excipient may include tricine, leucine, iso-leucine, glycine, glutamine, L-arginine, L-arginine hydrochloride, lysine, L-alanine, Tryptophan, Phenylalanine, Tyrosine, Valine, Cysteine, Glycine, Methionine, Proline, Serine, Threonine.
• hydrolyzed protein may include gelatin, lactalbumin hydrolysate, monosodium glutamate, collagen hydrolysate, keratin hydrolysate, peptides, Casein hydrolysate and whey protein hydrolysate, serum albumin.
• preservative examples include 2-phenoxyethanol, Benzethonium chloride (Phemerol), Phenol, m-cresol, Thiomersal, Formaldehyde, paraben esters (e.g. methyl-, ethyl, propyl- or butyl- paraben), benzalkonium chloride, benzyl alcohol, chlorobutanol, p-chlor-m- cresol, or benzyl alcohol or a combination thereof.
• a vaccine composition may include preservative for a single immunization or may include material for multiple immunizations (i.e. a ‘multidose’ kit). The inclusion of a preservative is preferred in multidose arrangements.
• the compositions may be contained in a container having an aseptic adaptor for removal of material.
• the amount of the preservative is in the range of 1 mg/mL to 10 mg/ml.
• the suitable liquid carriers may include WFI (water for injection) and saline.
• the vaccine formulation comprises: - at least one at least one recombinant protein/ at least one modified fHbp - aluminium hydroxide; - mannitol; - phosphate; and - polysorbate.
• the vaccine formulation comprises: - at least one modified fHbp of the amino acid sequence with at least 75% identity with from any one of amino acid sequences SEQ ID NO.6 to 10 or - at least one modified fHbp encoded the nucleic acid sequence is with at least 75% identity with by any one of nucleic acid sequence SEQ ID NO.1 to 5; and - aluminium hydroxide; - mannitol; - phosphate; and - polysorbate.
• the vaccine formulation is formulated to be in a dose in the range of 0.1 mL to 1 mL, for example from 0.2 mL to 0.8 mL, 0.4 mL to 0.6 mL.
• the vaccine formulation is formulated to be at a dose of 0.5 mL.
• the recombinant protein/ modified fHbp fusion protein is present in the vaccine formulation in an amount in the range of 15 ⁇ g/ml to 200 ⁇ g/ml, for example from 20 ⁇ g/ml to 200 ⁇ g/ml, 25 ⁇ g/ml to 200 ⁇ g/ml, 25 ⁇ g/ml to 150 ⁇ g/ml, 30 ⁇ g/ml to 200 ⁇ g/ml, 30 ⁇ g/ml to 150 ⁇ g/ml, 35 ⁇ g/ml to 200 ⁇ g/ml, 35 ⁇ g/ml to 150 ⁇ g/ml, 40 ⁇ g/ml to 200 ⁇ g/ml, 40 ⁇ g/ml to 150 ⁇ g/ml, 45 ⁇ g/ml to 200 ⁇ g/ml, 45 ⁇ g/ml to 150 ⁇ g/ml, 45 ⁇ g/ml to
• the recombinant protein/ modified fHbp fusion protein is present in the vaccine formulation in an amount of 15 ⁇ g/ml, 20 ⁇ g/ml, 25 ⁇ g/ml, 30 ⁇ g/ml, 35 ⁇ g/ml, 40 ⁇ g/ml, 45 ⁇ g/ml, 50 ⁇ g/ml, 55 ⁇ g/ml, 60 ⁇ g/ml, 65 ⁇ g/ml, 70 ⁇ g/ml, 75 ⁇ g/ml, 80 ⁇ g/ml, 85 ⁇ g/ml, 90 ⁇ g/ml, 95 ⁇ g/ml, 100 ⁇ g/ml, 105 ⁇ g/ml, 110 ⁇ g/ml, 115 ⁇ g/ml, 120 ⁇ g/ml, 125 ⁇ g/ml, 130 ⁇ g/ml, 135 ⁇ g/ml, 140 ⁇ g/ml, 145 ⁇
• the vaccine formulation comprises (i) at least one recombinant protein/ modified fHbp fusion protein; (ii) aluminium hydroxide in an amount in the range of 0.5 mg/ml to 4.5 mg/ml; (iii) mannitol in an amount in the range of 5 mg/ml to 100 mg/ml; (iv) phosphate buffer in an amount in the range of 1 mM to 10 mM; and (v) polysorbate 20 in an amount 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 ⁇ g/ml to 150 ⁇ g/ml.
• the vaccine formulation comprises (i) at least two recombinant proteins/ modified fHbp fusion proteins; (ii) aluminium hydroxide in an amount in the range of 0.5 mg/ml to 4.5 mg/ml; (iii) mannitol in an amount in the range of 5 mg/ml to 100 mg/ml; (iv) phosphate buffer in an amount in the range of 1 mM to 10 mM; and (v) polysorbate 20 in an amount 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 ⁇ g/ml to 150 ⁇ g/ml.
• the vaccine formulation comprises (i) at least three recombinant proteins/ modified fHbp fusion proteins; (ii) aluminium hydroxide in an amount in the range of 0.5 mg/ml to 4.5 mg/ml; (iii) mannitol in an amount in the range of 5 mg/ml to 100 mg/ml; (iv) phosphate buffer in an amount in the range of 1 mM to 10 mM; and (v) polysorbate 20 in an amount 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 ⁇ g/ml to 150 ⁇ g/ml.
• the vaccine formulation comprises (i) at least four recombinant proteins/ modified fHbp fusion proteins; (ii) aluminium hydroxide in an amount in the range of 0.5 mg/ml to 4.5 mg/ml; (iii) mannitol in an amount in the range of 5 mg/ml to 100 mg/ml; (iv) phosphate buffer in an amount in the range of 1 mM to 10 mM; and (v) polysorbate 20 in an amount 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 ⁇ g/ml to 150 ⁇ g/ml.
• the vaccine formulation comprises: - at least one modified fHbp of the amino acid sequence with at least 75% identity with from any one of amino acid sequences SEQ ID NO.6 to 10 or - at least one modified fHbp encoded the nucleic acid sequence is with at least 75% identity with by any one of nucleic acid sequence SEQ ID NO.1 to 5 and o aluminium hydroxide; o mannitol; o phosphate; and o polysorbate.
• vaccine formulation comprises: - at least one modified fHbp of the amino acid sequence with at least 75% identity with from any one of amino acid sequences SEQ ID NO.6 to 10, in an amount in the range of 15 ⁇ g/ml to 150 ⁇ g/ml, or - at least one modified fHbp encoded the nucleic acid sequence is with at least 75% identity with by any one of nucleic acid sequence SEQ ID NO.
• vaccine formulation comprises: - fHbpV3.45 M5:PorA316-320/exP1.14 (of SEQ ID NO.6 or encoded by SEQ ID NO.
• vaccine formulation comprises: - fHbpV3.45 M5:PorA316-320/exP1.14 (of SEQ ID NO.6 or encoded by SEQ ID NO.
• vaccine formulation comprises: - fHbpV3.45 M5:PorA316-320/exP1.14 (of SEQ ID NO.6 or encoded by SEQ ID NO.
• vaccine formulation comprises: - fHbpV3.45 M5:PorA316-320/exP1.14 (of SEQ ID NO. 6 or encoded by SEQ ID NO.1) in an amount in the range of 15 ⁇ g/mL to 150 ⁇ g/mL; - fHbpV2.19 M6:PorA316-320/exP1.4 (of SEQ ID NO. 7 or encoded by SEQ ID NO.2) in an amount in the range of 15 ⁇ g/mL to 150 ⁇ g/mL; - HbpV1.1:PorA307-311/exP1.9 (of SEQ ID NO.
• the vaccine formulation comprises 2-phenoxyethanol in an amount in range of 1 mg/mL to 10 mg/ml.
• the vaccine formulation comprises (i) at least one fusion protein comprising stable non-functional/ non-lipidated fHbp and PorA VR2 loop, and (ii) at least one polysaccharide-protein conjugate.
• the vaccine formulation comprises i) at least one fusion protein comprising stable non-functional /non-lipidated fHbp and PorA VR2 loop and at least one conjugate selected from (a) a conjugate of (i) the capsular saccharide of serogroup A N.
• the vaccine formulation comprises i) at least two fusion proteins, each consisting of one fHbp variant type coupled to one PorA VR2 loop and at least one conjugate selected from (a) a conjugate of (i) the capsular saccharide of serogroup A N. meningitidis and (ii) tetanus toxoid; (b) a conjugate of (i) capsular saccharide of serogroup C N. meningitidis and (ii) CRM197; (c) a conjugate of (i) capsular saccharide of serogroup Y N.
• the vaccine formulation comprises i) at least three fusion proteins, each consisting of one fHbp variant type coupled to two PorA VR2 loop and at least one conjugate selected from (a) a conjugate of (i) the capsular saccharide of serogroup A N.
• the vaccine formulation comprises i) at least four fusion proteins, each consisting of one fHbp variant type coupled to three PorA VR2 loop and at least one conjugate selected from (a) a conjugate of (i) the capsular saccharide of serogroup A N. meningitidis and (ii) tetanus toxoid; (b) a conjugate of (i) capsular saccharide of serogroup C N. meningitidis and (ii) CRM197; (c) a conjugate of (i) capsular saccharide of serogroup Y N.
• the recombinant protein/ modified fHbp fusion protein of the present invention is co-administered with one or more vaccine selected from BEXSERO, MENVEO, MENACTRA, NIMENRIX, MenQuadFi, MENFIVE, MenAfriVac, Men AC, and Men ACHib.
• the recombinant protein/ modified fHbp fusion protein of the present invention is co-administered with MENFIVE.
• the vaccine formulation comprises 2-phenoxyethanol in an amount in range of 1 mg/mL to 10 mg/ml.
• the vaccine formulation has Zeta potential in the range of -16 mV to -30 mV.
• the vaccine formulation has osmolality in the range of 200 mOsmol/kg to 500 mOsmol/kg.
• the vaccine formulation described above is manufactured by the following broad steps, which are further explained in detail in the succeeding paragraphs: - growing host cells comprising the expression vector in nutrient medium; - inducing the host cells for expressing protein; - harvesting and separating the host cells; - lysing the harvested cells and separating host cell debris to obtain tagged proteins; - purifying tagged proteins; - removing tags from the tagged proteins to obtain recombinant proteins/ modified fHbp fusion protein; - purifying the recombinant proteins/ modified fHbp fusion protein; and - preparing vaccine formulation comprising the purified recombinant proteins/ modified fHbp fusion protein.
• the upstream bioprocess is developed and optimized to increase cell density and recombinant protein/ modified fHbp fusion protein productivity for the lead cell lines.
• the upstream bioprocess may comprise of batch, fed-batch, continuous or perfusion mode of cultivation for the production of recombinant protein/ modified fHbp fusion protein.
• the upstream bioprocess may comprise of fed-batch method comprising following steps: (a) preparing an aqueous fermenter nutrient medium and feed solution; (b) inoculating the fermenter nutrient medium with the Host cell line; (c) continuous feeding with a feed solution; (d) inducing protein expression; (e) harvesting and cell separation.
• the aqueous fermenter nutrient medium may comprise of Undefined medium, Terrific Broth (TB) Medium, Lysogenia Broth, Luria Broth or Luria-Bertani medium, chemically defined medium, M9 Minimal Medium, Chemically Defined M9 Modified Salt Medium, 2xYT medium or Super Optimal broth with Catabolite repression (SOC) Medium or combination thereof.
• LB Media Composition of Luria Broth (LB Media) is summarized in Table-5. Table-5: LB medium Composition Sr No.
