CN118105474A - Recombinant B-type meningococcal vaccine - Google Patents

Abstract

The present disclosure relates to an immunogenic composition comprising a combination of meningococcal antigens, the combination comprising at least one factor H binding protein (fHBP) a protein, at least one fHBP B protein, at least one neisseria adhesion a (NadA) protein, and at least one detergent-extracted outer membrane vesicle (dOMV). The meningococcal antigen may be from neisseria meningitidis serogroup B. The combination of antigens provides broad coverage of bacterial strains. In addition, the present disclosure relates to the use of the immunogenic composition in a method of eliciting an immune response.

Description

Recombinant B-type meningococcal vaccine

The application is a divisional application of China patent application (application day: 2022, 2, 18 days, application name: recombinant B meningococcal vaccine) with application number 202280015689.8.

RELATED APPLICATIONS

The present application claims priority from European patent application Ser. No. 21305211.1 filed on month 19 of 2021 and U.S. provisional patent application Ser. No. 63/172,885 filed on month 4 of 2021, the disclosures of which are incorporated herein by reference in their entireties.

Technical Field

The present disclosure relates to the field of vaccines. The present disclosure relates to immunogenic compositions and vaccines for preventing meningococcal infections such as neisseria meningitidis (NEISSERIA MENINGITIDIS) (n.menningitidis or Nm) serogroup B (MenB) infections.

Background

Neisseria meningitidis is a gram-negative diplococcus, the only known natural host of which is human. Neisseria meningitidis is a common colonization of the human nasopharynx and oropharynx, but can also be found in other areas of the body, such as the anal mucosa, conjunctiva and genitourinary tract (Rouphael et al, methods Mol biol.2012;799:1-20; stephens, vaccine.2009;27 journal 2:B71-7; batista et al, asian Pac J Trop Med.2017;10 (11): 1019-29).

At least 12 different meningococcal serogroups have been divided according to immunochemistry of capsular Polysaccharide (PS). Some strains are more likely to cause infection than others. Worldwide, most meningococcal disease cases are caused by serogroups A, B, C, W, X and Y. Serogroup B is responsible for endemic disease and some outbreaks (Harrison et al, [ code ]Orenstein WA,Offit PA,Edwards KM Plotkin SA.Vaccines.7.Philadelphia(PA):Elsevier;2018.p.619-43;Borrow et al, expert Rev vaccines.2017;16 (4): 313-28; harrison et al, EMERG INFECT Dis.2013;19 (4): 566-73,Pollard,Pediatr Infect Dis J.2004;23 (12 supplement): S274-9; kvalsvig et al, J Clin Pathol.2003;56 (6): 417-22).

Neisseria meningitidis serogroup B is a respiratory tract-transmitted bacterium (via spray) that cannot survive in the environment and requires intimate and prolonged contact or direct physical contact (e.g., kissing) to effectively transmit. Asymptomatic carriers are present in less than 2% of children under 5 years and 20% -25% of adolescents and young adults, a major factor in the pathogen transmission pathway and its maintenance in nature, even during epidemic periods (Christensen et al LANCET INFECT Dis.2010;10 (12): 853-61; batista et al Asian Pac J Trop Med.2017;10 (11): 1019-29).

In general, the age groups with the highest incidence of carry over are teenagers and young adults who are prone to conduct activities that are considered risk factors for carrying over and eventual appearance of Invasive Meningococcal Disease (IMD) (Christensen et al LANCET INFECT Dis.2010;10 (12): 853-61; stephens, vaccine.2009;27 journal 2: B71-7; bruce et al, JAMA.2001;286 (6): 688-93; germinario et al, hum vaccine.2010; 6 (12): 1025-7; macLennan et al EMERG INFECT Dis.2006;12 (6): 950-7). Thus, vaccination of these age groups has the potential to affect IMD incidence in other age groups, which has been demonstrated in many european countries, whose vaccination activity with serogroup C conjugate vaccines has led to population protection in unvaccinated age groups (Maiden et al, J select dis.2008;197 (5): 737-43; trutter et al, lancet.2004;364 (9431): 365-7; bijlsma et al, CLIN INFECT dis.2014;59 (9): 1216-21).

Invasive Meningococcal Disease (IMD) is a severe disorder caused by neisseria meningitidis (including neisseria meningitidis serogroup B), and symptoms may include severe headache, fever, nausea, vomiting, photophobia, stiff neck, somnolence, myalgia, and characteristic petechiae rashes (Harrison et al, ed. [ code ] Orenstein WA, offit PA, edwards KM plotkin sa. Vaccine.7. Philiadelphia (PA): elsevier;2018. P.619-43). IMD may cause meningococcal meningoectomy and meningococcemia. Meningococcemia is probably the most rapidly fatal infectious condition to humans, with about 90% of deaths reported to occur within the first 2 days of hospitalization. Between 6% and 15% of IMD patients may develop inflammatory syndromes due to the deposition of antigen-antibody complexes consisting mainly of capsular polysaccharide, specific immunoglobulins and complement component C3. These reactions typically occur 4 to 12 days after onset of the disease and include arthritis (mainly monoarthritis (7% -14% of patients)), cutaneous vasculitis, iritis, episcleritis, pleurisy and pericarditis. At the same time, reheating, leukocytosis and elevated serum C-reactive protein can occur. Other complications that may occur in IMD patients include herpes simplex infection activation, distal symmetric necrosis, extensive ulcers in the appearance of vasculitis, digestive tract bleeding, subdural effusion, myocarditis, rhabdomyolysis, adult respiratory distress syndrome, acid-base and electrolyte disorders, cerebral infarction, and intracranial suppuration.

Sequelae may occur in IMD survivors. The risk of occurrence of neurological sequelae is 7% -12% (lower ratio compared to pneumococcal meningitis), occurring mainly in infants. Hearing loss (sustained or temporary) is the most common complication, occurring in about 4% of cases. Other sequelae include: visual impairment, hydrocephalus, ataxia, speech disorders, motor deficits, developmental retardation, arthritis, spasms, tics, renal failure, osteonecrosis, atrophic scars, partial loss of limbs, learning disorders, behavioral disorders, and the like (Batista et al, asian Pac J Trop Med.2017;10 (11): 1019-29; stephens et al, NEISSERIA MENINGITIDIS. [ code ] J.E.Bennett, R.Dolin and M.J. Blaser. Philadelphia: elsevier Saunders;2015.p.2425-45; campsall et al, crit Care Clin.2013;29 (3): 393-409; pace et al, vaccine.2012;30 increments 2:B3-9).

Serogroup B is an important cause of endemic disease and leads to a variety of long-term epidemics in several industrialized countries (Vuocolo et al, hum Vaccin immunother.2018;14 (5): 1203-15), including Goba (Rodriguez et al, mem Inst Oswaldo Cruz.1999;94 (4): 433-40), norway (Fredriksen et al, NIPH Ann.1991;14 (2): 67-79; discussed-80) and New Zealand (Martin et al, J Infect Dis.1998;177 (2): 497-500; dyet al, epidemiol Infect.2006;134 (2): 377-83). Smaller bursts caused by a single strain have also been reported in other countries such as France (from 2000 to 2003) (Grodet et al, microbiol Infect.2004;10 (9): 845-8; caron et al, LANCET INFECT Dis.2011;11 (6): 455-63) and the United states (from 2013 to 2017), some of which are related to colleges and universities (Folaranmi et al, 2015.MMWR Morb Mortal Wkly Rep.2015;64 (22): 608-12; atkinson et al, pharmacotherapy.2016;36 (8): 880-92).

Two broad protective protein-based vaccines targeting neisseria meningitidis group B have recently been licensed: 1) A 4-component MenB protein vaccine (4 CMenB; from GlaxoSmithKline company [ GSK ]Vaccine) is licensed as a two-dose regimen for individuals between 10 and 25 years old in the United States (US) and is used in individuals from 2 months up to 50 years old in some countries in europe, australia, canada and south america; 2) Vaccine based on bivalent recombinant fHBP protein (rLP 2086) (from Pfizer/>Vaccine) is licensed as a 2-dose or 3-dose regimen for individuals between 10 and 25 years of age in the united states and europe.

Clinical trials of these two licensed vaccines revealed that fever was an adverse event of particular concern to the pediatric population. It has been reported that up to 70% of infants receiving BEXSERO vaccine and conventional vaccine develop >38 ℃ fever (> 39 ℃ fever in 6% -12%) and therefore preventive acetaminophen is recommended for BEXSERO vaccine immunization (and within the first 24 hours after vaccination). During a TRUMENBA vaccine phase I/IIb study against pediatric populations, 64% and 90% fever (majority <39.0 ℃) was reported in participants receiving 20 or 60 μg rLP2086 doses, respectively, and the study was terminated prematurely (Martinon-Torres et al, vaccine.2014;32 (40): 5206-11).

Humans immunized with these vaccines will develop complement-mediated Serum Bactericidal Antibody (SBA) responses. However, for TRUMENBA and BEXSERO vaccines, low fHBP-associated SBA activity and coverage against some MenB strains was demonstrated, especially in toddlers and infants (Brunelli et al, vaccine.2011;29 (5): 1072-81; marshall et al, PEDIATR INFECT DIS J.2012;31 (10): 1061-8). It has been demonstrated that binding of host molecules to vaccine antigens can reduce immunogenicity by covering important epitopes or reducing vaccine uptake, which can lead to reduced antigen processing and presentation (Meri et al, vaccine.2008;26 journal 8: i 113-7). Preclinical studies have demonstrated that binding of factor H (fH) to wild-type recombinant fHBP antigen can impair the protective serum anti-fHBP antibody response in human fH transgenic mice and young rhesus monkeys (Costa et al, mBio.2014;5 (5): e01625-14; granoff et al, CLIN VACCINE immunol.2013;20 (8): 1099-107).

Thus, it appears that there is still a need for immunogenic compositions, such as vaccines, for neisseria meningitidis (e.g. MenB) that have a greater coverage in terms of bacterial strains.

There is a need for immunogenic compositions with a broader coverage of the MenB strain than BEXSERO or TRUMENBA.

There is also a need for immunogenic compositions with good reactogenic properties.

There is a need for immunogenic compositions with good reactogenic and safety characteristics for infant and pediatric use.

In addition, there is a need for immunogenic compositions having improved reactogenic characteristics compared to TRUMENBA.

There is a need for immunogenic compositions having improved reactivity characteristics compared to BEXSERO.

Furthermore, there is a need for immunogenic compositions against MenB that can be readily combined with other antigens, such as neisseria meningitidis ACWY antigens, for example ACWY polysaccharide conjugated to diphtheria or tetanus toxoid.

This disclosure is intended to satisfy all or a portion of these needs.

Disclosure of Invention

In one aspect, a multicomponent meningococcal immunogenic composition is disclosed that comprises at least 4 antigens and is intended to provide broad protection against meningococcal infection, such as Invasive Meningococcal Disease (IMD) caused by MenB, with acceptable infant indication safety. It consists of 3 major surface-exposed recombinant Neisseria proteins selected based on their key role in the pathogenesis of Neisseria meningitidis and their ability to induce Serum Bactericidal Antibodies (SBA) against homologous and heterologous MenB strains (Pizza et al, science.2000;287 (5459): 1816-20): 2 non-lipidated factor H binding proteins (fHBP) from subfamilies a and B and a neisseria adhesion protein a (NadA). To improve the immunogenicity and potential strain coverage of multicomponent MenB vaccines, outer Membrane Vesicles (OMVs) obtained by detergent extraction (detergent extracted OMVs, dwmv, also known as Outer Membrane Protein Complexes (OMPCs)) were added to the formulation. dOMVs are derived from MenB strains, e.g., strains that express PorA proteins such as PorA VR 2P 1.2. The dOMV efficacy is associated with bactericidal antibodies directed primarily against homologous PorA, which is very abundant on bacterial surfaces.

Unexpectedly, the inventors have observed that the combination of MenB antigens in an immunogenic composition as disclosed herein confers broad coverage to the composition in terms of MenB strains against which an immune response can be elicited, while inducing low reactivities and pro-inflammatory effects.

Indeed, as shown in the examples section, the immunogenic compositions of the present disclosure, such as vaccines, allow for a wider coverage of the MenB strain than TRUMENBA or BEXSERO. Notably, the immunogenic compositions disclosed herein elicit protective immune responses against the 6 MenB strains not covered by BEXSERO.