• Trace Element Solution Composition Sr No. Component Quantity (per Litre) 1 FeSO4,7H2O 2.8 g 2 MnSO4.H2O 2.68 g 3 CoCl 2 ,6H 2 O 1.42 g 4 CaCl2,2H2O 1.5 g 5 CuSO 4 .5H 2 O 0.18 g 6 ZnSO 4 ,7H 2 O 0.3 g 7 HCl 87 mL
• Composition of Trace Element Solution is summarized in Table-8.
• Table-8 TB medium Composition Sr No.
• the aqueous fermenter nutrient medium may additionally comprise of antibiotic selected from the group comprising of kanamycin, neomycin, streptomycin, tobramycin, paromomycin, amphotericin B, Ampicillin, Erythromycin, Gentamycin, Nystatin Penicillin-Streptomycin, Polymyxin B, Tetracyclin, Thiabendazole or Tylosin or a combination thereof.
• the Host cell line may comprise of bacterial expression host system.
• the bacterial expression host system is Escherichia coli (E. coli) and the E. coli strain is selected from the group comprising BL21 (DE3), BL21 (DE3) pLysS*, BL21 (DE3) pLysE*, BL21 star (DE3), BL21-A1, BLR (DE3), HMS174 (DE3)**, Tuner (DE3), Origami2 (DE3)**, Rosetta2 (DE3)*, Rosettagami (DE3), Lemo21 (DE3)*, T7 Express, m15 pREP4*, C41(DE3), C43(DE3) or B834(DE3).
• E. coli Escherichia coli
• the bacterial expression host system is Escherichia coli strain B834(DE3).
• the fermentation condition post inoculating the Host cell line may comprise of temperature in the range of 35°C to 39°C; pH in the range of 5.0 to 9.0; dissolved oxygen in the range of 10 to 100%; agitation in the range of 100–1800 rpm; gas flow rate 0-2 VVM (volume of gas per unit volume of liquid per minute).
• the inoculated fermenter nutrient medium may comprise of continuous feeding with a feed solution post depletion of carbon source.
• the composition of feed solution comprising glucose is summarized in Table-9.
• Table-9 Glucose Feed Composition Sr No. Component Quantity/L 1 Glucose 100-1000 g 2 MgSO4,7H2O 10-50 g 3 Thiamine HCL 1-200 mg 4 Trace Element solution 5-20 mL (Glucose/ dextrose/ dextrose monohydrate are used interchangeably in the present application)
• the composition of feed solution comprising glycerol is summarized in Table-10.
• Table-10 Glycerol Feed Composition Sr No.
• 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%. In one embodiment, the feeding rate of glucose feed is in the range of 0.3 to 3.0 mL/min/L of culture. In one embodiment, the glucose feed may be replaced with glycerol feed solution and subsequently the host cell line may be induced for recombinant protein/ modified fHbp fusion protein expression.
• the feeding rate of glycerol feed is in the range of 0.3 to 3.0 mL/min/L of culture.
• the host cell line is induced for recombinant protein/ modified fHbp fusion protein expression using an inducer selected from lactose or its non-hydrolyzable analog isopropyl ⁇ -D-1-thiogalactopyranoside (IPTG).
• the induction parameters may comprise of inducing at OD measured at 590/600 nm in the range of 20-100.
• the inducer is IPTG at a concentration in the range of 1mM to 10 mM.
• the inducer is IPTG at a concentration in the range of 5 mM to 10 mM. In an embodiment, the inducer is lactose at a concentration in the range of 1 g/L to 50 g/L. In another embodiment, the inducer is lactose at a concentration in the range of 5 g/L to 50 g/L. In an embodiment, the inducing/ induction temperature is in the range of 17°C to 37°C, and the induction hours is in the range of 3 to 24 hours. In one embodiment, during induction number of experiments were performed to optimize induction density and length, culture temperature, culture pH, temperature shift, and glucose/glycerol feed rates.
• Table-11 fed-batch methods/protocols for the production of chimeric proteins in fermenter is summarized in Table-11.
• Table-11 fed-batch methods/protocols for the production of chimeric proteins in fermenter Sr. no Carbon Source in Carbon Source in Feed Inducer Fermentation Media 1 Glucose Glucose IPTG 2 Glycerol Glycerol IPTG/ Lactose 3 Glucose Dual Feed IPTG/ Lactose A.
• Glycerol feed in induction/ production phase In one embodiment, the culture is harvested at 3-7 hours after induction. In one embodiment, post harvesting the cells are separated from the fermenter nutrient medium by centrifugation.
• the centrifugation parameters may comprise of Relative Centrifugal Force (rcf) 6000-8000 for 30 – 60 min at 1°C to 5°C.
• post harvesting the resulting wet cell mass (g/L) may comprise of 150 – 350 g/L of harvest broth.
• the downstream bioprocess is developed and optimized for production of recombinant protein/ modified fHbp fusion protein with high yield and high purity.
• the downstream bioprocess may comprise of any one of following steps: a) Cell Lysis/Cell disruption; b) Cell Separation and Clarification; c) Purification of tagged protein; d) Affinity Tag removal; e) Protein purification; f) Concentration/ diafiltration/ buffer exchange.
• a no of experiments were performed in order to achieve the high-yield and high purity required by the prospective commercial process.
• post harvesting and cell separation the cell culture is lysed or disrupted to make the intracellular product accessible.
• the cell lysis/cell disruption is carried out by a method selected from the group comprising of Chemical, Biological, Physical or Mechanical mode or a combination thereof.
• the chemical method of cell lysis may comprise of detergents, solvents, acid, or base or a combination thereof.
• the biological method of cell lysis may comprise of Lysozyme.
• the physical method of cell lysis may comprise of Freeze– thawing, Acoustic cavitation, Hydrodynamic cavitation or Osmotic shock or a combination thereof.
• the Mechanical mode of cell lysis may comprise of Grinding (e.g. bead mill) or High-pressure homogenization or a combination thereof.
• the cell lysis/ cell disruption is carried out by mechanical mode comprising high pressure homogenization.
• the cell lysis/ cell disruption is carried out by chemical lysis using a lysis buffer.
• the cell lysis/cell disruption is carried out by a combination of chemical lysis followed by mechanical mode comprising of high-pressure homogenization.
• high pressure homogenization comprises pressure in a range from 1000-1500 Bar, Cycles in a range from 3-8, flow rate of 9 L/hour +/- 5%, temperature in a range from 4 °C to15 °C.
• the flow rate is increased as the process is scaled up.
• the mechanical lysis is carried out using a homogenizer.
• the lysis buffer comprises sodium phosphate in the range of 10-100 mM, pH 6.0-8.0, Imidazole in the range of 10-50 mM, Sodium chloride (NaCl) in the range of 100-500 mM.
• Lysis buffer A lysis buffer is a buffer solution used for the purpose of breaking open cells for use.
• post cell lysis/cell disruption the intracellular product is evaluated for disruption efficiency and potential product loss by measuring either or all of the following parameters comprising of total protein release, Cell viability and Particle size distribution.
• the intracellular product is evaluated for recombinant protein/ modified fHbp fusion protein expression levels by a method selected from the group comprising of SDS-PAGE, Western blot, ELISA, enzyme assays.
• the recombinant protein/ modified fHbp fusion protein may comprise of fHbp- PorA chimeric protein.
• the fHbp-PorA chimeric protein may comprise of affinity tags selected from the group comprising of peptide tag and/or a large polypeptide.
• Affinity tag When devising a project where a purified soluble active recombinant protein/ modified fHbp fusion protein is needed (as is often the case), it is invaluable to have means to (i) detect it along the expression and purification scheme, (ii) attain maximal solubility, and (iii) easily purify it from the E. coli cellular milieu.
• the expression of a stretch of amino acids (peptide tag) or a large polypeptide (fusion partner) in tandem with the desired protein to form a tagged fusion protein may allow these three goals to be straightforwardly reached.
• the fHbp-PorA chimeric protein may comprise of small peptide tags selected from the group comprising of poly-Arg-, FLAG-, poly-His-, c-Myc-, S-, and Strep II-tags and a large polypeptide (fusion partner) selected from the group comprising of maltose-binding protein (MBP), N-utilization substance protein A (NusA), thioredoxin (Trx), glutathione S- transferase (GST), ubiquitin and SUMO.
• MBP maltose-binding protein
• NusA N-utilization substance protein A
• Trx thioredoxin
• GST glutathione S- transferase
• the fHbp-PorA chimeric protein may comprise of poly-His and MBP tag; wherein the expression vector may comprise of pET28a-His-MBP-TEV-fHbp-PorA.
• post cell lysis/cell disruption the cell mass is subjected to cell separation and clarification.
• the cell separation is carried out by centrifugation, wherein the intracellular product comprising of recombinant protein/ modified fHbp fusion protein is separated in the supernatant.
• centrifugation is carried out at 4000 – 10000 RPM for 30 - 60 min at 1°C to 5°C.
• the supernatant is subjected to clarification for removal of lysate proteins.
• the clarification is carried out by using filters selected from the group comprising of decreasing pore sizes (e.g., 6 ⁇ , 5 ⁇ , 0.8 ⁇ , 0.65 ⁇ , 0.45 ⁇ , 0.2 ⁇ ).
• filters selected from the group comprising of decreasing pore sizes (e.g., 6 ⁇ , 5 ⁇ , 0.8 ⁇ , 0.65 ⁇ , 0.45 ⁇ , 0.2 ⁇ ).
• filters selected from the group comprising of decreasing pore sizes (e.g., 6 ⁇ , 5 ⁇ , 0.8 ⁇ , 0.65 ⁇ , 0.45 ⁇ , 0.2 ⁇ ).
• Suitable commercially available filters and filtration devices are well known in the art and can be selected by those of skill.
• Exemplary filtration devices could be made of Polypropylene or Cellulose acetate or Polyethersulfone and the commercially available filters could be Millipak (Millipore), Kleenpak (Pall) and SartobranTM
• post cell separation and clarification the supernatant comprising of tagged recombinant protein/ modified fHbp fusion protein is subjected to purification comprising of chromatography-based purification methods, ultrafiltration, diafiltration or combination thereof.
• the chromatography-based purification methods comprise Ion-exchange or affinity chromatography or a combination of both.
• the chromatography-based purification methods comprise affinity chromatography-based purification wherein the tagged recombinant protein/ modified fHbp fusion protein is bound to an affinity column and is further subjected to washing with increasing concentration of buffer comprising sodium phosphate + NaCl, + Imidazol. The tagged protein is eluted with elution buffer.
• the affinity column may comprise of Immobilized metal affinity column resin.
• Immobilized metal affinity chromatography (IMAC) resin is a high binding-capacity resin for purifying his-tagged proteins wherein it relies on the affinity that His has for immobilized transition metals.
• the Immobilized metal affinity column resin comprises metal ions selected from copper, zinc, nickel, and the like.
• the Immobilized metal affinity column resin comprise of uncharged or precharged with Ni2+ resin. In an embodiment, the uncharged form is charged with the metal ion of choice for even greater purification flexibility.
• the Immobilized metal affinity column resin may comprise of Ni Sepharose 6 Fast Flow Column.
• the wash buffer comprises 20-50mM Sodium-Phosphate buffer, 50- 300mM Sodium Chloride (NaCl) pH 7.4 and increased concentration of Imidazol comprises 20 – 80 mM (each 5-6 CV).
• the elution of the target protein is carried out using elution buffer comprising of 20-50mM Sodium-Phosphate buffer, 50-120mM Sodium Chloride (NaCl) pH 7.4 and Imidazol comprising of 100 -300 mM.
• post elution the elute comprising the tagged recombinant protein/ modified fHbp fusion protein is further subjected to concentration and diafiltration.
• concentration and diafiltration is carried out using tangential flow filtration (TFF) typically through filters with a molecular weight cut off (MWCO) ranging in between 5KDa -50KDa and 10-50mM Sodium-Phosphate buffer pH7.4.