Advantageously, the antigens of the immunogenic compositions disclosed herein provide cross-protection between prevalent MenB strains that cause IMD.

Advantageously, the immunogenic compositions disclosed herein comprise 2 non-lipidated fHBP recombinant antigens: a05 (also known as variant 3.45 in Novartis nomenclature according to Pfizer classification, or peptide ID45 according to PubMLST nomenclature) and B01 (also known as variant 1.55 in Novartis nomenclature according to Pfizer classification, or peptide ID55 according to PubMLST nomenclature), which represent 1 variant antigen from each of the 2 genetically and immunologically distinct subfamilies of fHBP, this allows to ensure broad protection against all MenB strains and allows for infant indication from six weeks.

Another advantage is that the immunogenic compositions disclosed herein comprise a dOMV that can induce specific protective responses against strains expressing homologous PorA (such as PorA VR2 strains and e.g. PorA VR 2P 1.2 strains) as well as protective cross-reactivity against heterologous strains.

Another advantage of the immunogenic compositions disclosed herein is that they elicit an immune response against MenB strains from the ST-41/44, ST-32, ST-269, ST-213, ST-35, ST-461, ST-11 and ST-461 clone complexes. Another advantage is that the immunogenic compositions disclosed herein elicit an immune response to the emerging super virulence clone complex ST-11 clone complex.

In addition, as shown in the examples section, the immunogenic compositions disclosed herein exhibit a reactivity profile that is comparable to and even less than the overall reactivity profile of BEXSERO, BEXSERO has an improvement (i.e., less reactivity) over TRUMENBA. Notably, the disclosed immunogenic compositions exhibit a weaker pro-inflammatory cytokine response than TRUMENBA. In addition, they have minimal impact on cell viability.

According to one aspect thereof, the present disclosure relates to an immunogenic composition comprising a combination of meningococcal antigens, the combination comprising at least one factor H binding protein (fHBP) a protein, at least one fHBP B protein, at least one neisseria adhesion a (NadA) protein, and at least one detergent-extracted outer membrane vesicle (dOMV). The antigen is present in an immunologically effective amount.

In one embodiment, the meningococcal antigen may be from neisseria meningitidis serogroup B.

According to another aspect thereof, the present disclosure relates to an immunogenic composition comprising a combination of neisseria meningitidis serogroup B antigens, said combination comprising at least one factor H binding protein (fHBP) a protein, at least one fHBP B protein, at least one neisseria adhesin a (NadA) protein, and at least one detergent-extracted outer membrane vesicle (dOMV). The fHBP a protein and/or the fHBP B protein may be non-lipidated.

In another embodiment, the fHBP a protein may be a lipidated or non-lipidated protein, and for example is a non-lipidated protein.

In another embodiment, the fHBP B protein may be a lipidated or non-lipidated protein, and for example is a non-lipidated protein.

In another embodiment, the fHBP a protein and/or fHBP B protein may be non-lipidated. In one embodiment, both fHBP a protein and fHBP B protein may be non-lipidated.

In one embodiment, the fHBP a protein may be a non-naturally occurring fHBP.

In one embodiment, the fHBP B protein may be a non-naturally occurring fHBP.

In one embodiment, the fHBP a and/or fHBP B protein may be a non-naturally occurring fHBP.

In one embodiment, the fHBP a and/or fHBP B protein may be a mutated fHBP. In one embodiment, the fHBP a and/or fHBP B protein may be a mutant non-lipidated fHBP.

In one embodiment, the fHBP A protein can be a mutein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% identity to SEQ ID NO. 1.

In another embodiment, the fHBP a protein may comprise at least one amino acid substitution based on the numbering of SEQ ID NO:6 selected from at least one of: a) An amino acid substitution of asparagine (N115) at amino acid 115; b) Amino acid substitution of aspartic acid (D121) at amino acid 121; c) Amino acid substitution of serine at amino acid 128 (S128); d) Amino acid substitution of leucine (L130) at amino acid 130; e) Amino acid substitution of valine (V131) at position 131; f) Amino acid substitution of glycine (G133) at position 133; g) Amino acid substitution of lysine (K219) at position 219; and h) amino acid substitution of glycine (G220) at position 220.

In one embodiment, the fHBP a protein may be a mutein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or at least about 99.5% identity to SEQ ID No. 1 and comprising at least one amino acid substitution based on the numbering of SEQ ID No. 6 selected from at least one of: a) An amino acid substitution of asparagine (N115) at amino acid 115; b) Amino acid substitution of aspartic acid (D121) at amino acid 121; c) Amino acid substitution of serine at amino acid 128 (S128); d) Amino acid substitution of leucine (L130) at amino acid 130; e) Amino acid substitution of valine (V131) at position 131; f) Amino acid substitution of glycine (G133) at position 133; g) Amino acid substitution of lysine (K219) at position 219; and h) amino acid substitution of glycine (G220) at position 220.

In one embodiment, the fHBP A protein may comprise at least the amino acid substitution G220S based on the numbering of SEQ ID NO. 6. The fHBP a protein may be a non-lipidated protein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or at least about 99.5% identity with SEQ ID No. 1 and comprising at least the amino acid substitution G220S based on the numbering of SEQ ID No. 6.

In one embodiment, the fHBP A protein may comprise at least three amino acid substitutions selected from the group consisting of G220S, L R and G133D based on the numbering of SEQ ID NO. 6. In another embodiment, the fHBP A protein may comprise only three amino acid substitutions G220S, L R and G133D based on the numbering of SEQ ID NO. 6. The fHBP a protein may be a mutein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or at least about 99.5% identity to SEQ ID No. 1 and comprising at least or only three amino acid substitutions selected from G220S, L R and G133D based on the numbering of SEQ ID No. 6.

In another embodiment, the fHBP A protein may comprise or consist of SEQ ID NO. 2.

In one embodiment, the fHBP B protein can be a mutein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% identity to SEQ ID NO. 3.

In another embodiment, the fHBP B protein may comprise at least one amino acid substitution based on the numbering of SEQ ID NO:6 selected from at least one of: a) Amino acid substitution of glutamine (Q38) at amino acid 38; b) Amino acid substitution of glutamic acid (E92) at amino acid 92; c) Amino acid substitution of arginine (R130) at amino acid 130; d) Amino acid substitution of serine (S223) at amino acid 223; and e) an amino acid substitution of histidine (H248) at amino acid 248.

In one embodiment, the fHBP B protein may be a mutein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or at least about 99.5% identity to SEQ ID No. 3 and comprising at least one amino acid substitution based on the numbering of SEQ ID No. 6 selected from at least one of: a) Amino acid substitution of glutamine (Q38) at amino acid 38; b) Amino acid substitution of glutamic acid (E92) at amino acid 92; c) Amino acid substitution of arginine (R130) at amino acid 130; d) Amino acid substitution of serine (S223) at amino acid 223; and e) an amino acid substitution of histidine (H248) at amino acid 248.

In another embodiment, the fHBP B protein may comprise at least the amino acid substitution H248L based on the numbering of SEQ ID NO. 6. In another embodiment, the fHBP B protein may comprise only the amino acid substitution H248L based on the numbering of SEQ ID NO. 6. The fHBP B protein can be a mutein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or at least about 99.5% identity to SEQ ID NO. 3 and comprising at least or only the amino acid substitution H248L based on the numbering of SEQ ID NO. 6.

In another embodiment, the fHBP B protein may comprise or consist of SEQ ID NO. 4.

In one embodiment, fHBP a protein and/or fHBP B may be present in an amount ranging from about 20 μg/dose to about 200 μg/dose, or from about 25 μg/dose to about 180 μg/dose, or from about 40 μg/dose to about 140 μg/dose, or from about 50 μg/dose to about 120 μg/dose, or from about 75 μg/dose to about 100 μg/dose, or about 25 μg/dose, or about 50 μg/dose, or about 100 μg/dose.

As used herein, the term "dose" refers to the total amount or volume of a composition administered to an individual. The dosage may range from about 0.1ml to about 1ml, for example from about 0.2ml to about 0.8ml, from about 0.4ml to about 0.6ml, or may be about 0.5ml.

In one embodiment, the NadA protein may be a NadA1 protein.

In another embodiment, the NadA protein may comprise at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% identity to SEQ ID No. 5. In another embodiment, the NadA protein may comprise or consist of SEQ ID No. 5.

In one embodiment, nadA protein may be present in an amount ranging from about 20 μg/dose to about 200 μg/dose, or from about 25 μg/dose to about 180 μg/dose, or from about 40 μg/dose to about 140 μg/dose, or from about 50 μg/dose to about 120 μg/dose, or from about 75 μg/dose to about 100 μg/dose, or about 25 μg/dose, or about 50 μg/dose, or about 100 μg/dose. In one embodiment, the NadA protein may be present in an amount of about 50 μg/dose.

In one embodiment, the dOMV may comprise outer membrane protein porin A (PorA). In one embodiment, the dOMV may comprise outer membrane protein porin B (PorB). In another embodiment, the dOMV may comprise outer membrane protein porin A (PorA) and outer membrane protein porin B (PorB).

In another embodiment, the dOMV may comprise outer membrane protein porin A (PorA) and/or outer membrane protein porin B (PorB). The dOMV may comprise porin a (serotype PorA VR 2P 1.2), porin B (serotype PorB P2.2 a) and optionally an immune type LOS L3,7.PorA and PorB may represent about 50% of the dcmv protein.

The PorA may be present in an amount ranging from about 3% to about 15%, or in an amount of about 5% to about 9% or about 10%, relative to the total protein present in the dOMV. PorB may be present in an amount ranging from about 30% to about 70%, or about 35% to about 65%, or about 38% to about 58%, relative to the total protein present in the dOMV.

In one embodiment, the dOMV may comprise porin A (PorA). In one embodiment, the PorA may be a PorA of the VR2 family. In one example, the dOMV may comprise a PorA VR 2P 1.2 subtype.

In one embodiment, the dOMV may be obtained from MenB strain 99M expressing PorA VR2, P1.2.

In one embodiment, the dOMVs may comprise PorA VR 2P 1.2 and PorB P2.2a.

In one embodiment, the dOMVs may be obtained using a detergent extraction process using at least one deoxycholate treatment step.

In one embodiment, the dOMV may be present in an amount ranging from about 5 μg/dose to about 400 μg/dose, or from about 10 μg/dose to about 300 μg/dose, or from about 25 μg/dose to about 250 μg/dose, or from about 35 μg/dose to about 225 μg/dose, or from about 50 μg/dose to about 200 μg/dose, or from about 75 μg/dose to about 180 μg/dose, or from about 100 μg/dose to about 150 μg/dose, or from about 110 μg/dose to about 125 μg/dose, or about 25 μg/dose, or about 50 μg/dose, or about 125 μg/dose.

In another embodiment, the composition as disclosed herein may further comprise an adjuvant. In one embodiment, the adjuvant may be an aluminum-based adjuvant. In one embodiment, the aluminum-based adjuvant may be an aluminum-based adjuvant selected from the group comprising: aluminum hydroxide adjuvants, aluminum phosphate adjuvants, aluminum sulfate adjuvants, aluminum hydroxy phosphate sulfate adjuvants, aluminum potassium sulfate adjuvants, aluminum hydroxy carbonate, combinations of aluminum hydroxide and magnesium hydroxide, and mixtures thereof. In one embodiment, the adjuvant may be an aluminum phosphate adjuvant.

In another embodiment, the compositions as disclosed herein may further comprise a pharmaceutically acceptable excipient.

In another embodiment, the compositions as disclosed herein may further comprise a buffer. In one embodiment, the buffer may be selected from the group comprising: tris buffer, acetate buffer, citrate buffer, phosphate buffer, HEPES buffer or histidine buffer. In one embodiment, the buffer may be an acetate buffer. In one embodiment, the acetate buffer may be a sodium acetate buffer. In one embodiment, the sodium acetate buffer may be present at a concentration ranging from about 10mM to about 300mM, or ranging from about 10mM to about 250mM, or ranging from about 20mM to about 150mM, or about 20mM to about 130mM, or about 30mM to about 120mM, or about 40mM to about 100mM, or about 50mM to about 80mM, or about 50mM to about 60mM, or for example at a concentration of about 50 mM.