• concentration and diafiltration is carried out using tangential flow filtration (TFF) typically through filters with a molecular weight cut off (MWCO) ranging in between 5 kDa to 50 kDa and 10-50mM Sodium-Phosphate buffer pH 7.4 or Tris-HCl buffer pH 8.5.
• the tagged recombinant protein/ modified fHbp fusion protein is subjected to TEV protease digestion for removal of affinity tag.
• the recombinant protein/ modified fHbp fusion protein comprising of poly-His and MBP tag is subjected to TEV protease digestion for removal of His-MBP tag, wherein the TEV protease digestion comprises incubating the recombinant protein comprising of poly-His and MBP tag with TEV protease.
• Tobacco etch virus (TEV) protease is a 27 ⁇ kDa catalytic domain of the polyprotein nuclear inclusion a (NIa) in TEV, which recognizes the specific amino acid sequence ENLYFQG/S and cleaves between Q and G/S.
• the tagged recombinant protein/ modified fHbp fusion protein is subjected to TEV protease digestion for removal of affinity tag, wherein the HIS-GST-TEV protease with at least 75% identity with amino acid SEQ ID NO.12 or is encoded by nucleic acid sequence with at least 75.0% identity with sequence of SEQ ID NO.11.
• the His-GST-TEV protease has a molecular size of ⁇ 50 kDa.
• the tagged recombinant protein/ modified fHbp fusion protein is subjected to TEV protease digestion for removal of affinity tag, wherein the HIS-GST-TEV protease has at least 75% or 80.0%, or 85.0%, or 90.0% or 95.0% or 98.0%, or 99.0%, or 99.5% identity with amino acid SEQ ID NO.12 or is encoded by nucleic acid sequence with at least 75.0%, or 80.0%, or 85.0%, or 90.0% or 95.0% or 98.0%, or 99.0%, or 99.5% identity with sequence of SEQ ID NO.11.
• the ratio of tagged recombinant protein/ modified fHbp fusion protein: TEV protease is in the range of 5:1 to 30:1 or higher based on activity and purity of TEV protease.
• the tagged recombinant protein/ modified fHbp fusion protein (substrate): TEV protease ratio is 20:1.
• the temperature of incubation is in the range of 4°C to 35°C and time of incubation is in the range of 15 to 20 hours.
• the TEV protease digestion is performed in presence of 10-50mM Sodium-Phosphate buffer pH 7.4, followed by addition of dithiothreitol (DTT) at a final concentration of 0.5 mM to 5 mM.
• the TEV protease used for tag removal/ digestion is produced by a method comprising the following steps: - growing host cells comprising the expression vector in nutrient medium; - inducing the host cells for expressing TEV protease; - harvesting and separating the host cells; - lysing the harvested cells and separating host cells to obtain TEV protease; and - purifying TEV protease.
• the host cell may comprise of Escherichia coli (E. coli) and the E. coli strain may be selected from the group comprising 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), RosettaTM(DE3)pLysS or B834(DE3).
• E. coli Escherichia coli
• the host cell for expression of TEV protease is E. coli RosettaTM(DE3)pLysS.
• the TEV protease enzyme produced by the aforementioned method includes the TEV protease having SEQ ID NO.12 or encoded by SEQ ID NO.11.
• the TEV protease is purified using the following non-limiting steps: - lysing the harvested cells; - separating host cell debris and collecting the supernatant; - subjecting the supernatant to at least one wash using a wash buffer; - eluting purified TEV protease with an elution buffer; - concentrating the purified TEV protease.
• the wash buffer comprises sodium phosphate, sodium chloride and imidazole.
• the supernatant is subjected to at least 4 wash steps with wash buffer having pH 7.4. In another embodiment, the supernatant is subjected to at least 2 wash steps with wash buffer having pH 8.5.
• the method and parameters for the production and purification of TEV protease is similar to that of the tagged recombinant protein/ modified fHbp fusion protein described above and is explained in detail in succeeding examples.
• post tag removal the recombinant protein/ modified fHbp fusion protein is subjected to purification comprising of chromatography-based purification methods, ultrafiltration, diafiltration or combination thereof.
• 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, pseudoaffinity 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, reverse phase chromatography, and combinations thereof.
• HPLC high-pressure liquid chromatography
• the chromatography-based purification methods comprise Ion-exchange or affinity chromatography or a combination of both.
• the chromatography-based purification methods comprise Ion- exchange followed by affinity chromatography-based purification.
• the Ion-exchange chromatography comprises anion exchange chromatography.
• the anion exchange chromatography comprises Strong anion exchange chromatography.
• the affinity column may comprise Immobilized metal affinity column resin.
• Immobilized metal affinity chromatography (IMAC) resin is a high binding-capacity resin for purifying his-tagged proteins wherein it relies on the affinity that His has for immobilized transition metals.
• the Immobilized metal affinity column resin comprises uncharged or pre- charged with Ni2+ resin. In an embodiment, the uncharged form is charged with the metal ion of choice for even greater purification flexibility. In an embodiment, the Immobilized metal affinity column resin comprises Ni Sepharose 6 Fast Flow Column. Chromatography could be multimodal anion exchange resin CaptoTM Adhere, Capto adhere ImpRes, Capto MMC ImpRes or any other mixed mode combination of chromatography including Ion exchange, Electrostatic interaction, Hydrogen bonding and Hydrophobic interaction. In an embodiment, the concentration and diafiltration is carried out using tangential flow filtration (TFF) typically through filters with a molecular weight cut off (MWCO) ranging in between 5KDa -50KDa.
• TMF tangential flow filtration
• MWCO molecular weight cut off
• the elute comprising the recombinant protein/ modified fHbp fusion protein is sterilized by (Direct flow filtration (DFF) through at least one sterilization grade filter to obtain a filtrate comprising sterilized recombinant protein/ modified fHbp fusion protein.
• DFF Direct flow filtration
• the sterilization grade filter may be selected from the group comprising of 0.8 ⁇ , 0.45 ⁇ , 0.2 ⁇ .
• commercially available filters and filtration devices are well known in the art and can be selected by those of skill.
• Exemplary filtration devices could be made of Polypropylene or Cellulose acetate or Polyethersulfone or Polyvinylidene difluoride and the commercially available filters could be Millipak (Millipore), Kleenpak (Pall) and SartobranTM P filtration devices.
• the recombinant protein/ modified fHbp fusion protein additionally comprises inclusion bodies (IB’s).
• IBs inclusion bodies
• IBs inclusion bodies
• IBs The buildups of protein aggregates/insoluble proteins are known as IBs. IB formation results from an unbalanced equilibrium between protein aggregation and solubilization.
• the recombinant protein/ modified fHbp fusion protein comprising of inclusion bodies (IB’s) is subjected to: a) Inclusion body wash and recovery; b) Inclusion bodies (IBs) solubilization and Refolding;
• the recombinant protein/ modified fHbp fusion protein comprising of inclusion bodies (IB’s) is subjected to Urea denaturation and Inclusion bodies solubilized in urea are bound to an affinity column and is further subjected to washing with reducing concentration of urea and refolding buffer that promotes correct refolding.
• the target protein is eluted with elution buffer.
• denaturation is carried out using denaturation buffer composition comprising of 20-50mM Sodium-Phosphate buffer, 50-120mM Sodium Chloride (NaCl) pH 7.4 and 5-10M Urea.
• washing with reducing concentration of urea comprises washing with 20-50mM Sodium-Phosphate buffer, 50-120mM Sodium Chloride (NaCl) pH 7.4.
• reducing concentration of Urea comprises 8M, 6M, 4M, 2M, 1M (each 5-6 CV).
• elution of the target protein is carried out using elution buffer comprising imidazole 40mM-400mM.
• the affinity column comprises Immobilized metal affinity column resin.
• Immobilized metal affinity chromatography (IMAC) resin is a high binding-capacity resin for purifying his-tagged proteins wherein it relies on the affinity that His has for immobilized transition metals.
• the Immobilized metal affinity column resin comprises uncharged or pre- charged with Ni2+ resin.
• the uncharged form is charged with the metal ion of choice for even greater purification flexibility.
• the Immobilized metal affinity column resin comprises Ni Sepharose 6 Fast Flow Column.
• the supernatant comprising of inclusion bodies is subjected to protein precipitation prior to urea denaturation.
• the protein precipitation is carried out using Ammonium sulphate.
• post Urea denaturation the supernatant is subjected to filtration using 0.22 ⁇ M filter.
• post elution the elute comprising the target recombinant protein/ modified fHbp fusion protein is further subjected to concentration and diafiltration.
• concentration and diafiltration is carried out using tangential flow filtration (TFF) typically through filters with a molecular weight cut off (MWCO) ranging in between 5kDa -50kDa.
• the concentration of the recombinant protein/ modified fHbp fusion protein is more than 0.95 mg/ ml.
• the glucose feed is stopped, and glycerol feed is started when OD at 590/600 nm is 20-100 and inducing the culture by adding and/or maintaining lactose at 1-50 g/L in fed batch mode.
• the purified chimeric/ recombinant protein/ modified fHbp fusion protein is stored at 2°C to 8°C till further use.
• the purified chimeric/ recombinant protein/ modified fHbp fusion protein is stored at 2°C to 8°C in the presence of a stabilizer(s).
• the stabilizer is selected from TRIS, Tween/polysorbate, non-ionic detergent, such as polyethyleneglycol lauryl ether (BRIJ35), sucrose (up to 5%.), and the like.
• the recombinant protein/ modified fHbp fusion protein formulation is optimized for improving immunogenicity, improving stability and maintaining stability over long-term storage of recombinant protein/ modified fHbp fusion protein antigens.
• the optimized vaccine formulation has low viscosity, is devoid of aggregation, and has long-term stability across wide temperature ranges.
• the optimized vaccine formulation includes solid or liquid carriers. In one embodiment, the vaccine formulation is fully liquid.
• Suitable forms of liquid preparation include solutions, suspensions, emulsions, syrups, isotonic aqueous solutions, viscous compositions and elixirs that are buffered to a selected pH.
• the vaccine formulation comprises polymers or other agents to control the consistency of the composition, and/or to control the release of the antigen/ secreted protein from the composition.
• the vaccine formulation is in the form of transdermal preparations including lotions, gels, sprays, ointments or other suitable techniques. If nasal or respiratory (mucosal) administration is desired (e.g., aerosol inhalation or insufflation), compositions can be in a form and dispensed by a squeeze spray dispenser, pump dispenser or aerosol dispenser.
• Aerosols are usually under pressure by means of a hydrocarbon.
• Pump dispensers can preferably dispense a metered dose or a dose having a particular particle size.
• the immunogenic compositions contain a major amount of water (preferably purified water) in addition to the active ingredient(s).
• the vaccine formulation is stable at 2-8 °C from 12 to 36 months; at 25 °C from 2 to 6 months; at 37 °C from 1 week to 4 weeks, at 42 °C for 2-7 days, and at 55 °C for 2-7 days.
• the vaccine formulation is a lyophilized/ freeze dried formulation.
• the final pH of the formulation may be in the range of pH 6.0 to pH 8.0.
• a modified fHbp, nucleic acid, or formulation according to the invention for use in the treatment or prevention of a pathogenic infection or colonisation of a subject.
• a method of treatment or prevention of a pathogenic infection or colonisation of a subject comprising the administration of a modified fHbp, nucleic acid, or composition according to the invention to the subject.
• a method of vaccination comprising the administration of a modified fHbp, nucleic acid, or composition according to the invention to a subject.
• the modified fHbp is immunogenic involving administration of an immunologically effective amount of the immunogenic formulation on to a human subject via parenteral or subcutaneous or intradermal, intramuscular or intraperitoneal or intravenous administration or injectable administration or sustained release from implants or administration by eye drops or nasal or rectal or buccal or vaginal, peroral or intragastric or mucosal or perlinqual, alveolar or gingival or olfactory or respiratory mucosa administration or any other routes of immunization.
• co-administered means that the different immunogenic compositions/ vaccines can be administered either separately or as a combination. Where the vaccines are administered separately, they will typically be administered at different sites e.g.
• two vaccines may be administered contralaterally (e.g. both arms or both legs, or a contralateral arm and leg) or ipsilaterally (e.g. the arm and leg on the same side of the body).
• the vaccines are administered separately, they are administered at substantially the same time (e.g. during the same medical consultation or visit to a healthcare professional or vaccination centre), such as within 1 hour of each other.
• administration as a combination may be performed.
• co-immunisation may use a combination vaccine i.e. a single composition in which the different immunogens are admixed.
• compositions of the invention will generally be administered directly to a patient.
• Direct delivery may be accomplished by parenteral injection (e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly, or to the interstitial space of a tissue), or by rectal, oral, vaginal, topical, transdermal, intranasal, ocular, aural, pulmonary or other mucosal administration.
• Intramuscular administration to the thigh or the upper arm is preferred.
• Injection may be via a needle (e.g. a hypodermic needle), but needle-free injection may alternatively be used.
• a typical intramuscular dose is about 0.5 ml.
• the composition may further be provided in a ‘multidose’ kit, i.e., a single container containing sufficient composition for multiple immunisations. Multidoses may include a preservative, or the multidose container may have an aseptic adaptor for removal of individual doses of the composition.
• the subject who is immunized is a human being, who may be any age e.g.0-12 months old, 1 -5 years old, 5-18 years old, 18-55 years old, or more than 55 years old.
• the subject who is immunized is an adolescent (e.g. 12-18 years old) or an adult (18 years or older).
• the subject is an adolescent or adult who has been immunized against N. meningitidis in childhood (e.g. before 12 years of age), and who receives a booster dose of an immunogenic composition according to the invention.
• the vaccine formulation of the present invention elucidates cross protection against Neisseria gonorrhea strains and Neisseria meningitidis serogroups ACWYX.
• the administration may be provided in a therapeutically effective amount.
• a skilled person will be capable of determining an appropriate dosage and repetitions for administration.
• the vaccine formulation could be formulated as single dose vials or multidose vials (2 Dose or 5 Dose or 10 Dose vials) or multidose kit or as pre-filled syringes wherein the said vaccine formulation may be given in a single dose schedule, or preferably a multiple dose schedule in which a primary course of vaccination is followed by 1-3 separate doses given at subsequent time intervals after 1-3 years if needed.
• the dosage regimen will also, at least in part, be determined on the need of a booster dose required to confer protective immunity.
• the vaccine formulation is formulated for administration to a human subject elders, adolescents, adults or children less than 2 years of age or more than 2 years of age according to a one dose or two dose regimens or 3 dose regimens consisting of a first dose and/or a second dose to be administered between 3 months to 2 years after the first dose and/or a third dose to be administered between 3 months to 2 years after the second dose.
• the subject is a mammal, such as human.
• the infection is a bacterial infection.
• the infection is meningitis, such as Neisseria meningitidis, or Neisseria gonorrhoeae.
• the modified fHbp according to the invention and at least one other prophylactically or therapeutically active molecule.
• the at least one other prophylactically or therapeutically active molecule comprises a vaccine or antigen different to the modified fHbp according to the invention herein.
• the antigen is selected from but not limited to Diphtheria toxoid (D), Tetanus toxoid (T), Whole cell pertussis (wP), hepatitis B virus surface antigen (HBsAg), Haemophilus influenzae b PRP-Carrier protein conjugate ( ⁇ ib), Haemophilus influenzae (a, c, d, e, f serotypes and the unencapsulated strains), Neisseria meningitidis A antigen(s), Neisseria meningitidis C antigen(s), Neisseria meningitidis W-135 antigen(s), Neisseria meningitidis Y antigen(s), Neisseria meningitidis X antigen(s), Streptococcus Pneumoniae antigen(s), Neisseria meningitidis B bleb or purified antigen(s), Staphylococcus
• the present invention envisages compositions comprising antigens for immunising against other diseases or infections in addition to fHbp-PorA chimeric antigens.
• the composition comprises the following additional antigens: - a protein antigen from PorB, Fet A, OmpC, NHBA, NadA, meningococcal antigen 287, NspA, HmbR, NhhA, App, 936, - a saccharide antigen from N.
• a tetanus antigen such as a tetanus toxoid
• an antigen from Bordetella pertussis such as a tetanus toxoid
• a saccharide antigen from Haemophilus influenzae B such as IPV, - measles, mumps and/or rubella antigens
• - influenza antigen(s) such as the haemagglutinin and/or neuraminidase surface proteins
• - antigen (protein or saccharide) from Streptococcus agalactiae group B streptococcus
• - antigen (protein or saccharide) from Streptococcus pyogenes group A streptococcus
• - antigen (protein or saccharide) from Staphylococcus aureus such as Salmonella Spp.
• the at least one other prophylactically or therapeutically active molecule comprises a monovalent capsule polysaccharide- protein conjugate vaccine.
• the monovalent protein capsule polysaccharide vaccine may comprise any of Neisseria meningitidis serogroup C or A capsule with bacterial toxoids, bi-valent vaccines (with serogroup C and A capsular polysaccharide conjugated to bacterial toxoids), quadrivalent (serogroups A, C, Y, W) or pentavalent (A, C, Y, W, X) conjugate vaccines.
• the at least one other prophylactically or therapeutically active molecule may comprise a conjugate vaccine, wherein antigen(s) comprising the fHbp scaffold bearing exogenous peptide loops (such as PorA loops) may be incorporated as the protein carrier molecule in the conjugate vaccine.
• the conjugate vaccine may comprise any of serogroup capsular polysaccharides from A, C, Y, W, or X strains individually or in combination.
• Combination Vaccines could be selected from Hexavalent (ACWYX-B), Quadrivalent (AC- Hib-B), Trivalent (AC-B), Bivalent (A-B, X-B, C-B).
• the liquid N meningitidis serogroup B vaccine is reconstituted with Lyophilized ACWYX conjugate vaccine for a bed side administration.
• instant invention relates to a method of inducing an immune response against Neisseria meningitidis in a mammal. The method includes administering to the mammal an effective amount of an immunogenic composition including i) at least one fusion protein comprising stable non-functional /non-lipidated fHbp and PorA VR2 loop of PorA and at least one conjugate selected from (a) a conjugate of (i) the capsular saccharide of serogroup A N.
• An aspect of the present invention relates to a method of inducing an immune response against Neisseria meningitidis in a mammal.
• the method includes administering to the mammal an effective amount of an immunogenic composition including i) at least two fusion proteins, each consisting of one fHbp variant type coupled to one PorA VR2 loop and at least one conjugate selected from (a) a conjugate of (i) the capsular saccharide of serogroup A N. meningitidis and (ii) tetanus toxoid; (b) a conjugate of (i) capsular saccharide of serogroup C N.
• Another aspect of the invention relates to a method of inducing an immune response against Neisseria meningitidis in a mammal.
• the method includes administering to the mammal an effective amount of an immunogenic composition including i) at least three fusion proteins, each consisting of one fHbp variant type coupled to two PorA VR2 loop and atleast one conjugate selected from (a) a conjugate of (i) the capsular saccharide of serogroup A N. meningitidis and (ii) tetanus toxoid; (b) a conjugate of (i) capsular saccharide of serogroup C N. meningitidis and (ii) CRM197; (c) a conjugate of (i) capsular saccharide of serogroup Y N.
• an immunogenic composition including i) at least three fusion proteins, each consisting of one fHbp variant type coupled to two PorA VR2 loop and atleast one conjugate selected from (a) a conjugate of (i) the capsular saccharide of serogroup A N. meningitidis and (ii) tetanus to
• Yet another aspect of the instant invention relates to a method of inducing an immune response against Neisseria meningitidis in a mammal.
• the method includes administering to the mammal an effective amount of an immunogenic composition including i) at least four fusion proteins, each consisting of one fHbp variant type coupled to three PorA VR2 loop and at least one conjugate selected from (a) a conjugate of (i) the capsular saccharide of serogroup A N. meningitidis and (ii) tetanus toxoid; (b) a conjugate of (i) capsular saccharide of serogroup C N. meningitidis and (ii) CRM197; (c) a conjugate of (i) capsular saccharide of serogroup Y N.
• fHbp-PorA Chimeric antigens for use with the invention comprise an amino acid sequence: having 50% or more identity (e.g.
• H44/76 ⁇ fHbp ⁇ PorA:fHbp V1.1 2. H44/76 ⁇ fHbp ⁇ PorA:fHbp V1.14 3. H44/76 ⁇ fHbp ⁇ PorA:fHbp V2.19 4. H44/76 ⁇ fHbp ⁇ PorA:fHbp V3.45
• meningitidis strains M15240912, M16240272, M15240460 or any other appropriate strain may be used for SBA.
• factor H binding protein fHbp
• the use as an epitope display scaffold may comprise the use of a factor H binding protein (fHbp) comprising any of the modifications described herein.
• compositions or modified fHbps according to the invention may be useful as diagnostic reagents and as a measure of the immune competence of a vaccine.
• the immune response elicited by the modified fHbp of the invention may affect the ability of Neisseria meningitidis (Nm) to infect a subject immunised with the modified fHbp of the invention.
• Nm Neisseria meningitidis
• the ability of Nm to infect a subject immunised with the modified fHbp of the invention is impeded or prevented.
• the immune response elicited may recognise and destroy Nm. Alternatively, or additionally, the immune response elicited may impede or prevent replication of Nm. Alternatively, or additionally, the immune response elicited may impede or prevent Nm causing disease in the human or non-human animal.
• TECHNICAL ADVANTAGES 1.
• the present invention provides an efficient platform process for manufacturing an effective vaccine against Neisseria meningitidis that meets multiple criterion including improved immunogenicity, safety, and affordability. 2.
• the present invention provides a method to develop and optimize upstream bioprocess to increase cell density and recombinant protein/ modified fHbp fusion protein productivity for the lead cell lines. 3.
• the present invention provides a method to develop and optimize downstream bioprocess for production of recombinant protein/ modified fHbp fusion protein with high yield and high purity. 4.
• the improved formulation overcomes the limitations of the prior-art and shows low viscosity, devoid of aggregation; showing long-term stability across wide temperature ranges indirectly preserving the desired characteristics of the recombinant protein/ modified fHbp fusion protein, including high stability and immunogenicity. 5.
• ChAs exploit fHbp (non- lipidated) as a molecular scaffold to present the surface exposed PorA VR2 loop, which is achieved by inserting the VR2 loop (“10-20 amino acid” PorA VR2 loops instead of a “whole PorA protein) into a ⁇ -turn region in fHbp. ChAs retain epitopes from both fHbp and PorA and are found to elicit functional immune responses against both antigens. The integration of a VR2 loop does not alter the overall architecture of fHbp and that the VR2 loop folds into a conformation recognized by a bactericidal mAb. 7.
• the chimeric proteins that are soluble and high yielding are stable, wherein fHbp and the PorA VR2 loop both are immunogenic.
• the Chimeras generated are composed of the most prevalent fHbp and PorA antigens to maximize vaccine coverage as Chimera composition mirrors the prevalent fHbp and PorA antigens circulating within a given geographical area.
• PorA loop at a specific position in fHbp results in desired reduction in Factor H binding (reduced by at least 10 %, at least 50%; preferably >70% as compared to wild type) & having Molecular weight in the range of 20 kDa to 40 kDa, while preserving immunogenic epitopes of both fHbp & PorA (Table-12).
• Table-12 fHbp-PorA chimeras Sr. No.