In another embodiment, the compositions as disclosed herein may further comprise a salt, such as a sodium, calcium, or magnesium salt. The sodium salt may be, for example, a sodium salt selected from the group comprising: sodium chloride, sodium phosphate. In one embodiment, the sodium salt may be sodium chloride. The calcium salt may be a calcium chloride salt. The magnesium salt may be a magnesium chloride salt. In one embodiment, the sodium salt may be present at a concentration ranging from about 10mM to about 300mM, or about 30mM to about 280mM, or about 50mM to about 250mM, or about 60mM to about 220mM, or about 80mM to about 200mM, or about 100mM to about 180mM, or about 120mM to about 160mM, or may be, for example, at a concentration of about 150 mM. The calcium or magnesium salt may be present in an amount ranging from about 1mM to about 15mM, or from about 5mM to about 10 mM.

In another embodiment, the compositions as disclosed herein may have a pH in the range of about 4.0 to about 9.0. In one embodiment, the pH of the compositions as disclosed herein may range from about 4.5 to about 8.5, or from about 4.8 to about 8.2, or from about 5.0 to about 8.0, or from about 5.2 to about 7.5, or from about 5.4 to about 7.0, or from about 5.5 to about 6.8, or from about 5.7 to about 6.5, or from about 5.8 to about 6.2, or may be about 6.0. In another embodiment, a composition as disclosed herein may comprise or consist of: a non-lipidated fHBP a protein comprising or consisting of SEQ ID No. 2, a non-lipidated fHBP B protein comprising or consisting of SEQ ID No. 4, nadA protein, and a dOMV from MenB expressing PorA protein. In the composition, the NadA may be NadA1 or may comprise or consist of at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5% or about 100% amino acid sequence identity with SEQ ID NO:5, and/or the dOMV may comprise or consist of PorA VR2 subtype or PorA VR 2P 1.2 and optionally PorA P2.2a, or may be obtained from MenB strain 99M. The compositions as disclosed herein may comprise an aluminum phosphate adjuvant. The composition as disclosed herein may comprise 50mM acetate buffer and pH 6.0.

In another embodiment, a composition as disclosed herein may comprise or consist of: a non-lipidated mutant fHBP a protein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% identity with SEQ ID No.1, a non-lipidated mutant fHBP B protein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% identity with SEQ ID No.3, nadA protein, and a dOMV from MenB expressing PorA protein. In the composition, the NadA may be NadA1 or may comprise or consist of at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5% or about 100% amino acid sequence identity with SEQ ID NO:5, and/or the dOMV may comprise or consist of PorA VR2 subtype or PorA VR 2P 1.2 and optionally PorA P2.2a, or may be obtained from MenB strain 99M. The compositions as disclosed herein may comprise an aluminum phosphate adjuvant. The composition as disclosed herein may comprise 50mM acetate buffer and pH 6.0.

In another embodiment, a composition as disclosed herein may comprise or consist of: a non-lipidated mutant fHBP a protein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% identity to SEQ ID NO:1 and comprising at least one amino acid substitution based on numbering of SEQ ID NO:6, a non-lipidated mutant fHBP B protein, nadA protein, and a dOMV from MenB expressing a PorA protein: a) An amino acid substitution of asparagine (N115) at amino acid 115; b) Amino acid substitution of aspartic acid (D121) at amino acid 121; c) Amino acid substitution of serine at amino acid 128 (S128); d) Amino acid substitution of leucine (L130) at amino acid 130; e) Amino acid substitution of valine (V131) at position 131; f) Amino acid substitution of glycine (G133) at position 133; g) Amino acid substitution of lysine (K219) at position 219; and h) an amino acid substitution of glycine (G220) at position 220, the non-lipidated mutant fHBP B protein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% identity to SEQ ID No. 3 and comprising at least one amino acid substitution based on the numbering of SEQ ID No. 6 selected from at least one of: a) Amino acid substitution of glutamine (Q38) at amino acid 38; b) Amino acid substitution of glutamic acid (E92) at amino acid 92; c) Amino acid substitution of arginine (R130) at amino acid 130; d) Amino acid substitution of serine (S223) at amino acid 223; and e) an amino acid substitution of histidine (H248) at amino acid 248. In the composition, the NadA may be NadA1 or may comprise or consist of at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5% or about 100% amino acid sequence identity with SEQ ID NO:5, and/or the dOMV may comprise or consist of PorA VR2 subtype or PorA VR 2P 1.2 and optionally PorA P2.2a, or may be obtained from MenB strain 99M. The compositions as disclosed herein may comprise an aluminum phosphate adjuvant. The composition as disclosed herein may comprise 50mM acetate buffer and pH 6.0.

In another embodiment, a composition as disclosed herein may comprise or consist of: a non-lipidated mutant fHBP a protein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% identity to SEQ ID NO:1 and comprising at least one amino acid substitution based on numbering of SEQ ID NO: 6: G220S, L R and G133D, said non-lipidated mutant fHBP B protein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% identity to SEQ ID NO.3 and comprising at least the amino acid substitution H248L based on the numbering of SEQ ID NO. 6. In the composition, the NadA may be NadA1 or may comprise or consist of at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5% or about 100% amino acid sequence identity with SEQ ID NO:5, and/or the dOMV may comprise or consist of PorA VR2 subtype or PorA VR 2P 1.2 and optionally PorA P2.2a, or may be obtained from MenB strain 99M. The compositions as disclosed herein may comprise an aluminum phosphate adjuvant. The composition as disclosed herein may comprise 50mM acetate buffer and pH 6.0.

In another embodiment, a composition as disclosed herein may comprise or consist of: a non-lipidated mutant fHBP a protein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% identity to SEQ ID NO:1 and numbering based on SEQ ID NO:6 comprising at least three amino acid substitutions selected from the group consisting of: G220S, L R and G133D, said non-lipidated mutant fHBP B protein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% identity to SEQ ID NO.3 and comprising at least the amino acid substitution H248L based on the numbering of SEQ ID NO. 6. In the composition, the NadA may be NadA1 or may comprise or consist of at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5% or about 100% amino acid sequence identity with SEQ ID NO:5, and/or the dOMV may comprise or consist of PorA VR2 subtype or PorA VR 2P 1.2 and optionally PorA P2.2a, or may be obtained from MenB strain 99M. The compositions as disclosed herein may comprise an aluminum phosphate adjuvant. The composition as disclosed herein may comprise 50mM acetate buffer and pH 6.0.

In another embodiment, a composition as disclosed herein may comprise or consist of: a non-lipidated mutant fHBP a protein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% identity to SEQ ID NO:1 and comprising only three amino acid substitutions based on numbering of SEQ ID NO: 6: G220S, L R and G133D, said non-lipidated mutant fHBP B protein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% identity to SEQ ID NO. 3 and comprising only amino acid substitution H248L based on the numbering of SEQ ID NO. 6. In the composition, the NadA may be NadA1 or may comprise or consist of at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least 99%, at least about 99.5% or about 100% amino acid sequence identity with SEQ ID NO:5, and/or the dOMV may comprise or consist of PorA VR2 subtype or PorA VR 2P 1.2 and optionally PorA P2.2a, or may be obtained from MenB strain 99M. The compositions as disclosed herein may comprise an aluminum phosphate adjuvant. The composition as disclosed herein may comprise 50mM acetate buffer and pH 6.0.

In another embodiment, a composition as disclosed herein may comprise or consist of: a non-lipidated fHBP a protein comprising or consisting of SEQ ID No.2, a non-lipidated fHBP B protein comprising or consisting of SEQ ID No.4, a NadA protein comprising or consisting of SEQ ID No. 5, a dOMV from MenB expressing PorA VR 2P 1.2 and optionally PorA P2.2a or obtained from MenB strain 99M. The compositions as disclosed herein may comprise an aluminum phosphate adjuvant. The composition as disclosed herein may comprise 50mM acetate buffer and pH 6.0.

In another embodiment, a composition as disclosed herein may comprise or consist of: the non-lipidated fHBP a protein consisting of SEQ ID No.2, the non-lipidated fHBP B protein consisting of SEQ ID No. 4, the NadA protein consisting of SEQ ID No. 5, and the dOMV from MenB expressing PorA VR 2P 1.2 and optionally PorA P2.2a or obtained from MenB strain 99M. The compositions as disclosed herein may comprise an aluminum phosphate adjuvant.

In another embodiment, a composition as disclosed herein may comprise or consist of: non-lipidated fHBP a protein consisting of SEQ ID No. 2, non-lipidated fHBP B protein consisting of SEQ ID No. 4, nadA protein consisting of SEQ ID No. 5, d omv from MenB expressing PorA VR 2P 1.2 and optionally PorA B P2.2a or obtained from MenB strain 99M, aluminium phosphate adjuvant, 50mM acetate buffer and pH 6.0.

In another embodiment, a composition as disclosed herein may comprise or consist of: an mRNA encoding a fHBP a protein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5% or about 100% amino acid sequence identity to SEQ ID No. 2, an mRNA encoding a fHBP B protein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99.5% or about 100% amino acid sequence identity to SEQ ID No. 4, an mRNA encoding a NadA protein comprising at least about 85%, at least about 90%, at least about 99.5% or about 100% amino acid sequence identity to SEQ ID No. 5, and a dOMV from MenB expressing PorA. The PorA may be a PorA VR2 subtype or PorA VR 2P 1.2.

In another embodiment, a composition as disclosed herein may comprise or consist of: about 25 to about 100 μg/dose of a non-lipidated fHBP a protein comprising SEQ ID No. 2, about 25 to about 100 μg/dose of a non-lipidated fHBP B protein comprising SEQ ID No. 4, about 25 to about 100 μg/dose of NadA protein comprising SEQ ID No. 5, about 20 to about 150 μg/dose of a dOMV from a MenB strain expressing PorA VR 2P 1.2, about 100 to about 600 μg/dose of an aluminum phosphate adjuvant, 50mM acetate buffer and pH 6.0.

In another embodiment, a composition as disclosed herein may comprise or consist of: about 25 to about 100 μg/dose of non-lipidated fHBP a protein consisting of SEQ ID No. 2, about 25 to about 100 μg/dose of non-lipidated fHBP B protein consisting of SEQ ID No. 4, about 25 to about 100 μg/dose of NadA protein consisting of SEQ ID No. 5, about 20 to about 150 μg/dose of dOMV from the MenB strain expressing PorA VR 2P 1.2, about 100 to about 600 μg/dose of aluminium phosphate adjuvant, 50mM acetate buffer and pH 6.0.

In another aspect, the present disclosure relates to a vaccine comprising a composition as disclosed herein.

In another aspect, the present disclosure relates to a composition as disclosed herein as a medicament, particularly as a vaccine.

In another aspect, the present disclosure relates to a composition as disclosed herein for use in preventing meningococcal infection, and in one exemplary embodiment, for use in preventing neisseria meningitidis serogroup B infection.

In another aspect, the present disclosure relates to a composition as disclosed herein for inducing an immune response against meningococcal bacteria, and in one exemplary embodiment against neisseria meningitidis serogroup B bacteria.

In another aspect, the present disclosure relates to a composition as disclosed herein for inducing an immune response against neisseria meningitidis serogroup B bacteria from ST-41/44, ST-32, ST-269, ST-213, ST-35, ST-461, ST-11, and/or ST-461 clone complexes.

In another aspect, the disclosure relates to a composition as disclosed herein for inducing an immune response against neisseria meningitidis serogroup B bacteria from an ST-11 clone complex.

In another aspect, the present disclosure relates to a method of protecting an individual from meningococcal infection and in one exemplary embodiment from neisseria meningitidis serogroup B infection, the method comprising at least the step of administering to the individual an immunogenic composition as disclosed herein or a vaccine as disclosed herein.

In another aspect, the present disclosure relates to a method for reducing the risk of developing an invasive meningococcal disease caused by a meningococcal infection in an individual, and in one exemplary embodiment by a neisseria meningitidis serogroup B infection, the method comprising at least the step of administering to the individual an immunogenic composition as disclosed herein or a vaccine as disclosed herein.

In another aspect, the present disclosure relates to a method of eliciting an immune response against neisseria meningitidis serogroup B bacteria in an individual, and in one exemplary embodiment, the method comprising at least the step of administering to the individual an immunogenic composition as disclosed herein or a vaccine as disclosed herein.