• a modified factor H binding protein comprising wild type fHbp variant and at least one exogenous peptide loop(s), wherein a. the modified factor H binding protein (fHbp) is selected from amino acid sequence with at least 75% identity with any one of sequences of SEQ ID Nos 6 to 10, b. wherein the at least one exogenous peptide loop(s) is immunogenic, c. wherein the at least one exogenous peptide loop(s) is derived from a bacterial membrane protein, d. wherein the modified fHbp is a fusion protein, e.
• the fHbp variant is selected from v1, v2 and v3, modified with at least one PorA loop comprising at least 10 amino acids inserted into a ⁇ -turn region in fHbp; and f. wherein the PorA loop is selected from VR1, and VR2.
• a nucleic acid sequence encoding the modified fHbp wherein the nucleic acid sequence is with at least 75% identity with any one of sequences of SEQ ID NO.1 to 5.
• V. An immunogenic composition comprising at least one modified fHbp as disclosed in any one of previous embodiments or the nucleic acid sequence encoding the modified fHbp as disclosed in embodiment IV.
• composition as disclosed in any one of the previous embodiments, wherein the composition further comprises at least one other prophylactically or therapeutically active molecule comprising a monovalent protein: capsule polysaccharide vaccine.
• composition further comprises at least one other prophylactically or therapeutically active molecule comprising a monovalent protein: capsule polysaccharide vaccine.
• fHbp scaffold bearing exogenous peptide loops is incorporated as the protein carrier molecule in the conjugate vaccine.
• the immunogenic composition as disclosed in any one of the previous embodiments comprising a recombinant protein/ modified fHbp fusion protein in combination with atleast one additional antigen selected from: - a protein antigen from PorB, Fet A, OmpC, NHBA, NadA, meningococcal antigen 287, NspA, HmbR, NhhA, App, 936, - a saccharide antigen from N. meningitidis serogroup A, C, W, Y and/or X, - a saccharide antigen from Streptococcus pneumoniae, - a diphtheria antigen, such as a diphtheria toxoid e.g.
• a tetanus antigen such as a tetanus toxoid
• an antigen from Bordetella pertussis such as a tetanus toxoid
• a saccharide antigen from Haemophilus influenzae B such as IPV, - measles, mumps and/or rubella antigens
• - influenza antigen(s) such as the haemagglutinin and/or neuraminidase surface proteins
• - antigen (protein or saccharide) from Streptococcus agalactiae group B streptococcus
• - antigen (protein or saccharide) from Streptococcus pyogenes group A streptococcus
• - antigen (protein or saccharide) from Staphylococcus aureus such as Salmonella Spp.
• XII The immunogenic composition as disclosed in any one of the previous embodiments, wherein the fHbp scaffold bearing exogenous peptide loops is incorporated as the protein carrier molecule in the polysaccharide conjugate vaccine selected from (A, X, C, W, Y), Bivalent (A-B, X-B, C-B), Trivalent (AC-B, AC-Hib), Quadrivalent (AC- Hib-B), Pentavalent (ACWYX) or Hexavalent (ACWYX-B).
• the polysaccharide conjugate vaccine selected from (A, X, C, W, Y), Bivalent (A-B, X-B, C-B), Trivalent (AC-B, AC-Hib), Quadrivalent (AC- Hib-B), Pentavalent (ACWYX) or Hexavalent (ACWYX-B).
• XIV. A combination of the modified fHbp as disclosed in any one of embodiments I to III, a nucleic acid as disclosed in embodiment IV, or a composition as disclosed in any one of embodiments V to IX, and at least one other prophylactically or therapeutically active molecule.
• the at least one other prophylactically or therapeutically active molecule comprises a conjugate vaccine, comprising any of serogroup capsular polysaccharides selected from A, C, Y, W, or X strains, or combinations thereof.
• a conjugate vaccine comprising any of serogroup capsular polysaccharides selected from A, C, Y, W, or X strains, or combinations thereof.
• capsule polysaccharide vaccine comprises any of serogroup C or A capsule with bacterial toxoids, bivalent vaccines (with serogroup C and A capsular polysaccharide conjugated to bacterial toxoids), quadrivalent (serogroups A, C, Y, W polysaccharides conjugated to bacterial toxoids) or pentavalent (serogroups A, C, Y, W, X polysaccharides conjugated to bacterial toxoid) conjugate vaccines.
• fHbp factor H binding protein as disclosed in embodiments I to III as and when used as an epitope display scaffold.
• a vaccine formulation comprising at least one recombinant protein/ modified fHbp fusion protein an adjuvant and one or more pharmaceutically acceptable excipient, wherein the recombinant protein/ modified fHbp fusion protein is at least one selected from the group consisting of Transferrin Binding Protein, Neisserial Heparin Binding Protein, Neisserial Surface Protein A, PorA, meningococcal enterobactin receptor FetA, Neisserial Adhesin A, the factor H binding protein (fHbp) as disclosed in any one of the previous embodiments or a combination thereof.
• fHbp the factor H binding protein
• fHbp has molecular weight in the range of 10 kDa to 200 kDa, preferably up to 50 kDa.
• the adjuvant is selected from the group consisting of aluminum hydroxide, aluminum phosphate, aluminum hydroxyphosphate, and potassium aluminum sulfate, MF-59, a liposome, a lipopolysaccharide, a saponin, lipid A, lipid A derivatives, Monophosphoryl lipid A, GLA, 3–deacylated monophosphoryl lipid A, AS01, AS03, AF3, IL-2, RANTES, GM- CSF, TNF-a, IFN-g, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L and 41 BBL, an oligonucleotide, an oligonucleotide comprising at least one unmethylated CpG and/or a liposome, Freund’s adjuvant, Freund’s complete adjuvant, Freund’s incomplete adjuvant, polymers, co-polymers such as polyoxyethylene-polyoxy
• XXIII The vaccine formulation as disclosed in any one of the previous embodiments, wherein the adjuvant is aluminium hydroxide having particle size >500 nm.
• XXIV The vaccine formulation as disclosed in any one of the previous embodiments, wherein the one or more pharmaceutically acceptable excipient is a. a buffering agent selected from carbonate, phosphate, acetate, HEPES, Succinate, TRIS, borate, citrate, lactate, gluconate, tartrate, or a combination thereof; b.
• a sugar selected from trehalose, mannose, raffinose, lactobionic acid, glucose, maltulose, iso- maltulose, maltose, lactose, dextrose, fructose, or a combination thereof;
• a sugar alcohol or polyol selected from mannitol, lactitol, sorbitol, glycerol, xylitol, maltitol, lactitol, erythritol, isomalt and hydrogenated starch hydrolysates or a combination thereof; d.
• a surfactant selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 85, nonylphenoxypolyethoxethanol, octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate, soy lecithin, a poloxamer, copolymers of ethylene oxide (EO), propylene oxide (PO), and/or butylene oxide (BO), octoxynols, phospholipids, nonylphenol ethoxylates, polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols, sorbitan esters or a combination thereof; e.
• EO ethylene oxide
• PO propylene oxide
• BO butylene oxide
• hydrolysed protein selected from gelatin, lactalbumin hydrolysate, monosodium glutamate, collagen hydrolysate, keratin hydrolysate, peptides, Casein hydrolysate, whey protein hydrolysate, serum albumin or a combination thereof; i. a preservative selected from phenoxyethanol, Benzethonium chloride (Phemerol), Phenol, m-cresol, Thiomersal, Formaldehyde, paraben esters, benzalkonium chloride, benzyl alcohol, chlorobutanol, p-chlor-m-cresol, benzyl alcohol or a combination thereof; and j.
• a preservative selected from phenoxyethanol, Benzethonium chloride (Phemerol), Phenol, m-cresol, Thiomersal, Formaldehyde, paraben esters, benzalkonium chloride, benzyl alcohol, chlorobutanol,
• a liquid carrier selected from water for injection (WFI) or saline.
• WFI water for injection
• saline a liquid carrier selected from water for injection (WFI) or saline.
• XXV The vaccine formulation as disclosed in any one of the previous embodiments, wherein the vaccine formulation comprises: - at least one at least one recombinant protein/ at least one modified fHbp as disclosed in any one of previous embodiments; - aluminium hydroxide; - mannitol; - phosphate; and - polysorbate.
• XXVI The vaccine formulation as disclosed in any one of the previous embodiments, wherein the vaccine formulation comprises: - at least one at least one recombinant protein/ at least one modified fHbp as disclosed in any one of previous embodiments; - aluminium hydroxide; - mannitol; - phosphate; and - polysorbate.
• the vaccine formulation as disclosed in any one of the previous embodiments comprising (i) at least one recombinant protein/ modified fHbp fusion protein; (ii) aluminium hydroxide in an amount in the range of 0.5 mg/ml to 4.5 mg/ml; (iii) mannitol in an amount in the range of 5 mg/ml to 100 mg/ml; (iv) phosphate buffer in an amount in the range of 1 mM to 10 mM; and (v) polysorbate 20 in an amount in the range of 0.01 mg/ml to 2 mg/ml; wherein each recombinant protein/ modified 5 fHbp fusion protein is present in an amount in the range of 15 ⁇ g/ml to 150 ⁇ g/ml.
• the vaccine formulation as disclosed in any one of the previous embodiments comprising (i) at least two recombinant proteins/ modified fHbp fusion proteins; (ii) aluminium hydroxide in an amount in the range of 0.5 mg/ml to 4.5 mg/ml; (iii)0 mannitol in an amount in the range of 5 mg/ml to 100 mg/ml; (iv) phosphate buffer in an amount in the range of 1 mM to 10 mM; and (v) polysorbate 20 in an amount 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 ⁇ g/ml to 150 ⁇ g/ml.
• the vaccine formulation as disclosed in any one of the previous embodiments comprising (i) at least three recombinant proteins/ modified fHbp fusion proteins; (ii) aluminium hydroxide in an amount in the range of 0.5 mg/ml to 4.5 mg/ml; (iii) mannitol in an amount in the range of 5 mg/ml to 100 mg/ml; (iv) phosphate buffer in an amount in the range of 1 mM to 10 mM; and (v) polysorbate 20 in an amount in0 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 ⁇ g/ml to 150 ⁇ g/ml.
• the vaccine formulation as disclosed in any one of the previous embodiments comprising (i) at least four recombinant proteins/ modified fHbp fusion proteins; (ii)5 aluminium hydroxide in an amount in the range of 0.5 mg/ml to 4.5 mg/ml; (iii) mannitol in an amount in the range of 5 mg/ml to 100 mg/ml; (iv) phosphate buffer in an amount in the range of 1 mM to 10 mM; and (v) polysorbate 20 in an amount 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 ⁇ g/ml to 150 ⁇ g/ml.0 XXXI.
• the vaccine formulation as disclosed in any one of the previous embodiments, wherein the vaccine formulation comprises: - at least one modified fHbp of the amino acid sequence with at least 75% identity with from any one of amino acid sequences SEQ ID NO.6 to 10; or - at least one modified fHbp encoded the nucleic acid sequence is with at least 75% identity with by any one of nucleic acid sequence SEQ ID NO.1 to 5; and - aluminium hydroxide in an amount in the range of 0.5 mg/ml to 4.5 mg/ml; 5 - mannitol in an amount in the range of 5 mg/ml to 100 mg/ml; - phosphate buffer in an amount in the range of 1 mM to 10 mM; and - polysorbate 20 in an amount in the range of 0.01 mg/ml to 2 mg/ml.
• the vaccine formulation as disclosed in any one of the previous embodiments,0 comprising: a. fHbpV3.45 M5:PorA316-320/exP1.14 (of SEQ ID NO. 6 or encoded by SEQ ID NO.1) in an amount in the range of 15 ⁇ g/ml to 150 ⁇ g/ml; or b. fHbpV2.19 M6:PorA316-320/exP1.4 (of SEQ ID NO. 7 or encoded by SEQ ID NO.2) in an amount in the range of 15 ⁇ g/ml to 150 ⁇ g/ml; or 5 c. fHbpV1.14:PorA307-311/exP1.9 (of SEQ ID NO.
• polysorbate 20 in an amount in the range of 0.01 mg/ml to 2 mg/ml.
• XXXIII The vaccine formulation as disclosed in any one of the previous embodiments,5 comprising: a. fHbpV3.45 M5:PorA316-320/exP1.14 (of SEQ ID NO. 6 or encoded by SEQ ID NO.1) in an amount in the range of 15 ⁇ g/ml to 150 ⁇ g/ml; b. fHbpV2.19 M6:PorA316-320/exP1.4 (of SEQ ID NO. 7 or encoded by SEQ0 ID NO.2) in an amount in the range of 15 ⁇ g/ml to 150 ⁇ g/ml; c.
• fHbpV1.14:PorA307-311/exP1.9 (of SEQ ID NO. 8 or encoded by SEQ ID NO. 3) in an amount in the range of 15 ⁇ g/ml to 150 ⁇ g/ml; d. fHbpV1.1:PorA307-311/exP1.4 (SEQ ID NO. 4 or 9) in an amount in the range of 15 ⁇ g/ml to 150 ⁇ g/ml; e. aluminium hydroxide in an amount in the range of 0.5 mg/ml to 4.5 mg/ml; f. mannitol in an amount in the range of 5 mg/ml to 100 mg/ml; 5 g.
• phosphate buffer in an amount in the range of 1 mM to 10 mM; and h. polysorbate 20 in an amount in the range of 0.01 mg/ml to 2 mg/ml.
• XXXIV The vaccine formulation as disclosed in any one of the previous embodiments, comprising: 0 a. fHbpV3.45 M5:PorA316-320/exP1.14 (of SEQ ID NO. 6 or encoded by SEQ ID NO.1) in an amount in the range of 15 ⁇ g/mL to 150 ⁇ g/mL; or b. fHbpV2.19 M6:PorA316-320/exP1.4 (of SEQ ID NO.
• aluminium hydroxide in an amount in the range of 0.5 mg/mL to 4.5 mg/mL
• mannitol in an amount in the range of 5 mg/mL to 100 mg/mL
• phosphate buffer in an amount in the range of 1 mM to 10 mM
• polysorbate 20 in an amount in the range of 0.01 mg/mL to 2 mg/mL.
• XXXV The vaccine formulation as disclosed in any one of the previous embodiments, comprising: 5 a. fHbpV3.45 M5:PorA316-320/exP1.14 (of SEQ ID NO.
• fHbpV1.14:PorA307-311/exP1.9 (of SEQ ID NO. 8 or encoded by SEQ ID NO. 3) in an amount in the range of 15 ⁇ g/mL to 150 ⁇ g/mL; e. aluminium hydroxide in an amount in the range of 0.5 mg/mL to 4.5 mg/mL; f. mannitol in an amount in the range of 5 mg/mL to 100 mg/mL; g. phosphate buffer in an amount in the range of 1 mM to 10 mM; and h. polysorbate 20 in an amount in the range of 0.01 mg/mL to 2 mg/mL.
• XXXVII The vaccine formulation as disclosed in any one of the previous embodiments, wherein the vaccine composition is stable at 2-8°C, 25°C and 40°C for over a period 10 of six months.
• XXXVIII The vaccine formulation as disclosed in any one of the previous embodiments having Zeta potential in the range of -16 mV to -30 mV; osmolality in the range of 200 mOsmol/kg to 500 mOsmol/kg. 15 XXXIX.
• the vaccine formulation as disclosed in any one of the previous embodiments comprising (i) at least one fusion protein comprising stable non-functional/ non- lipidated fHbp and PorA VR2 loop, and (ii) at least one polysaccharide-protein conjugate.
• XLI The vaccine formulation as disclosed in any one of the previous embodiments, wherein the recombinant protein/ modified fHbp fusion protein is co-administered with one or more vaccine selected from BEXSERO, MENVEO, MENACTRA, NIMENRIX, MenQuadFi, MENFIVE, MenAfriVac, Men AC, and Men ACHib.
• the vaccine formulation as disclosed in any one of the previous embodiments comprising i) at least one fusion protein comprising stable non-functional /non- lipidated fHbp and PorA VR2 loop and at least one conjugate selected from (a) a conjugate of (i) the capsular saccharide of serogroup A N. meningitidis and (ii) tetanus toxoid; (b) a conjugate of (i) capsular saccharide of serogroup C N.
• the vaccine formulation as disclosed in any one of the previous embodiments comprising i) at least two fusion proteins, each consisting of one fHbp variant type coupled to one PorA VR2 loop and at least one conjugate selected from (a) a conjugate of (i) the capsular saccharide of serogroup A N. meningitidis and (ii) tetanus toxoid; (b) a conjugate of (i) capsular saccharide of serogroup C N. meningitidis and (ii) CRM197; (c) a conjugate of (i) capsular saccharide of serogroup Y N.
• the vaccine formulation as disclosed in any one of the previous embodiments comprising i) at least three fusion proteins, each consisting of one fHbp variant type coupled to two PorA VR2 loop and at least one conjugate selected from (a) a conjugate of (i) the capsular saccharide of serogroup A N.
• the vaccine formulation as disclosed in any one of the previous embodiments comprising i) at least four fusion proteins, each consisting of one fHbp variant type coupled to three PorA VR2 loop and at least one conjugate selected from (a) a conjugate of (i) the capsular saccharide of serogroup A N. meningitidis and (ii) tetanus toxoid; (b) a conjugate of (i) capsular saccharide of serogroup C N. meningitidis and (ii) CRM197; (c) a conjugate of (i) capsular saccharide of serogroup Y N.
• L The vaccine formulation as disclosed in any one the previous embodiments, wherein percent adsorption of fHbp V3.45 M5 PorA 316-320 exP1.14 on to an adjuvant is in the range of 80 % to 100 % LI.
• the vaccine formulation as disclosed in any one of the previous embodiments, wherein the percent adsorption of fHbp V1.14 PorA 307-311 exP1.9 on to an adjuvant is in the range of 80 % to 90 %.
• LII The vaccine formulation as disclosed in any one of the previous embodiments, wherein the percent adsorption of fHbp V2.19 PorA 316-320 exP1.4 on to an adjuvant is in the range of 80 % to 90 %.
• LIII The vaccine formulation as disclosed in any one of the previous embodiments, wherein the percent adsorption of fHbp V1.1 PorA 307-311 exP1.4 on to an adjuvant is in the range of 80 % to 90 %.
• LIV The vaccine formulation as disclosed in any one of the previous embodiments, wherein the percent adsorption of fHbp V1.1 PorA 307-311 exP1.4 on to an adjuvant is in the range of 80 % to 90 %.
• a method for manufacturing a vaccine formulation as disclosed in any one of the preceding embodiments comprising the following steps: a. growing host cells comprising the expression vector in nutrient medium; b. inducing the host cells for expressing protein; c. harvesting and separating the host cells; d. lysing the harvested cells and separating host cell debris to obtain tagged proteins; e. purifying the tagged proteins; f.
• LVI The method as disclosed in any one of the previous embodiments, wherein the host cell is a bacterial expression host system. LVII.
• 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).
• 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)**
• the nutrient medium is selected from Undefined medium, Terrific Broth (TB) Medium, Lysogenia Broth, Luria Broth or Luria-Bertani medium, chemically defined medium, M9 Minimal Medium, Chemically Defined M9 Modified Salt Medium, 2xYT medium or Super Optimal broth with Catabolite repression (SOC) Medium and combinations thereof.
• LIX The method as disclosed in any one of the previous embodiments, wherein the concentration of L-methionine during growth of host cells in step (a) is maintained in the range of 1 mM to 10 mM, wherein the fermentation is in fed-batch mode.
• LX concentration of L-methionine during growth of host cells in step (a) is maintained in the range of 1 mM to 10 mM, wherein the fermentation is in fed-batch mode.
• 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, pseudoaffinity 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, reverse phase chromatography, and combinations thereof.
• LXXI The method as disclosed in any one of the previous embodiments, wherein the tagged proteins are expressed as inclusion bodies (IB) and are purified by urea unfolding of protein and on column refolding of the protein.
• LXXII The method as disclosed in any one of the previous embodiments, wherein the vaccine formulation is prepared by adsorbing individual recombinant protein/ modified fHbp fusion protein on to an adjuvant, followed by addition to an excipient mixture comprising a sugar alcohol, a buffering agent, a stabilizer, and a liquid carrier.
• LXXIII The method as disclosed in any one of the previous embodiments, wherein the excipient mixture comprises a preservative.
• LXXIV The method as disclosed in any one of the previous embodiments, wherein the excipient mixture comprises a preservative.
• TEV protease is produced by a method comprising the following steps: a. growing host cells comprising the expression vector in nutrient medium; b. inducing the host cells for expressing TEV protease; c. harvesting and separating the host cells; d. lysing the harvested and separating host cells to obtain TEV protease; and e. purifying the TEV protease; and f. concentrating/ diafiltration and storage of purified TEV protease. LXXV.
• the host cell for expressing 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), RosettaTM(DE3)pLysS or B834(DE3).
• 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 (DE
• LXXVI The method as disclosed in any one of the previous embodiments, wherein the5 glucose feed is stopped, and glycerol feed is started when OD at 590/600 nm is 20- 100 and inducing the culture by adding and/or maintaining lactose at 1-50 g/L in fed batch mode.
• LXXVII A method for inducing an immune response against Neisseria meningitidis serogroup0 B strain in an individual by administering to the individual a vaccine formulation as disclosed in any one of the previous embodiments, wherein the step of administration induces an immune response against the Neisseria meningitidis serogroup B strain.
• the method for manufacturing the vaccine formulation in accordance with the present5 disclosure comprises the following broad steps, which are further explained in detail in the succeeding paragraphs: - growing host cells comprising the expression vector in nutrient medium; - inducing the host cells for expressing protein; - harvesting and separating the host cells; 0 - lysing the harvested cells and separating host cell debris to obtain tagged proteins; - purifying the tagged proteins; - removing tags from the tagged proteins to obtain recombinant proteins/ modified fHbp fusion protein; - purifying the recombinant proteins/ modified fHbp fusion proteins; - preparing vaccine formulation comprising the purified recombinant proteins/ modified fHbp fusion protein.
• Example-1 Expression of tagged fHbp proteins and TEV protease
• Host Cell E. coli B834(DE3) was used for the expression of recombinant protein; Cat. No. D48175, Sigma Aldrich (Transferred to Serum Institute of India Pvt. Ltd. from Oxford University) 2) Plasmid*: pET-28a(+) DNA; Cat. No. D48556, EMD Millipore (Transferred to Serum Institute of India Pvt. Ltd. from Oxford University) 3) Host Cell: RosettaTM(DE3)pLysS Competent Cells was used for the expression of TEV protease; Cat. No.
• Plasmid* pET-28a(+) DNA; Cat. No. D48556, EMD Millipore (Transferred to Serum Institute of India Pvt. Ltd. from Oxford University) (*The genes of interest are cloned/ inserted into the backbone of pET-28a(+) plasmid) 1A: Expression of tagged fHbp proteins 33 clone constructs expressing fHbp-PorA chimeric protein were received from University of Oxford. Out of the 33 clones received, 24 clones were screened for protein expression, details of which are provided in Table-3.
• Table-14 Vector map and nucleotide sequence of the clones used for expression of fHbp tagged proteins
• Table-14 Vector map and nucleotide sequence of the clones used for expression of fHbp tagged proteins
• Table-14 Vector map Nucleotide sequence fHbp V3.45
• M5 PorA 316-320/exP1.14
• M6 PorA 316-320/exP1.4
• Figure-2 SEQ ID NO.2 fHbp V1.14: PorA 307-311/exP1.9 Figure-3
• SEQ ID NO.3 fHbp V1.1 PorA 307-311/exP1.4
• Figure-4 SEQ ID NO.4 fHbp V1.1:PorA 307-311/exP1.9
• Figure-5 SEQ ID NO.5 fHbp tagged proteins production was carried out at 10 L scale fermentation batch.