In another aspect, the present disclosure relates to a method of eliciting an immune response against neisseria meningitidis serogroup B bacteria from a ST-41/44, ST-32, ST-269, ST-213, ST-35, ST-461, ST-11 and/or ST-461 clone complex, the method comprising at least the step of administering to the individual an immunogenic composition as disclosed herein or a vaccine as disclosed herein.

In another aspect, the present disclosure relates to a method of eliciting an immune response against neisseria meningitidis serogroup B bacteria from an ST-11 clone complex, the method comprising at least the step of administering to the individual an immunogenic composition as disclosed herein or a vaccine as disclosed herein.

In another aspect, the disclosure relates to a method of preparing an immunogenic composition as disclosed herein or a vaccine as disclosed herein, the method comprising at least the step of mixing a meningococcal antigen comprising at least one factor H binding protein (fHBP) a protein, at least one fHBP B protein, at least one neisseria adhesion agent a (NadA) protein, and at least one detergent-extracted outer membrane vesicle (dOMV), and optionally an aluminium salt.

In one embodiment, the step of mixing may include blending a first mixture of at least one factor H binding protein (fHBP) a protein optionally adsorbed on AlPO ₄ and at least one fHBP B protein optionally adsorbed on AlPO ₄ salt with a second mixture of at least one NadA protein and dOMV optionally adsorbed on AlPO ₄.

In one embodiment, the step of mixing may include blending a first mixture of at least one factor H binding protein (fHBP) a protein adsorbed on AlPO ₄ and at least one fHBP B protein adsorbed on AlPO ₄ salt with a second mixture of at least one NadA protein and dOMV adsorbed on AlPO ₄.

In another embodiment, the antigen and aluminum salt used in the preparation methods as disclosed herein may be in a buffer.

In another aspect, the disclosure relates to a multicomponent kit comprising a plurality of containers, wherein each of the containers comprises at least one meningococcal antigen or a combination of at least two meningococcal antigens selected from the group comprising: fHBP a protein, fHBP B protein, nadA protein and detergent-extracted outer membrane vesicles (dOMV).

In one embodiment, at least one of the antigens of the multicomponent kit may be in dry form. In one embodiment, at least one of the antigens may be in the form of lyophilized or dried pellets. In one embodiment, the kit may optionally comprise a container containing a physiologically injectable vehicle.

Drawings

FIGS. 1A and 1B show specific anti-A05 tmN (FIG. 1A) or anti-B01 smN (FIG. 1B) IgG titers (LLOQ means a specified lower limit) measured by ELISA in sera collected from rabbits immunized with TRUMENBA or with formulations F1 to F6 or F1 co-administered with MENQUADFI at D0 (open/white symbols) and D42 (solid/black symbols) at D0, D28 and D56.

Fig. 2A and 2B fig. 2A shows specific anti-NadA IgG titers measured by ELISA in D0 (open/white symbols) and D42 (solid/black symbols) from sera collected from rabbits immunized with BEXSERO or with formulations F1-F5 and F1 co-administered with MENQUADFI at D0 and D28. Figure 2B shows specific anti-dcmv IgG measured by ELISA in sera collected at D0 and D42 from rabbits immunized with formulations F1 to F5 and F1 co-administered with MENQUADFI at D0, D28 and D56.

Figure 3 shows hSBA against closely related MenB B44 strain (strain n°3) measured at D0 (open/white symbols) and D42 (solid/black symbols) from IgG purified from rabbits immunized with TRUMENBA, BEXSERO or formulations F1 to F6 or F1 co-administered with MENQUADFI at D0, D28 and D56.

Fig. 4 shows hSBA against a heterologous MenB B24 strain (strain n° 4) measured at D0 (open/white symbols) and D42 (solid/black symbols) from IgG purified from rabbits immunized with TRUMENBA, BEXSERO or formulations F1 to F6 or F1 co-administered with MENQUADFI at D0, D28 and D56.

Fig. 5 shows hSBA against a heterologous MenB B24 strain (strain n°18) measured at D0 (open/white symbols) and D42 (solid/black symbols) from IgG purified from rabbits immunized with TRUMENBA, BEXSERO or formulations F1 to F6 or F1 co-administered with MENQUADFI at D0, D28 and D56.

Fig. 6 shows hSBA against the closely related MenB a56 strain (strain n°1) measured at D0 (open/white symbols) and D42 (solid/black symbols) from IgG purified from rabbits immunized with TRUMENBA, BEXSERO or formulations F1 to F6 or F1 co-administered with MENQUADFI at D0, D28 and D56.

Fig. 7 shows hSBA against a heterologous MenB a22 strain (strain n°2) measured at D0 (open/white symbols) and D42 (solid/black symbols) from IgG purified from rabbits immunized with TRUMENBA, BEXSERO or formulations F1 to F6 or F1 co-administered with MENQUADFI at D0, D28 and D56.

Fig. 8 shows hSBA against the MenB NadA1 strain (strain n°6) measured at D0 (open/white symbols) and D42 (solid/black symbols) from IgG purified from rabbits immunized with TRUMENBA, BEXSERO or formulations F1 to F6 or F1 co-administered with MENQUADFI at D0, D28 and D56.

Fig. 9 shows hSBA against MenB OMV PorA 1.2 strain (strain n°5) measured at D0 (open/white symbols) and D42 (solid/black symbols) from IgG purified from rabbits immunized with TRUMENBA, BEXSERO or formulations F1 to F6 or F1 co-administered with MENQUADFI at D0, D28 and D56.

FIG. 10 shows cytokine production (IL-6, TNF. Alpha., MIP-1. Beta. And IL-1. Beta.) of Dendritic Cells (DCs) derived from adult PTE treated with different doses BEXSERO and TRUMENBA: culture supernatants from PTEs were harvested 48 hours post-treatment and cytokine secretion was assessed using multiple arrays. The graph shows the Geometric Mean (GMV) of cytokine production by IL-6, TNF alpha, IL-1β and MIP-1β in Dendritic Cells (DCs). Treatment was performed at 10-fold dilution of human dose (n=20 donors). N/A is a mimetic control without antigen.

FIG. 11 shows cytokine production (IL-6, TNF. Alpha., MIP-1. Beta. And IL-1. Beta.) of neonatal PTE-derived DCs treated with different doses BEXSERO and TRUMENBA: culture supernatants from PTE were harvested 48 hours post-treatment and cytokine secretion was assessed using a multiplex array, graphically representing GMV for cytokine production by IL-6, TNF alpha, IL-1β and MIP-1β in DCs. Treatment was performed at 10-fold dilution of human dose (n=20 donors).

Figures 12A and 12B show forest plots of cytokine secretion triggered by BEXSERO in adult and neonatal PTE at a dilution of 1:10000 compared to geometric mean ratio (with 95% confidence interval) in the case of TRUMENBA. The dashed line set to a value of 1, a value and confidence of the interval below 1 means TRUMENBA is better than BEXSERO, whereas BEXSERO is better than TRUMENBA if the value and interval are above 1.

FIG. 13 shows secretion of cytokines (IL-6, TNF. Alpha., MIP-1. Beta. And IL-1. Beta.) induced by F1-F5 formulations in adult PTE modules. Adult PTE was simulated with different doses of F1-F5 formulation and control (BEXSERO) treatments. Thereafter, culture supernatants were collected and cytokine secretion was assessed by multiplex arrays. Mean values of IL-6, TNF alpha, IL-1 beta and MIP-1 beta.+ -. 95% CI; n=16 to 24.

Figures 14A, 14B and 14C represent forest plots of geometric mean ratio (with 95% confidence interval) of cytokine secretion induced by BEXSERO at 1:10000 dilution in adult PTE compared to the case of formulations F1, F2, F3, F4 and F5. The dashed line being set to a value of 1, a value of the interval and confidence below 1 means that the process is better than BEXSERO, while if the value and interval are above 1, BEXSERO is better than the process. Fig. 14A shows the results obtained with F2 and F3 formulations, and fig. 14B and 14C show the results obtained with F1, F4 and F5 formulations.

Figure 15 shows immunocytotoxicity (or percentage of living cells after treatment) after treatment with F1-F5 formulation and BEXSERO in adult simulated PTE.

FIG. 16 shows an increase in cytokine (IL-6, TNFα, MIP-1β, and IL-1β) secretion levels from F1-F5 formulations compared to BEXSERO in neonatal PTE modules. Treatment with different doses of F1-F5 formulation and control (BEXSERO) mimics neonatal PTE. Thereafter, culture supernatants were collected and cytokine secretion was assessed by multiplex arrays. Mean values of IL-6, TNF alpha, IL-1b and MIP-1b (n=16 to 24). + -95% CI. N/A is a mimetic control without antigen.

Figures 17A and 17B show forest plots of geometric mean ratio (with 95% confidence intervals) of cytokine secretion induced by BEXSERO at 1:10000 dilution in neonatal PTE compared to the case of formulations F1, F2, F3, F4 and F5. The dashed line is set to a value of 1, and values and confidence levels of the interval below 1 mean that the process is better thanWhereas if the value and interval is higher than 1, then/>Is superior to the treatment. Fig. 17A shows the results obtained with F2 and F3 formulations, and fig. 17B shows the results obtained with F1, F4 and F5 formulations.

Figure 18 shows immunocytotoxicity (or percentage of viable cells after treatment) after treatment with F1-F5 formulation and BEXSERO in neonatal mock PTE.

Fig. 19 shows a summary of hSBA results, demonstrating different clonal complexes covered or uncovered by the different formulations tested: f3, TRUMENBA and BEXSERO against 18 MenB strains from different cloning complexes. Black arc: BEXSERO. Dotted arc: TRUMENBA. Gray arc: f3 formulation. The numbers in the circular arc represent the number of MenB strains covered by the formulation in the total number of MenB strains tested for each clone complex.

DESCRIPTION OF THE SEQUENCES

SEQ ID NO. 1 shows the wild-type sequence of fHBP A05 without the signal peptide responsible for lipidation.

CSSGSGSGGGGVAADIGTGLADALTAPLDHKDKGLKSLTLEDSISQNGTL TLSAQGAEKTFKVGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPSGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELASAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIREKVHEIGIAGKQ

SEQ ID NO.2 shows the mutated fHBP A05 sequence (numbering is determined with respect to the sequence SEQ ID NO.6 (fHBP B24)) without the signal peptide responsible for lipidation and with the mutation G220S, L, R, G D.

CSSGSGSGGGGVAADIGTGLADALTAPLDHKDKGLKSLTLEDSISQNGTLT LSAQGAEKTFKVGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFRVSDLGGEHTAFNQLPSGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELASAELKADEKSHAVILGDTRYGSEEKSTYHLALFGDRAQEIAGSATVKIREKVHEIGIAGKQ

SEQ ID NO. 3 shows the wild-type sequence of fHBP B01 without the signal peptide responsible for lipidation.

CSSGGGGSGGGGVTADIGTGLADALTAPLDHKDKGLKSLTLEDSISQNG TLTLSAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQTEQEQDPEHSEKMVAKRRFRIGDIAGEHTSFDKLPKDVMATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAVAYIKPDEKHHAVISGSVLYNQDEKGSYSLGIFGEKAQEVAGSAEVETANGIHHIGLAAKQ

SEQ ID NO. 4 shows the mutated fHBP B01 sequence (numbering is determined with respect to the sequence SEQ ID NO. 6 (fHBP B24)) without the signal peptide responsible for lipidation and with the mutation H248L.

CSSGGGGSGGGGVTADIGTGLADALTAPLDHKDKGLKSLTLEDSISQNG TLTLSAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQTEQEQDPEHSEKMVAKRRFRIGDIAGEHTSFDKLPKDVMATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAVAYIKPDEKHHAVISGSVLYNQDEKGSYSLGIFGEKAQEVAGSAEVETANGIHLIGLAAKQ

SEQ ID NO. 5 shows the NadA1 sequence from the MenB MC58 strain in which 23 amino acids of the N-terminal signal peptide and the last 55 amino acids of the C-terminal have been deleted.

MTSDDDVKKAATVAIVAAYNNGQEINGFKAGETIYDIGEDGTITQKDA TAADVEADDFKGLGLKKVVTNLTKTVNENKQNVDAKVKAAESEIEKLTTKLADTDAALADTDAALDETTNALNKLGENITTFAEETKTNIVKIDEKLEAVADTVDKHAEAFNDIADSLDETNTKADEAVKTANEAKQTAEETKQNVDAKVKAAETAAGKAEAAAGTANTAADKAEAVAAKVTDIKADIATNKADIAKNSARIDSLDKNVANLRKETRQGLAEQAALSGLFQPYNVG

SEQ ID NO. 6 shows the wild-type sequence of fHBP B24 on the basis of which the numbering of the mutation positions in A05 and B01 is determined.

CSSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKL AAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKMVAKRQFRIGDIAGEHTSFDKLPEGGRATYRGTAFGSDDAGGKLTYTIDFAAKQGNGKIEHLKSPELNVDLAAADIKPDGKRHAVISGSVLYNQAEKGSYSLGIFGGKAQEVAGSAEVKTVNGIRHIGLAAKQ

SEQ ID NO. 7 shows the wild-type NadA1 sequence from the MenB MC58 strain.

MKHFPSKVLTTAILATFCSGALAATSDDDVKKAATVAIVAAYNNGQE INGFKAGETIYDIGEDGTITQKDATAADVEADDFKGLGLKKVVTNLTKTVNENKQNVDAKVKAAESEIEKLTTKLADTDAALADTDAALDETTNALNKLGENITTFAEETKTNIVKIDEKLEAVADTVDKHAEAFNDIADSLDETNTKADEAVKTANEAKQTAEETKQNVDAKVKAAETAAGKAEAAAGTANTAADKAEAVAAKVTDIKADIATNKADIAKNSARIDSLDKNVANLRKETRQGLAEQAALSGLFQPYNVGRFNVTAAVGGYKSESAVAIGTGFRFTENFAAKAGVAVGTSSGSSAAYHVGVNYEW

Detailed Description

Definition of the definition

It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antigen" includes a plurality of such antigens, and reference to "a protein" includes reference to one or more proteins, and so forth.

The term "about" or "approximately" as used herein refers to a common error range for the corresponding value as readily known to those of skill in the art. References herein to "about" a value or parameter include (and describe) implementations directed to the value or parameter itself. In some embodiments, the term "about" refers to ± 10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, ±1% of the given value. However, as long as the value in question refers to an indivisible object, such as a molecule or other object that loses its identity once subdivided, "about" refers to + -1 of the indivisible object.

The term "antigen" includes any molecule, such as a peptide, protein, polysaccharide or glycoconjugate, that comprises at least one epitope against which an immune response is to be elicited and/or at least one epitope against which an immune response is to be elicited. For example, an antigen is a molecule that, optionally after processing, induces an immune response, e.g., specific for the antigen or cells expressing the antigen. After processing, the antigen can be presented by MHC molecules and react specifically with T lymphocytes (T cells). Thus, an antigen or fragment thereof should be recognizable by a T cell receptor and should be able to induce clonal expansion of T cells carrying T cell receptors specifically recognizing the antigen or fragment in the presence of an appropriate co-stimulatory signal, which results in an immune response against the antigen or antigen expressing cells. Any suitable antigen that is a candidate for an immune response is contemplated in accordance with the present disclosure. The antigen may correspond to or may be derived from a naturally occurring antigen.

It should be understood that the aspects and embodiments of the present disclosure described herein include, consist of, and consist essentially of the "having", "comprising" aspects and embodiments. The terms "having" and "comprising" or variations such as "having", "including" or "comprising" are to be construed as implying that one or more of the elements such as a composition of matter or method steps is included, but not excluding any other elements. The term "consisting of … …" implies inclusion of one or more of the recited elements, excluding any additional elements. The term "consisting essentially of … …" implies inclusion of the recited element, and possibly one or more other elements, wherein the one or more other elements do not materially affect one or more of the basic and novel features of the present disclosure. It is to be understood that the various embodiments of the disclosure that use the term "comprising" or equivalent terms contemplate embodiments in which the term is replaced with "consisting of … …" or "consisting essentially of … ….

The phrase "a disease caused by a strain of neisseria meningitidis" includes any clinical symptom or combination of clinical symptoms that exist when a human is infected with neisseria meningitidis. These symptoms include, but are not limited to: the upper respiratory tract (e.g., the mucosa of the nasopharynx and tonsils) is colonized by pathogenic strains of neisseria meningitidis, bacterial infiltration into the mucosa and submucosal vascular bed, sepsis, septic shock, inflammation, hemorrhagic skin injury, activation of fibrinolysis and coagulation, organ dysfunction (e.g., kidney, lung and heart failure), adrenal hemorrhage and muscle infarction, capillary leakage, oedema, peripheral limb ischemia, respiratory distress syndrome, pericarditis and meningitis.

As used herein, the terms "individual" or "subject" or "patient" are used interchangeably and are intended to refer to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some exemplary embodiments, the individual or subject is a human.

In the context of the present disclosure, the expression "pharmaceutically acceptable carrier" refers to a carrier or vehicle that is physiologically acceptable for administration to a mammal, such as a human, while retaining the physiological activity of the immunogenic composition as disclosed herein, i.e. its ability to induce an immune response with a hypoallergenic effect.

As used herein, the term "preventing" or "delay of progression" (and grammatical variants thereof) in relation to a disease or disorder relates to the prophylactic treatment of the disease or disorder, for example, in an individual suspected of having or at risk of having the disease. Prevention may include, but is not limited to, preventing or delaying the onset or progression of a disease and/or maintaining one or more symptoms of a disease or disorder at a desired level or sub-pathological level. The term "preventing" does not require 100% elimination of the possibility or likelihood of occurrence of an event. Rather, it means that the likelihood of an event occurring has been reduced in the presence of a composition or method as described herein.

The term "protective immunity" means that a vaccine or immunization regimen administered to a mammal induces an immune response that prevents, delays the progression of, or reduces the severity of a disease caused by neisseria meningitidis, or reduces or completely eliminates symptoms of the disease. Protective immunity may be accompanied by the production of bactericidal antibodies. It should be noted that the production of bactericidal antibodies against neisseria meningitidis is accepted in the art as a predictor of the protective effect of the vaccine in humans. (Goldschneider et al (1969) J.Exp. Med.129:1307).

In this disclosure, the term "significant" as used in relation to a change is intended to mean that the observed change is apparent and/or that it has statistical significance.

In this disclosure, the term "substantially" as used in connection with a feature of this disclosure is intended to define a set of embodiments related to that feature that are largely analogous to, but not entirely analogous to, the feature. The distinction between a set of embodiments associated with a given feature and a given feature is such that the nature and function of the given feature is not materially affected in this set of embodiments.

The phrase "in an amount sufficient to elicit an immune response" or "immunologically effective amount" as used with respect to an antigen or combination of an antigen and an adjuvant is intended to refer to an amount effective to elicit an immune response against an antigen when administered to a subject. The amount may vary depending on various factors such as the health or physical condition of the subject, age, ability of the subject's immune system to produce antibodies, degree of protection desired, formulation of the antigen-containing composition, assessment of the medical condition by the treating physician. The amount may be determined by conventional methods known to the skilled person. Immune response indicators include, but are not limited to: antibody titer or specificity as detected by an assay such as: enzyme-linked immunosorbent assay (ELISA), bactericidal assay, flow cytometry, immunoprecipitation, ouchterlonyl immunodiffusion; binding detection assays such as spots, western blots or antigen arrays; cytotoxicity measurement, and the like.

As used herein, the terms "treat," "treatment," "therapy," and the like in the context of eliciting an immune response refer to the administration or consumption of a composition as disclosed herein for the purpose of curing, healing, alleviating, altering, remedying, ameliorating, improving, or affecting the symptoms of a disease or disorder, condition, or preventing or delaying the onset of symptoms, complications, or otherwise preventing or inhibiting the further development of a disorder in a statistically significant manner. Furthermore, as used herein, in the context of the present disclosure, the terms "treatment," "treatment," and the like refer to the alleviation or alleviation of pathological processes mediated by neisseria meningitidis infection. In the context of the present disclosure, the terms "treat," "treatment," and the like, when referring to any other disorder described herein, refer to alleviating or alleviating one or more symptoms associated with such disorder.

As used herein, the term "vaccine" is intended to mean an immunogenic composition against a pathogen that is administered to a subject to induce an immune response, intended to protect or treat the subject from a disorder caused by the pathogen. The vaccine as disclosed herein is intended for use as a prophylactic (preventative) vaccine for administration to a subject prior to infection, intended to prevent or reduce the likelihood of an initial (and/or recurrent) infection occurring.

As used herein, the term "messenger RNA" or "mRNA" refers to a polynucleotide encoding at least one polypeptide. As used herein, mRNA includes both modified and unmodified RNAs. An mRNA may comprise one or more coding and non-coding regions. The coding region may alternatively be referred to as an Open Reading Frame (ORF). The non-coding region in the mRNA comprises a 5' cap, a 5' untranslated region (UTR), a 3' UTR, and a poly (A) tail. mRNA can be purified from a natural source, produced using a recombinant expression system (e.g., in vitro transcription) and optionally purified, or chemically synthesized.

The mRNA disclosed herein may be modified or unmodified. In some embodiments, the mRNA comprises at least one chemical modification. In some embodiments, the mRNA disclosed herein can comprise one or more modifications that generally enhance RNA stability. Exemplary modifications may include backbone modifications, sugar modifications, or base modifications. In some embodiments, the disclosed mRNA can be synthesized from naturally occurring nucleotides and/or nucleotide analogs (modified nucleotides), including but not limited to purines (adenine (a) and guanine (G)) or pyrimidines (thymine (T), cytosine (C), and uracil (U)). In certain embodiments, the disclosed mRNA can be synthesized from modified nucleotide analogs or derivatives of purines and pyrimidines, such as, for example, 1-methyl-adenine, 2-methylsulfanyl-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2, 6-diaminopurine, 1-methyl-guanine, 2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5- (carboxyhydroxymethyl) -uracil, 5-fluoro-uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-methyl-uracil, 5-oxo-methyl-uracil, 5-methoxy-amino-methyl-uracil, 5-thio-methyl-uracil, 5' -methoxycarbonylmethyl-uracil, 5-methoxy-uracil, methyl uracil-5-oxyacetate, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, pigtail glycoside, beta-D-mannosyl-pigtail glycoside, phosphoramidate, phosphorothioate, peptide nucleotide, methylphosphonate, 7-deazaguanosine, 5-methylcytosine and inosine.

In some embodiments, the disclosed mRNA may comprise at least one chemical modification including, but not limited to, pseudouridine, N1-methyl pseudouridine, 2-thiouridine, 4 '-thiouridine, 5-methylcytosine, 2-thiol-methyl-1-deaza-pseudouridine, 2-thiol-methyl-pseudouridine, 2-thio5-aza-uridine, 2-thiodihydro-pseudouridine, 2-thio-dihydro-uridine, 2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydro-pseudouridine, 5-methyluridine, 5-methoxy-uridine, and 2' -O-methyl-uridine.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

A list of sources, ingredients, and components as described below are listed, as are combinations and mixtures thereof and are contemplated and within the scope of the present disclosure.

It is to be understood that each maximum numerical limit set forth throughout this specification includes each lower numerical limit as if such lower numerical limit were explicitly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

All item lists, such as component lists, are intended and should be construed as markush groups. Thus, all lists can be read and interpreted as "items selected from the list of items" and combinations and mixtures thereof.

Cited herein may be trade names for components including the various ingredients used in the present disclosure. The inventors herein do not intend to be limited by the materials under any particular trade name. Materials equivalent to those cited under trade name (e.g., materials obtained from different sources under different names or reference numbers) may be substituted and used in the description herein.

Antigens

The immunogenic composition as disclosed herein comprises at least a combination of meningococcal antigens. The combination of antigens may comprise at least one factor H binding protein (fHBP) a protein, at least one fHBP B protein, at least one neisseria adhesion protein a (NadA) protein and at least one detergent-extracted outer membrane vesicle (dOMV).

In one embodiment, the meningococcal antigen may be from neisseria meningitidis serogroup B.

fHBP

Meningococcal fHBP, also known in the art as lipoprotein 2086 (LP 2086), ORF2086, genome-derived neisserial antigen (GNA) 1870 or "741", is a lipoprotein expressed on the bacterial surface of almost all invasive meningococcal isolates. fHBP is an important virulence factor because it binds to human complement factor H (fH), which is a negative regulator of the alternative complement pathway (Seib et al, expert Rev vaccines.2015;14 (6): 841-59). Binding of fHBP to human fH allows the pathogen to escape alternative complement-mediated killing of the host's innate immune system and survive in human serum and blood.