• Figures 9c and 9d illustrate the growth profile at 10 L scale fermentation batch and SDS PAGE gel images, respectively for fHbpV2.19 M6:PorA316-320/exP1.4 (SEQ ID NO. 2, 7); Figures 9e and 9f illustrate the growth profile at 10 L scale fermentation batch and SDS PAGE gel images, respectively for fHbpV1.14:PorA307-311/exP1.9 (SEQ ID NO. 3, 8); and Figures 9g and 9h illustrate the growth profile at 10 L scale fermentation batch and SDS PAGE gel images, respectively for fHbpV1.1:PorA307-311/exP1.4 (SEQ ID NO.4, 9).
• TEV protease The expression vector used for the expression of the TEV protease is His-Gst-TEV protease (pET28a) (Kan/Chloramphenicol). Provided below in Table-20 and Figure are the detail of the vector map and nucleotide sequence.
• Table-20 Vector map and nucleotide sequence of the clones used for expression of TEV protease Clone
• Vector map Nucleotide sequence HIS-GST-TEV protease Figure-6 SEQ ID NO.11 TEV protease production was carried out at 10L scale fermentation batch. The details of media used are provided in Tables 21 to 24.
• Stock solutions for media and feed preparation 1) Base Solution: 14% Ammonia Solution 2) Acid Solution: 6% Ortho Phosphoric Acid 3) Antifoam Solution: 10% Antifoam (STRUKTOL® J 673 A)
• Table-21 Composition of Trace Element Solution (TES) Sr. No.
• Example-2 Purification of tagged fHbp proteins and TEV protease
• the harvested cells obtained in Example-1 are subjected to purification using the following general steps in any order: - cell lysis by chemical and mechanical means; - chromatographic separation; - one or more washing steps; - elution of the protein; - concentration and diafiltration of the protein - storage of the protein till further use.
• the tagged proteins are expressed in inclusion bodies (IB), additional steps of ammonium sulphate precipitation, urea unfolding of protein and on column refolding of the protein were carried out.
• coli cell pellet (for expression of tagged fHbp protein and TEV protease) was dissolved in lysis buffer (25 mM Na-Phosphate buffer+100mM NaCl + 20mM Imidazol, pH 7.4) and the cell were lysed with Panda Homogenizer at 1000-1200 Bar for 6 cycles.1 st cycle was run without pressure and next 5 cycles were run at 1000-1200 Bar pressure. After each cycle lysate was collected and optical density determined using spectrophotometer at 600 nm. Results are displayed in Table-25. Table-25: Optical density of lysate after each cycle of homogenization Sr. No.
• TEV protease cleavage process ⁇ Enzyme: Substrate concentration optimization: His-MBP tagged protein (FHBP V1.1: PorA 307-311/exP1.4 ) was used to optimize TEV protease Enzyme: Substrate concentration (substrate here is the tagged fHbp proteins).
• substrate concentration Substrate here is the tagged fHbp proteins.
• Table-28 Densitometric analysis of TEV cleavage products Temperature Incubation Band Fold change Time Intensity 5°C 1hr 27,69,543 1 10°C 1hr 31,71,273 1.14 20°C 1hr 45,21,956 1.63 30°C 1hr 69,56,920 2.51 40°C 1hr 55,25,733 1.99 50°C 1hr 20,03,602 0.72 Conclusion: It is seen from Table-28 and Figure-17 that at 0 hour, no cleavage was observed. After 1 hour product/ chimera formed was higher at 30°C, which is 2.5 fold than that at 5°C. Hence, optimum temperature for TEV protease cleavage was found to be around 30°C.
• Example-2A Purification of TEV protease
• the TEV protease harvested in Example-1B was purified using the process as per Figure-18, wherein lysis buffer had pH of 7.4.
• the isoelectric point (pI) of a protein is defined as the pH at which the net charge of a protein molecule is zero.
• pI isoelectric point
• the surface of the protein is predominantly negatively charged, and therefore like-charged molecules will exhibit repulsive forces.
• a solution pH that is below the pI the surface of the protein is predominantly positively charged, and repulsion between proteins occurs.
• the negative and positive charges are balanced, reducing repulsive electrostatic forces, and the attraction forces predominate, causing aggregation and precipitation.
• HIS-GST-TEV protease has a pI of 7.8 and hence, working pH of 7.4 did not result in the desired purity. Accordingly, the working pH was increased to 8.5 resulting in enhanced purity of HIS-GST-TEV protease as per the process illustrated in Figure-19.
• Figures 20a and 20b illustrate the purification profiles of TEV protease at pH 8.5 and pH 7.4, respectively. Lane marked with Star symbol ( ⁇ ) represents the final elution fraction of TEV protease. Purity of TEV protease produced using the process with pH 8.5 is higher as compared to TEV protease produced using process with pH 7.4.
• Example-2B Purification of tagged fHbp proteins
• the tagged fHbp proteins harvested in Example-1A were individually purified as per Figure- 21.
• the tagged fHbp proteins are soluble at the working pH used for the purification and the above mentioned process ( Figure-21) results in tagged fHbp proteins with desired purity.
• the purification process illustrated in Figure-21 was used for 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.
• the tagged fHbp proteins expressed at high rate may have solubility issues due to protein aggregation into inclusion bodies (IB).
• the IBs are formed as a result of the aggregation of partially folded and misfolded protein molecules.
• Example-2C Process for tag removal from purified tagged fHbp proteins The His-MBP tag from purified proteins (tagged fHbp proteins) was removed using TEV protease enzyme (SEQ ID NO. 12 or encoded by SEQ ID NO. 11) as per the process in Figure-23.
• the chimeric protein obtained in the digestion mixture was further purified by 2-step chromatography using ion exchange chromatography ( Figure-24) followed by affinity chromatography ( Figure-25).
• the characterization of the chimeric proteins produced using the method of the present disclosure is provided in Table-29.
• the fHbp specifically binds to human factor H (fH), which down-regulates complement activation and enhances resistance to bactericidal activity.
• fH human factor H
• the modification(s) introduced 5 into fHbp antigen results in decreased fH binding and can increase protective antibody responses. It is seen from Table-29, that the recombinant fHbp have > 85% decreased fH binding compared to wild type fHbp, which will aid in improving the immunogenicity of the vaccine formulations containing these recombinant fHbp.
• Previously multiple steps of chromatography were used for purification of the digestion mixture including Metal affinity chromatography, Cation exchange, Anion exchange and Gel filtration chromatography (GFC).
• Example-3 Formulations comprising recombinant proteins/ modified fHbp fusion protein/ chimeric proteins Different formulations of recombinant proteins/ modified fHbp fusion protein were prepared and evaluated to obtain formulation(s) with optimum physicochemical characteristics, stability, and immunogenicity.
• Adjuvant optimization To enhance immunogenic responses and physicochemical properties, formulation development started with adsorbing proteins on different adjuvants like Aluminium hydroxide (Alhydrogel), Aluminium phosphate (Adjuphos), double mutant heat-labile toxin (dmLT), etc. These formulations were evaluated on the basis of different physicochemical parameters. The summary of trials, its outcome and conclusion are provided below. Table-30: Adjuvant Optimization Batch no.290121-A Sr. No.
• Alhydrogel has an isoelectric point (pI) at about 11.4 while Adjuphos has pI value in between 4.5-6.0. All the fHbp proteins have pI in the range of 5.5 to 6.5. Thus, to achieve optimum adsorption of all proteins on adjuvant in the vaccine formulations, Alhydrogel was used for further studies.
• Buffer and Sugar optimization Table-31: Buffer and sugar optimization Batch no.051021-A Sr. No.
• Formulation comprising multiple chimeric proteins: Similar to the individual protein formulation, physicochemical properties of formulation comprising the combination of proteins were determined. The formulation detail and result obtained are summarized in Table-34. The proteins were individually adsorbed on the adjuvant and then added to the excipient mixture to obtain the formulation. Table-34: Vaccine formulation comprising optimized excipients Batch No.161021 Sr. No.
• Example-4 Stability studies of the vaccine formulations comprising recombinant protein/ modified fHbp fusion protein Stability studies at 2-8°C for 2 batches for 6 months and 10 months were carried out. The formulation detail and the result obtained are summarized in Table-44. Table-44: Stability studies at 2-8°C Sr. Formulation Concentration No.
• N. meningitidis serogroup B strains Glycerol stocks of N. Meningitidis were received from professor Chris Tang (Sir William Dunn School of Pathology, UK). Four strains of N. meningitidis serogroup B were received. The strain details are provided below and further summarized in Table-45. 1.
• N. meningitidis M08.240157 (fHbp V1.1:PorA VR216) 2.
• N. meningitidis M17.240832 (fHbp 1.4: PorA VR24) 3.
• Table-46 Vaccine formulations for immunogenicity studies Sr. No. Formulation Formulation 1 Formulation 2 Placebo Batch No. Batch No.
• Purpose of the study was to compare immunogenic response for individual Men B formulations – Formulation 1 and Formulation 2 along with Trumenba against 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 obtained are illustrated in Table-48.
• the formulations (Formulation 1/ Quad 1 and Formulation 2/ Quad 2) comprising the recombinant proteins/ modified fHbp fusion proteins of the present disclosure are highly immunogenic and capable of eliciting protective SBA titer against different (M 08240157, M 17240832, M 17240156, and M 18240043) strains of N. meningitides.
• PURPOSE ELISA for Assessing the binding of human complement factor-H to fHbp-PorA chimeric proteins as compared to the recombinant wild-type of Men B samples
• PURPOSE The purpose of this study is to describe the method for assessing the binding of human complement factor-H to fHbp-PorA chimeric proteins as compared to the recombinant wild- type of Men B samples in Monoclonal Antibodies IPQC Laboratory (MAb IPQC).
• PROCEDURE Chemicals and Reagents required are provided in Table-49. Table-49: List of Chemicals and reagents Sr. No. Chemical Name Vendor/ Brand Catalog No. 1. Complement Factor H from human plasma Sigma Aldrich C5813 2.
• OX24 anti-factor H monoclonal antibody Abcam ab118820 3. Polyclonal goat anti-mouse HRP secondary antibody Sigma Aldrich A4416 4. Tween20 Sigma Aldrich P7949 5. Bovine Serum Albumin (BSA) Sigma Aldrich A7030 6. Development reagent R&D systems DY999 7. Stop solution R&D systems DY994 8. PBS buffer tablet (pH 7.4 ⁇ 0.05) Gibco 18912-014 For all the above chemicals, equivalent make from other brands can also be used. Equipment required: - ELISA plate reader capable of measuring wavelengths at 450 nm and 630 nm.
• 1X PBST (0.05% Tween20 in 1X PBS pH 7.4) Prepare 1 L of PBS as mentioned above. Add 0.5 mL of Tween 20 solution to 999.5 mL of 1X PBS and mix using a magnetic stirrer. Store at room temperature (RT), use before 1 month of date of preparation. This 1X PBST is also used as diluent for blocking solution and antibody preparation.
• Blocking 4% BSA in PBST Accurately weigh 4.0 g of BSA and dissolve it in 100 mL of 1X PBST.
• TMB solution 2 component mixture Accurate measure equal volumes of solution A and B (provided as development reagent). Equilibrate to RT (store in dark).