Three major genetic and immunological fHBP variants have been described: variant 1 corresponding to subfamily B, and variants 2 and 3 both divided in subfamily A (Seib et al, expert Rev vaccines.2015;14 (6): 841-59). In addition to the nomenclature provided by Pfizer (fHBP a and B) and Novartis (variants 1,2 and 3), fHBP is identified in PubMLST database using a unique ID number. Although there is significant antigenic variability between fHBP subfamilies a and B, protein sequences within one subfamily are highly conserved across different strains, with >86% sequence identity. Each unique fHBP found in Neisseria meningitidis is also assigned a fHBP peptide ID according to Neisseria. Org or pubmlst. Org/neisseria/fHBP/website. Because the lengths of variant 2 (v.2) fHBP protein (from strain 8047, fHBP ID 77) and variant 3 (v.3) fHBP (from strain M1239, fHBP ID 28) differ by-1 and +7 amino acid residues, respectively, from the length of MC58 (fHBP ID 1), the numbering of the residues used to refer to v.2 and v.3fHBP proteins differs from the numbering based on the actual amino acid sequence of these proteins. Thus, for example, reference to leucine residue (L) at position 166 of the v.2 or v.3fhbp sequence refers to residues at position 165 of the v.2 protein and at position 173 of the v.3 protein. Members of variants 1,2 and 3 were present in approximately 65%, 25% and 10% of MenB clinical isolates leading to invasive disease, respectively. The ten most prevalent fHBP variants present in the global MenB strain population account for approximately 80% of the total invasive pathogenic strains in the united states and europe (Bambini et al ,Vaccine.2009;27(21):2794-803;Chang,J Infect2019;S0163-4453(19):30272-5;Lucidarme,Clin Vaccine Immunol 2010;17(6):919-29; and Murphy et al The Journal of infectious diseases 2009;200 (3): 379-89; wang et al, vaccine.2011;29 (29-30): 4739-44).

FHBP to be used according to the present disclosure may be a wild-type (or naturally occurring) polypeptide or may be modified (non-naturally occurring) by amino acid substitutions, insertions or deletions, provided that the polypeptide can elicit an immune response.

The fHBP to be used according to the present disclosure may be lipidated or non-lipidated fHBP. Lipidated proteins typically comprise a specific peptide sequence for lipidation at their N-terminal sequence. The sequence may be cleaved at the mature stage of the protein. Those lipidated signal peptides are specific to each protein and the cell of the host producing the protein.

The ORF2086 polypeptide is expressed in neisseria meningitidis as a precursor protein with a lipoprotein signal motif. During processing, the motif is cleaved leaving an N-terminal cysteine residue which is co-translationally modified to have a lipid anchor that tethers the protein to the Neisseria outer membrane (McNeil et al (2013) MMBR (2): 234-252).

To avoid lipidation of the recombinant protein, various techniques known in the art may be used. As an example, it is possible to delete the lipidated peptide signal or replace it with another peptide signal that is not recognized by the cells producing the protein. US10,300,122B2 describes the use of this technique for ORF 2086.

Furthermore, the codon encoding the N-terminal cysteine may be replaced with a codon encoding another amino acid, or the codon encoding the N-terminal cysteine may be deleted. For example, the fusion of the ATG (methionine) codon directly to the second 5' terminal codon of ORF2086 encoding the mature polypeptide, resulting in the deletion of the cysteinylation site (or substitution of cysteine with methionine) is described with respect to fHBP, US10,300,122B2. Furthermore, for example, US 9,724,402 B2 or US 11,077,180B2 disclose that non-lipidated fHBP is obtained in which the N-terminal Cys is substituted with an amino acid other than a Cys residue.

FHBP to be used according to the present disclosure may be a naturally occurring or non-naturally occurring protein. Non-naturally occurring proteins are referred to as "artificial proteins" and include fHBP with heterologous components that are not found in nature, unlike naturally occurring proteins. The non-naturally occurring protein may be a chimeric protein or a mutein. In the context of the present disclosure, "chimeric protein" is intended to refer to a protein comprising two or more different components, each component derived from a different fHBP (e.g., variants 1,2, or 3). Mutations in muteins may include amino acid substitutions, insertions or deletions. In one embodiment, the mutation is an amino acid substitution.

Non-naturally occurring fHBP suitable for use in the immunogenic compositions as disclosed herein are still capable of eliciting an immune response against fHBP. In one embodiment, the non-naturally occurring fHBP to be used according to the present disclosure may be a mutant fHBP. Mutations, such as amino acid substitutions, may be introduced to reduce or inhibit the binding of fHBP antigen to coagulation factor H (fH) normally found in the blood of an individual. Thus, preventing fH from binding to fHBP antigens used in the immunogenic compositions of the present disclosure can increase the amount of antigens accessible to the immune system and improve the efficacy and efficiency of immune responses against those antigens. Advantageously, the mutated fHBP can elicit a pool of anti-fHBP antibodies against fHBP epitopes within the fH binding site, which results in a stronger protective complement deposition activity compared to antibodies raised by wild-type (WT) fHBP antigens targeting fHBP epitopes outside the fH binding site.

It is contemplated that non-naturally occurring fHBP for use in the immunogenic compositions as disclosed herein may exhibit reduced affinity for fH or improved thermostability compared to the corresponding naturally occurring fHBP. Affinity for fH protein and thermostability may be measured as disclosed in WO 2016/014719 A1 (as in example 1 or 3 of this document).

For convenience and clarity, the naturally or naturally occurring amino acid sequence of fHBP B24 (or fHBP ID 1 or v.1fhbp of neisseria meningitidis strain MC 58) having the sequence SEQ ID No. 6 is chosen as a reference sequence for all naturally occurring and non-naturally occurring fHBP amino acid sequences herein, unless otherwise specifically indicated. Thus, when referring to the amino acid residue position in fHBP, the position number used herein corresponds to the amino acid residue number of SEQ ID NO:6 (fHBP B24). Thus, position number 1 refers to the first amino acid residue shown in SEQ ID NO. 6, which is cysteine. This is true even in the case of the N-terminus of SEQ ID NO. 6, where a further amino acid is added before this cysteine.

In one embodiment, mutations (e.g., amino acid substitutions) that may be introduced in the fHBP a or B antigen to be used in the present disclosure may be as disclosed in WO 2011/126863 A1, WO 2015/017817 A1 or WO 2016/014719 A1.

The immunogenic compositions as disclosed herein can comprise non-naturally occurring fHBP that differs in amino acid sequence from wild-type neisseria meningitidis fHBP by 1 to 10 amino acids (e.g., by 1, 2, 3,4, 5, 6, 7, 8, 9, or 10 amino acids), 10 amino acids to 15 amino acids, 15 amino acids to 20 amino acids, 20 amino acids to 30 amino acids, 30 amino acids to 40 amino acids, or 40 amino acids to 50 amino acids.

In some embodiments, fHBP may comprise an amino acid sequence that has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% amino acid sequence identity with the reference fHBP sequence.

Identity (e.g., percent homology) may be determined using various known sequence comparison tools, such as any homology comparison software that computes pair sequence alignments, e.g., by using default parameters, including, for example, blast software from the National Center for Biotechnology Information (NCBI). Identity is global identity, i.e. identity over the whole amino acid or nucleic acid sequence, but not over a part thereof. Paired global alignment is defined by Needleman et al Journal of Molecular Biology,1970, pages 443-53, volume 48). For example, the EMBOSS-6.0.1needleman-Wunsch algorithm (available from http:// EMBOSS. Sourceforge. Net/apps/cvs/EMBOSS/apps/needle. Html) can be used to find the best alignment of two sequences along their entire length, the "global alignment", when starting from a polypeptide sequence and comparing with other polypeptide sequences.

FHBP antigens for use in the immunogenic compositions disclosed herein may be obtained as disclosed in WO 2016/014719 A1. fHBP can be obtained as a recombinant protein from a recombinant expression vector (or construct) transfected into a host cell (e.g., e.coli strain) for production. Suitable vectors for transferring and expressing nucleic acids encoding fHBP may vary in composition. The integrating vector may be a conditionally replicable or suicide plasmid, phage, or the like.

Constructs may include various elements including, for example, promoters, selectable genetic markers (e.g., genes that confer resistance to antibiotics (e.g., kanamycin, erythromycin, chloramphenicol, or gentamicin)), origins of replication (to promote replication in host cells such as bacterial host cells), and the like. The choice of vector will depend on various factors such as the type of cell in which proliferation is desired and the purpose of proliferation. Certain vectors are useful for amplifying and producing large quantities of desired DNA sequences. Other vectors are suitable for expression in cultured cells. The selection of an appropriate carrier is well within the skill of the art. Many such carriers are commercially available.

In one example, the vector may be an episomal plasmid-based expression vector comprising a selectable marker of resistance and elements that provide autonomous replication in different host cells (e.g., in both E.coli and Neisseria meningitidis). An example of such a "shuttle vector" is plasmid pFPIO (Pagotto et al (2000) Gene 244:13-19). The vector may be provided for extrachromosomal maintenance in the host cell or may be provided for integration into the host cell genome. Vectors are well described in numerous publications known to those skilled in the art, including, for example, short Protocols in Molecular Biology, (1999) F.Ausubel, et al, eds., wiley & Sons. The vector may provide for expression of a nucleic acid encoding the subject fHBP, may provide for proliferation of the subject nucleic acid, or both.

Examples of vectors that may be used include, but are not limited to, those derived from recombinant phage DNA, plasmid DNA, or cosmid DNA. For example, plasmid vectors such as those of pBR322, pUC 19/18, pUC 118, 119 and M13 mp series can be used. pET21 is also an expression vector that can be used. Phage vectors may include phage vectors of the lambda gtl0, lambda gtll, lambda gtl8-23, lambda ZAP/R and EMBL series. Additional vectors that may be used include, but are not limited to, vectors of the pJB8, pCV 103, pCV 107, pCV 108, pTM, pMCS, pNNL, pHSG274, COS202, COS203, pWE15, pWE16 and Carborundum 9 series.

The recombinant expression vector may comprise a nucleotide sequence encoding fHBP operably linked to a transcriptional control element, e.g., a promoter. Promoters may be constitutive or inducible. Promoters may be engineered for use in prokaryotic or eukaryotic host cells.

Expression vectors provide transcriptional and translational regulatory sequences and may be provided for inducible or constitutive expression in which a coding region is operably linked under the transcriptional control of a transcription initiation region and a transcription and translation termination region. These control regions may be natural regions of fHBP from which the subject fHBP is derived, or may be derived from exogenous sources. In general, transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ribosome binding sites, transcriptional initiation and termination sequences, translational initiation and termination sequences, and enhancer or activator sequences. Promoters may be constitutive or inducible, and may be strong constitutive promoters (e.g., T7, etc.).

Expression vectors typically have convenient restriction sites located near the promoter sequence to provide for insertion of the nucleic acid sequence encoding the protein of interest. Constructs (recombinant vectors) may be prepared, for example, by inserting the polynucleotide of interest into the backbone of the construct (typically by DNA ligase attachment to a restriction enzyme site cut in the vector). Alternatively, the desired nucleotide sequence may be inserted by homologous recombination or site-specific recombination. In general, homologous recombination can be accomplished by attaching homologous regions to the vector flanking the desired nucleotide sequence, while site-specific recombination can be accomplished by using sequences that promote site-specific recombination (e.g., cre-lox, att sites, etc.). Nucleic acids comprising such sequences may be added by, for example, ligation of oligonucleotides or by polymerase chain reaction using primers comprising a homology region and a portion of the desired nucleotide sequence.

Furthermore, the expression construct may comprise further elements. For example, the expression vector may have one or two replication systems, thereby enabling it to be maintained in an organism, e.g., expressed in mammalian or insect cells, and cloned and amplified in a prokaryotic host. Furthermore, the expression construct may comprise a selectable marker gene to allow selection of transformed host cells. Selection genes are well known in the art and will vary with the host cell used.

Amino acid substitutions may be introduced into the fHBP nucleotide sequence by any technique known in the art. Amino acid substitutions may be obtained, for example, as disclosed in WO 2011/126863 A1, WO 2015/017817 A1 or WO 2016/014719 A1. In other exemplary embodiments, amino acid substitutions may be obtained as disclosed in WO 2015/128480, WO 2010/046715, WO 2016/008960, WO 2020/030782 or WO 2011/051893.

Recombinant fHBP can be obtained in purified form from the culture by any purification method known in the art, as described, for example, in the examples section.

In one embodiment, fHBP a protein and/or fHBP B may be present in an immunogenic composition as disclosed herein in an amount from about 20 μg/dose to about 200 μg/dose, or from about 25 μg/dose to about 180 μg/dose, or from about 40 μg/dose to about 140 μg/dose, or from about 50 μg/dose to about 120 μg/dose, or from about 75 μg/dose to about 100 μg/dose. In one embodiment, fHBP a protein and/or fHBP B may be present in an amount of about 25 μg/dose, or about 50 μg/dose, or about 100 μg/dose.

fHBP A

In one embodiment, an immunogenic composition as disclosed herein may comprise at least one fHBP a variant antigen. The at least one fHBP a protein may be a lipidated or non-lipidated protein. In one exemplary embodiment, the fHBP a protein may be a non-lipidated protein.

In one embodiment, the fHBP a protein may be naturally occurring or non-naturally occurring fHBP. In one embodiment, the fHBP a protein may be naturally occurring fHBP. In another embodiment, the fHBP a protein may be a non-naturally occurring fHBP.

In one embodiment, the fHBP A protein can be one comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5% or about 100% amino acid sequence identity to SEQ ID NO. 1. The at least one fHBP a protein may be a lipidated or non-lipidated protein and/or may be a naturally or non-naturally occurring fHBP (the non-naturally occurring fHBP a05 protein is not 100% identical to fHBP a05 or SEQ ID NO: 1).

The non-naturally occurring fHBP a protein may be a chimeric protein as disclosed in WO 2011/126863 A1 or WO 2015/017817 A1 or a mutant fHBP a protein as disclosed in WO 2016/014719 A1, WO 2011/051893, WO 2016/008960 or WO 2015/128480. In one exemplary embodiment, the fHBP a protein may be a mutein.

In one exemplary embodiment, the non-naturally occurring fHBP a protein may be a mutein. The non-naturally occurring fHBP a protein may be a non-lipidated protein. In an exemplary embodiment, the fHBP a protein may be non-naturally occurring, such as a mutant, non-lipidated fHBP a protein.

In one embodiment, the non-naturally occurring fHBP a protein may differ from a wild-type neisseria meningitidis fHBP a protein, such as fHBP a05, by 1 to 10 amino acids (e.g., by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids), 10 amino acids to 15 amino acids, 15 amino acids to 20 amino acids, 20 amino acids to 30 amino acids, 30 amino acids to 40 amino acids, or 40 amino acids to 50 amino acids.

In one embodiment, the non-naturally occurring fHBP A protein can be a mutein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, or at least about 99.5% amino acid sequence identity to SEQ ID NO. 1. The non-naturally occurring fHBP A05 protein is not 100% identical to fHBP A05 or SEQ ID NO: 1.

In another embodiment, the numbering of the non-naturally occurring or mutated fHBP a protein based on SEQ ID No. 6 may comprise at least one amino acid substitution selected from at least one of: a) An amino acid substitution of asparagine (N115) at amino acid 115; b) Amino acid substitution of aspartic acid (D121) at amino acid 121; c) Amino acid substitution of serine at amino acid 128 (S128); d) Amino acid substitution of leucine (L130) at amino acid 130; e) Amino acid substitution of valine (V131) at position 131; f) Amino acid substitution of glycine (G133) at position 133; g) Amino acid substitution of lysine (K219) at position 219; and h) amino acid substitution of glycine (G220) at position 220.

In another embodiment, numbering of the non-naturally occurring or mutated fHBP a protein with respect to the fHBP sequence (mature lipoprotein form of fHBP a 19) as identified in WO 2011/051893 as SEQ ID NO:5 may comprise at least one amino acid deletion or substitution at any of the following positions as disclosed in WO 2011/051893: d37, K45, T56, E83, E95, E112, S122, I124, R127, T139, F141, N142, Q143, L197, D210, R212, K218, N43, N116, K119, T220, and/or 240. In one embodiment, the amino acid deletion or substitution is as disclosed in WO 2011/051893.

In another embodiment, numbering of the non-naturally occurring or mutated fHBP a protein with respect to the fHBP sequence (a 124 or variant 3.28 or ID 28) as identified in WO 2016/008960 as SEQ ID NO:17 may comprise at least one amino acid deletion or substitution at any of the following positions as disclosed in WO 2016/008960: s32, L126 and/or E243. One, two or three residues may be deleted. Alternatively, they may be substituted with different amino acids. For example, leu-126 can be substituted with any of the other 19 naturally occurring amino acids. When substituted, the substituted amino acid may be a simple amino acid, such as glycine or alanine, in some embodiments. In other embodiments, the substituted amino acid is a conservative substitution, e.g., it is made within the following four groups: (1) acidic, i.e., aspartic acid, glutamic acid; (2) basic, i.e., lysine, arginine, histidine; (3) Nonpolar, i.e., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar, i.e., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. In other embodiments, the substitutions are non-conservative. In one embodiment, the substitutions at the specified residues are as follows: S32V; L126R; and/or E243A.

In another embodiment, numbering of the non-naturally occurring or mutated fHBP a protein with respect to the fHBP sequence (mature lipoprotein form) as identified in WO 2016/008960 as SEQ ID NO:5 may comprise at least one amino acid deletion or substitution at any of the following positions as disclosed in WO 2016/008960: s32, L123 and/or E240. One, two or three residues may be deleted. Alternatively, they may be substituted with different amino acids. For example, leu-123 can be substituted with any of the other 19 naturally occurring amino acids. When substituted, the substituted amino acid may be a simple amino acid, such as glycine or alanine, in some embodiments. In other cases, the substituted amino acid is a conservative substitution, e.g., it is made within the following four groups: (1) acidic, i.e., aspartic acid, glutamic acid; (2) basic, i.e., lysine, arginine, histidine; (3) Nonpolar, i.e., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar, i.e., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. In other embodiments, the substitutions are non-conservative. In one embodiment, the substitution at a given residue may be as follows: S32V; L123R; and/or E240A.

In another embodiment, numbering of the non-naturally occurring or mutated fHBP a protein with respect to the fHBP sequence (fHBP a19 or v2.16 or ID 16) identified as SEQ ID NO:5 in WO 2015/128480 may comprise at least one amino acid deletion or substitution at any one of the following positions as disclosed in WO 2015/128480: s32, V33, L39, L41, F69, V100, 1113, F122, L123, V124, S125, G126, L127, G128, S151, H239 and/or E240. In one embodiment, the mutated residue may be S32, V100, L123, V124, S125, G126, L127, G128, H239 and/or E240. Mutations at these residues produce proteins with good stability compared to wild-type fHBP a. In one embodiment, the mutated residue may be S32, L123, V124, S125, G126, L127 and/or G128. In one embodiment, the mutated residue may be S32, L123, V124, S125, G126, L127 and/or G128. In another embodiment, residues S32 and/or L123 may be mutated, e.g., S32V and/or L123. In the case where one or more of V100, S125 and/or G126 is mutated, mutation of residues other than the three may be introduced.

The specified residues may be deleted but are preferably substituted with different amino acids. For example, ser-32 can be substituted with any of the other 19 naturally occurring amino acids. When substituted, the substituted amino acid may be a simple amino acid, such as glycine or alanine, in some embodiments. In other embodiments, the substituted amino acid is a conservative substitution, e.g., it is made within the following four groups: (1) acidic, i.e., aspartic acid, glutamic acid; (2) basic, i.e., lysine, arginine, histidine; (3) Nonpolar, i.e., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar, i.e., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. In other embodiments, the substitutions are non-conservative. In some embodiments, the substitution does not use alanine.

For example, substitutions at specified residues may be as follows: S32V; V33C; L39C; L41C; F69C; V100T; I113S; F122C; L123R; V124I; S125G or S125T; G126D; L127I; G128A; S151C; H239R; or E240H.

In another embodiment, numbering of the non-naturally occurring or mutated fHBP a protein with respect to the fHBP sequence (a 124 or variant 3.28 or ID 28) identified as SEQ ID No. 17 as in WO 2015/128480 may comprise at least one amino acid deletion or substitution at any one of the following positions as disclosed in WO 2015/128480: s32, V33, L39, L41, F72, V103, T116, F125, L126, V127, S128, G129, L130, G131, S154, H242 and/or E243. In one embodiment, the mutated residue may be S32, V103, L126, V127, S128, G129, L130, G131, H242 and/or E243. In one embodiment, the mutated residue may be S32, L126, V127, S128, G129, L130 and/or G131. In another embodiment, residues S32, L126, V127, S128, G129, L130 and/or G131 may be mutated, for example residues S32 and/or L126, for example S32V and/or L126R.

The specified residues may be deleted but are preferably substituted with different amino acids. For example, ser-32 can be substituted with any of the other 19 naturally occurring amino acids. When substituted, the substituted amino acid may be a simple amino acid, such as glycine or alanine, in some embodiments. In other embodiments, the substituted amino acid is a conservative substitution, e.g., it is made within the following four groups: (1) acidic, i.e., aspartic acid, glutamic acid; (2) basic, i.e., lysine, arginine, histidine; (3) Nonpolar, i.e., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar, i.e., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. In other embodiments, the substitutions are non-conservative. In some embodiments, the substitution does not use alanine.

For example, substitutions at specified residues may be as follows: S32V; I33C; L39C; L41C; F72C; V103T; T116S; F125C; L126R; V127I; S128G or S128T; G129D; L130I; G131A; S154C; H242R; and E243H.

The amino acid substitution of asparagine (N115) at amino acid 115 may be an N115I substitution (I: isoleucine). Other amino acids having nonpolar, positively charged or aromatic side chains, such as valine, leucine, lysine, arginine, histidine, phenylalanine, tyrosine or tryptophan, may also be substituted at this position. Thus, in some cases, fHBP may comprise an N115V substitution, an N115L substitution, an N115K substitution, an N115R substitution, an N115H substitution, an N115F substitution, an N115Y substitution, or an N115W substitution.

The amino acid substitution of aspartic acid (D121) at amino acid 121 may be a D121G substitution (G: glycine). Other amino acids having nonpolar, positively charged or aromatic side chains, such as leucine, isoleucine, valine, lysine, arginine, histidine, phenylalanine, tyrosine or tryptophan, may also be substituted at this position. Thus, for example, in some cases, a fHBP variant may comprise a D121L substitution, a D121I substitution, a D121V substitution, a D121K substitution, a D121R substitution, a D121H substitution, a D121F substitution, a D121Y substitution, or a D121W substitution.

The amino acid substitution of serine at amino acid 128 (S128) may be an S128T substitution (T: threonine). Other amino acids having polar, charged or aromatic side chains, such as methionine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylalanine, tyrosine or tryptophan, may also be substituted at this position. Thus, for example, in some cases, a fHBP variant may comprise an S128M substitution, an S128N substitution, an S128D substitution, an S128E substitution, an S128K substitution, an S128R substitution, an S128H substitution, an S128F substitution, an S128Y substitution, or an S128W substitution.

FHBP a may comprise an amino acid substitution of leucine (L130) at amino acid 130. The amino acid substitution of leucine (L130) at amino acid 130 may be an L130R substitution (R: arginine).

FHBP a may comprise an amino acid substitution of valine (V131) at amino acid 131. Other amino acids having charged or aromatic side chains, such as glutamic acid, lysine, arginine, histidine, phenylalanine, tyrosine, or tryptophan, may also be substituted at this position. Thus, for example, in some cases fHBP may comprise a V131E substitution, a V131K substitution, a V131R substitution, a V131H substitution, a V131F substitution, a V131Y substitution, or a V131W substitution.

FHBP a may comprise an amino acid substitution of glycine (G133) at amino acid 133. The amino acid substitution of glycine (G133) at amino acid 133 may be a G133D substitution (D: aspartic acid).

FHBP a may comprise an amino acid substitution of lysine (K219) at position 219. Other amino acids having polar, negatively charged or aromatic side chains, such as glutamine, aspartic acid, glutamic acid, phenylalanine, tyrosine or tryptophan, may also be substituted at this position. Thus, for example, in some cases fHBP may comprise a K219Q substitution, a K219D substitution, a K219E substitution, a K219F substitution, a K219Y substitution, or a K219W substitution.