• Human complement factor-H dilution scheme Dilution Volume from stock ( ⁇ L) Volume of Final Resulting number diluent ( ⁇ L) Volume ( ⁇ L) concentration ( ⁇ g/mL) 1 30.0 ⁇ L from 1.0 mg/mL 1470.0 ⁇ L 1500.0 ⁇ L 20.0 ⁇ g/mL 2 250.0 ⁇ L from 20.0 ⁇ g/mL 250.0 ⁇ L 500.0 ⁇ L 10.0 ⁇ g/mL 3 200.0 ⁇ L from 20.0 ⁇ g/mL 300.0 ⁇ L 500.0 ⁇ L 8.0 ⁇ g/mL 4 150.0 ⁇ L from 20.0 ⁇ g/mL 350.0 ⁇ L 500.0 ⁇ L 6.0 ⁇ g/mL 5 100.0 ⁇ L from 20.0 ⁇ g/mL 400.0 ⁇ L 500.0 ⁇ L 4.0 ⁇ g/mL 6 50.0 ⁇ L from 20.0 ⁇ g/mL 450.0 ⁇ L 50
• Example-6 Combination formulations comprising recombinant protein/ modified fHbp fusion protein formulation
• the vaccine formulation of the present invention comprising recombinant proteins/ modified fHbp fusion proteins from N. meningitidis serogroup B was found to be stable and 10 immunogenic. Further, combination formulations comprising the vaccine formulation along with other N. meningitidis serogroups were studied to find out the impact on physicochemical characterization, stability, and immunogenicity. Applicant’s MenFive formulation comprising polysaccharide-protein conjugates against N. meningitidis serogroups ACYWX was used for the study.
• the lyophilized MenFive vaccine 15 was reconstituted with the MenB formulation and stored at room temperature for 2 hours to study the impact on physicochemical parameters of overall reconstituted solution. The study was done to evaluate stability of reconstituted solution during animal injections and mimic routine clinical practice.
• Table-52 Physicochemical characterization of combination formulations Sr. No. Admixture Test Parameters Initial 2 hrs at RT 1 MenFive (B#2352M001) + Placebo pH 7.30 7.30 for MenB (B#090823) Zeta Potential (mV) -33.3 -32.8 PSD (Zavg, nm) 1550 1938 2 MenFive (B#2352M001) + pH 7.64 7.63 MenB (B#090823-Formulation 1) Zeta Potential (mV) -41.3 -41.6 PSD (Zavg, nm) 1912 2302 MenFive (B#2352M001) + pH 7.65 7.66 MenB (B#090823-Formulation 2) Zeta Potential (mV) -35.6 -35.8 PSD (Zavg, nm) 2421 2447 Conclusion: It is seen from Table-52 that the reconstituted solution was stable physico- chemically for 2 hours and also visually no particle aggregation / settling was
• N. meningitidis serogroup B strains The N. meningitidis serogroup B strains used were same as those used in Example-5.
• N. meningitidis serogroup A, C, W, Y and X strains Glycerol stocks of N. Meningitidis were received from Professor Ray Borrow (Health Protection Agency, Manchester Laboratory UK) in July 2012. Five serogroups of N. Meningitidis were received as follows: 1. N. meningitidis serogroup A, F8238 2. N.
• Procedure for hSBA against MenFive serogroups (A, C, W, Y and X): Preparation by working assay stocks for SBA The master stock vial was retrieved from the deep freezer, culture was quickly streaked onto an already-dried blood agar plate (Columbia blood agar with 5% Horse blood). Plate was incubated at 37°C with 5 % CO2 overnight. Approximately 50 colonies were picked with a sterile loop and re-suspended (thus creating a heavy suspension) in a sterile 50 ml centrifuge tube containing 15 ml of BHI broth with glycerol. Suspension was aliquoted as 0.25 ml volumes into 1.8 ml cryo vials.
• Drug Product Meningococcal (A-TT, C-CRM, Y-CRM, W-CRM, X-TT) Polysaccharide Conjugate Vaccine – 5 Dose vial (Men5) (2352M001) reconstituted with Meningococcal B vaccine (MenB) (090823-Formulation 1 and 2) Study Design: The study design is illustrated in Table-53. Table-53: Treatment Groups in Immunogenicity Study Sr. Group Test Dose (mcg) / No. Inj. Inj. Day of Sera Collection Day No. ID Compound inj. Rou Vol.
• Purpose and Scope 10 Purpose of the study was to compare immunogenic response for individual MenFive+MenB, admix formulations, against MenFive serogroups (A, C, W, Y and X) and MenB strains - M 08240157(fhbp 1.1), M 17240832 (fhbp 1.4), M 17240156 (fhbp 3.45) and M 18240043 (fhbp 1.15) on using hSBA.
• the scope also includes, to check for any interference/cross protection in response between the two individual vaccines formulations 15 (MenFive and MenB). s n , n t u o . i t o i b .
• Example-7 Quantification of recombinant protein/ modified fHbp fusion protein using ELISA Bicinchoninic Acid (BCA) method is routinely used for estimating total recombinant protein/ modified fHbp fusion protein content in the final vaccine formulation.
• the vaccine formulation of the present invention comprises more than one recombinant protein/ modified fHbp fusion protein.
• BCA provides the total recombinant protein/ modified fHbp fusion protein quantification and is not able to provide the individual content of each protein present in the vaccine formulation.
• ELISA was used to quantify the content of individual recombinant protein/ modified fHbp fusion protein in the vaccine formulation. This test may also be used as a batch release test.
• the material used for ELISA for quantifying individual recombinant protein/ modified fHbp fusion protein is summarized in Table-56.
• Table-56 Material used for protein quantification Material Name Make Catalog Number Clear Flat-Bottom Immuno Nonsterile Thermo Scientific 442404 96-Well Plates BSA Sigma A7030 PBS Gibco 18912-014 Tween-20 Sigma P-7949 Anti-Factor H binding protein [JAR41], Absolute Antibody Ab03894-23.0 Rabbit IgG, Kappa Anti-Factor H binding protein from N.
• the coating incubation will be from 2.5 hrs to 18 hrs at either room temperature or 2-8°C.
• STEP-2 Blocking: 300 to 320 ⁇ L of 1% BSA in PBS will be added to 96 well plate and incubated from 1 hr to 2.5 hrs at room temperature.
• STEP-3 Standard / Test Sample Addition: The reference standard chimeras will be serially diluted from 0.5mg/mL to 1ng/mL using 0.1 % BSA in PBS and 50 ⁇ L to 150 ⁇ L will be added to respective wells. The test samples will be diluted using 0.1 % BSA in PBS such that they will fall in the quantification range by loading from 50 ⁇ L to 150 ⁇ L.
• the detection antibody (Anti-POR-A P1.14 mAb / Anti-POR-A P1.4 mAb / Anti-POR-A P1.9 mAb / JAR-11 mAb) will be diluted anywhere between 100 fold and 100000 fold and load 100 ⁇ L per well on 96 well plate. The detection antibody incubation will be from 0.5 hrs to 2.0 hrs at room temperature.
• STEP-5 Secondary Detection Antibody Addition
• the secondary detection antibody (goat anti-mouse HRP antibody) will be diluted anywhere between 100 fold and 100000 fold and load 100 ⁇ L per well on 96 well plate. The secondary detection antibody incubation will be from 0.5 hrs to 2.0 hrs at room temperature.
• STEP-6 Substrate Addition & Stopping: The 100 ⁇ L of TMB substrate will be added to each well and incubated from 10 to 45 min at room temperature followed by stopping with addition of 100 ⁇ L of 1N HCl to each well. Immediately the plate will be read for absorbance at 450 /630.
• Example-8 Estimation of protein in recombinant protein/ modified fHbp fusion protein using BCA PURPOSE: The purpose of this study is to provide the procedure for estimation of protein in Men B samples by 96 well plate Micro Bicinchoninic acid (BCA) method.
• BCA is a method for quantifying the concentration of protein in a sample. Under alkaline conditions, Cu 2+ reacts with proteins yielding Cu + . This Cu + generated reacts with bicinchoninic acid to form a purple coloured complex. The colour is developed after incubation at 37 °C. The coloured complex is detected using spectrophotometer at 562nm.
• MATERIAL & EQUIPMENTS Micro BCA Protein Assay Kit (Thermoscientific, 23235) containing a. Micro BCA Reagent A (MA) b. Micro BCA Reagent B (MB) c. Micro BCA Regent C (MC) d. Bovine Serum Albumin Standard ampules (2 mg/ml) - Water for injection (WFI) - Microfuge tubes (1.5 mL Eppendorf tubes) - Pipettes and tips - 96 well plate - 96 well plate reader (TECAN, Infinite M200) - Plate incubator (Biosan Thermoshaker) TEST PROCEDURE: Preparation of standards and working reagent: - Set the incubator at 37°C.
• Sample without centrifugation The formulation samples are either taken as neat or diluted so as to fit in the standard curve.
• Supernatant 500 ⁇ l of each sample is taken and centrifuged at 8000 rpm for 5 minutes. After centrifugation, the supernatant is carefully transferred into a fresh tube. The collected supernatant is taken as a neat sample or diluted so as to fit in the curve.
• Pellet The pellet formed after centrifugation is re-suspended by adding 500 ⁇ l of WFI. These samples are diluted same as per Sample as such (a). - Prepare the dilutions in duplicate for each sample and standards. - Transfer 150 ⁇ l of standards and samples into the plate wells.
• the percent adsorption is provided in Table-59.
• Table-59 Percent adsorption calculation Sample Type Sample Dilution Abs @ Abs @ Normalized Conc. Conc. Std CV % Name Factor 562 562 Absorbance Protein ( ⁇ g/mL) Dev % Adsor nm_Read nm_Read ption 1 2 Sample 090224- 12 0.7068 0.7158 0.607 43.798 525.6 0.01 0.00 90.8 without F3 centrifugation Soluble Form 090224- 1 0.7846 0.7805 0.678 48.604 48.6 0.00 0.01 F3 Pellet 090224- 12 0.6394 0.6439 0.537 39.100 469.2 0.00 0.00 F3

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Genetics & Genomics (AREA)
• Organic Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Medicinal Chemistry (AREA)
• Engineering & Computer Science (AREA)
• Biochemistry (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Microbiology (AREA)
• Wood Science & Technology (AREA)
• Zoology (AREA)
• General Engineering & Computer Science (AREA)
• Biotechnology (AREA)
• Biophysics (AREA)
• Biomedical Technology (AREA)
• Molecular Biology (AREA)
• Animal Behavior & Ethology (AREA)
• Pharmacology & Pharmacy (AREA)
• Plant Pathology (AREA)
• Physics & Mathematics (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Oncology (AREA)
• Communicable Diseases (AREA)
• General Chemical & Material Sciences (AREA)
• Immunology (AREA)
• Mycology (AREA)
• Epidemiology (AREA)
• Gastroenterology & Hepatology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Priority Applications (5)

Applications Claiming Priority (2)

Publications (1)

Family

ID=92903953

Family Applications (1)

Country Status (9)

Cited By (2)

Citations (4)

• 2023
  • 2023-03-24 IN IN202321021121A patent/IN202321021121A/en unknown
• 2024
  • 2024-03-22 WO PCT/IN2024/050300 patent/WO2024201502A1/en active Pending
  • 2024-03-22 TW TW113110870A patent/TW202444740A/zh unknown
  • 2024-03-22 KR KR1020257035689A patent/KR20250166279A/ko active Pending
  • 2024-03-22 CN CN202480028977.6A patent/CN121038809A/zh active Pending
  • 2024-03-22 AU AU2024249503A patent/AU2024249503A1/en active Pending
  • 2024-03-25 AR ARP240100707A patent/AR132199A1/es unknown
• 2025
  • 2025-09-24 MX MX2025011286A patent/MX2025011286A/es unknown
  • 2025-09-25 IL IL323559A patent/IL323559A/en unknown

Patent Citations (4)

Also Published As

Similar Documents

Legal Events