The amino acid substitution of glycine (G220) at amino acid 220 may be a G220S substitution (S: serine). Other amino acids having polar, charged or aromatic side chains, such as asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylalanine, tyrosine or tryptophan, may also be substituted at this position. Thus, for example, in some cases fHBP may comprise a G220N substitution, a G220Q substitution, a G220D substitution, a G220E substitution, a G220K substitution, a G220R substitution, a G220H substitution, a G220F substitution, a G220Y substitution, or a G220W substitution.

In one exemplary embodiment, the amino acid substitution of leucine (L130) at amino acid 130 may be an L130R substitution (R: arginine).

In one exemplary embodiment, the amino acid substitution of glycine (G133) at amino acid 133 may be a G133D substitution (D: aspartic acid).

In one exemplary embodiment, the amino acid substitution of glycine at position 220 (G220) may be a G220S substitution (S: serine).

In another embodiment, the non-naturally occurring or mutated fHBP A protein can comprise at least one amino acid substitution selected from the group consisting of G220S, L R and G133D based on the numbering of SEQ ID NO. 6. In another embodiment, the non-naturally occurring fHBP A protein may comprise at least three amino acid substitutions selected from the group consisting of G220S, L R and G133D based on the numbering of SEQ ID NO. 6. In another embodiment, the fHBP A protein may comprise only three amino acid substitutions G220S, L R and G133D based on the numbering of SEQ ID NO. 6.

In one embodiment, the non-naturally occurring (or mutated) fHBP a protein may be a non-lipidated mutant fHBP a protein comprising at least one amino acid substitution based on the numbering of SEQ ID NO:6 selected from the group consisting of: a) An amino acid substitution of asparagine (N115) at amino acid 115; b) Amino acid substitution of aspartic acid (D121) at amino acid 121; c) Amino acid substitution of serine at amino acid 128 (S128); d) Amino acid substitution of leucine (L130) at amino acid 130; e) Amino acid substitution of valine (V131) at position 131; f) Amino acid substitution of glycine (G133) at position 133; g) Amino acid substitution of lysine (K219) at position 219; and h) amino acid substitution of glycine (G220) at position 220.

In another embodiment, the non-naturally occurring (or mutated) fHBP a protein may be a non-lipidated mutant fHBP a protein comprising at least one of the amino acid substitutions selected from the group consisting of SEQ ID NO: 6: N115I substitution, N115V substitution, N115L substitution, N115K substitution, N115R substitution, N115H substitution, N115F substitution, N115Y substitution, N115W substitution, D121G substitution, D121L substitution, D121I substitution, D121V substitution, D121K substitution, D121R substitution, D121H substitution, D121F substitution, D121Y substitution, D121W substitution, S128T substitution, S128M substitution, S128N substitution, S128D substitution, S128E substitution, S128K substitution, S128R substitution, S128H substitution, S128F substitution S128Y substitution, S128W substitution, L130R substitution, V131E substitution, V131K substitution, V131R substitution, V131H substitution, V131F substitution, V131Y substitution, V131W substitution, G133D substitution, K219Q substitution, K219D substitution, K219E substitution, K219F substitution, K219Y substitution, K219W substitution, G220S substitution, G220N substitution, G220Q substitution, G220D substitution, G220E substitution, G220K substitution, G220R substitution, G220H substitution, G220F substitution, G220Y substitution, or G220W substitution.

In one exemplary embodiment, the non-naturally occurring (or mutated) fHBP a protein may be a non-lipidated mutant fHBP a protein comprising at least one amino acid substitution based on the numbering of SEQ ID NO:6 selected from the group consisting of: G220S, L R and G133D. In another embodiment, the non-naturally occurring or mutated non-lipidated fHBP A protein may comprise at least three amino acid substitutions selected from the group consisting of G220S, L R and G133D based on the numbering of SEQ ID NO. 6. In another exemplary embodiment, the non-lipidated mutant fHBP a protein may comprise only three amino acid substitutions G220S, L R and G133D based on the numbering of SEQ ID No. 6.

In one exemplary embodiment, the fHBP a protein may be a non-lipidated and mutated protein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or at least about 99.5% amino acid sequence identity to SEQ ID No. 1, and comprising at least one of the amino acid substitutions selected from the group consisting of G220S, L R and G133D based on the numbering of SEQ ID No. 6. The numbering of the mutant non-lipidated fHBP a protein based on SEQ ID No. 6 may comprise at least three amino acid substitutions selected from G220S, L R and G133D. In another exemplary embodiment, the non-lipidated mutant fHBP a protein may comprise only three amino acid substitutions G220S, L R and G133D based on the numbering of SEQ ID No. 6.

In one embodiment, the fHBP A protein can comprise at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% amino acid sequence identity to SEQ ID NO. 2.

In another embodiment, the fHBP A protein may comprise or consist of SEQ ID NO. 2.

Another embodiment relates to mRNA encoding a fHBP A protein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or at least about 99.5% amino acid sequence identity to SEQ ID NO. 2. The mRNA may encode a fHBP A protein comprising or consisting of SEQ ID NO. 1 or a fHBP A protein comprising or consisting of SEQ ID NO. 2.

Amino acid substitutions may be introduced into the fHBP a nucleotide sequence by any technique known in the art. For example, amino acid substitutions may be obtained as disclosed in WO 2016/014719 A1.

In one embodiment, the fHBP a protein may be present in an immunogenic composition as disclosed herein in an amount of about 20 μg/dose to about 200 μg/dose, or about 25 μg/dose to about 180 μg/dose, or about 40 μg/dose to about 140 μg/dose, or about 50 μg/dose to about 120 μg/dose, or about 75 μg/dose to about 100 μg/dose. In one embodiment, fHBP a protein and/or fHBP B may be present in an amount of about 25 μg/dose, or about 50 μg/dose, or about 100 μg/dose.

fHBP B

In one embodiment, an immunogenic composition as disclosed herein may comprise at least one fHBP B variant antigen. The at least one fHBP B protein may be a lipidated or non-lipidated protein. In one exemplary embodiment, the fHBP B protein may be a non-lipidated protein.

In one embodiment, the fHBP a and fHBP B proteins may be lipidated. In one embodiment, the fHBP a and fHBP B proteins may be non-lipidated. Alternatively, the fHBP a protein may be lipidated and the fHBP B protein may be non-lipidated. Still alternatively, the fHBP a protein may be non-lipidated and the fHBP B protein may be lipidated.

In one embodiment, the fHBP B protein may be naturally occurring or non-naturally occurring fHBP. In one embodiment, the fHBP B protein may be naturally occurring fHBP. In another embodiment, the fHBP B protein may be a non-naturally occurring fHBP.

In one embodiment, the fHBP a and fHBP B proteins may be naturally occurring fHBP. In one embodiment, the fHBP a and fHBP B proteins may be non-naturally occurring fHBP. Alternatively, the fHBP a protein may be naturally occurring fHBP, while the fHBP B protein may be non-naturally occurring fHBP. Still alternatively, the fHBP a protein may be a non-naturally occurring fHBP, and the fHBP B protein may be a naturally occurring fHBP.

In one embodiment, the fHBP B protein can be one comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5% or about 100% amino acid sequence identity to SEQ ID NO 3. The at least one fHBP B protein may be a lipidated or non-lipidated protein and/or may be a naturally or non-naturally occurring fHBP (the non-naturally occurring fHBP B01 protein is not 100% identical to fHBP B01 or SEQ ID NO: 3).

The non-naturally occurring fHBP B protein may be a chimeric protein as disclosed in WO 2011/126863 A1 or WO 2015/017817 A1 or a mutant fHBP B protein as disclosed in WO 2016/014719 A1, WO 2011/051893 or WO 2020/030782. In one exemplary embodiment, the fHBP B protein may be a mutein.

In one exemplary embodiment, the non-naturally occurring fHBP B protein may be a mutein. The non-naturally occurring fHBP B protein may be a non-lipidated protein. In an exemplary embodiment, the fHBP B protein may be non-naturally occurring, such as a mutant, non-lipidated fHBP B protein.

In one embodiment, the non-naturally occurring fHBP B protein may differ from a wild-type neisseria meningitidis fHBP B protein, such as fHBP B01, by 1 to 10 amino acids (e.g., by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids), 10 amino acids to 15 amino acids, 15 amino acids to 20 amino acids, 20 amino acids to 30 amino acids, 30 amino acids to 40 amino acids, or 40 amino acids to 50 amino acids.

In one embodiment, the non-naturally occurring fHBP B protein can be a mutein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, or at least about 99.5% amino acid sequence identity to SEQ ID NO. 3. The non-naturally occurring fHBP B protein is not 100% identical to fHBP B01 or SEQ ID NO: 3.

In another embodiment, the numbering of the non-naturally occurring or mutated fHBP B protein based on SEQ ID No. 6 may comprise at least one amino acid substitution selected from at least one of: a) Amino acid substitution of glutamine (Q38) at amino acid 38; b) Amino acid substitution of glutamic acid (E92) at amino acid 92; c) Amino acid substitution of arginine (R130) at amino acid 130; d) Amino acid substitution of serine (S223) at amino acid 223; and e) an amino acid substitution of histidine (H248) at amino acid 248.

In another embodiment, numbering of the non-naturally occurring or mutated fHBP B protein with respect to the fHBP sequence (mature lipoprotein form of fHBP B24) as identified in WO 2011/051893 as SEQ ID NO:4 may comprise at least one amino acid deletion or substitution at any of the following positions as disclosed in WO 2011/051893: d37, K45, T56, E83, E95, E112, K122, V124, R127, T139, F141, D142, K143, I198, S211, L213, K219, N43, D116, H119, S221, and K241.

In another embodiment, numbering of the non-naturally occurring or mutated fHBP B protein with respect to the fHBP sequence (mature lipoprotein form of fHBP B09) identified as SEQ ID NO:2 as in WO 2020/030782 may comprise at least one amino acid substitution at any of the following positions as disclosed in WO 2020/030782: e211, S216 or E232.

In another embodiment, numbering of the non-naturally occurring or mutated fHBP B protein with respect to the fHBP sequence (mature lipoprotein form of fHBP B09) as identified as SEQ ID NO:2 in WO 2020/030782 may comprise at least one of the following amino acid substitutions at any of the following positions as disclosed in WO 2020/030782: E211A, S216R or E232A.

In another embodiment, numbering of the non-naturally occurring or mutated fHBP B protein with respect to the fHBP sequence (mature lipoprotein form of fHBP B44) identified as SEQ ID NO:6 as in WO 2020/030782 may comprise at least one amino acid substitution at any one of the following positions as disclosed in WO 2020/030782: e214, S219 or E235.

In another embodiment, numbering of the non-naturally occurring or mutated fHBP B protein with respect to the fHBP sequence (mature lipoprotein form of fHBP B44) identified as SEQ ID NO:2 as in WO 2020/030782 may comprise at least one of the following amino acid substitutions at any of the following positions as disclosed in WO 2020/030782: E214A, S219R or E235A.

In another embodiment, numbering of the non-naturally occurring or mutated fHBP B protein with respect to the fHBP sequence (mature lipoprotein form of fHBP 24) as identified as SEQ ID NO:1 in WO 2010046715 may comprise at least one of the following amino acid substitutions ：103、106、107、108、109、145、147、149、150、154、156、157、180、181、182、183、184、185、191、193、194、195、196、199、262、264、266、267、268、272、274、283、285、286、288、289、302、304、306、311 and 313 at any of the following positions as disclosed in WO 2010046715. In one embodiment, the numbering of the one or more amino acids that may be altered in the factor H binding protein with respect to the fHBP sequence identified as SEQ ID No. 1 in WO 2010046715 may be selected from the group comprising: amino acid numbers 103, 106, 107, 108, 180, 181, 183, 184, 185, 191, 193, 195, 262, 264, 266, 272, 274, 283, 286, 304, and 306.

The amino acid substitution of glutamine (Q38) at amino acid 38 can be a Q38R substitution (R: arginine). Other amino acids having positively charged or aromatic side chains, such as lysine, histidine, phenylalanine, tyrosine or tryptophan, may also be substituted at this position. Thus, in some cases, fHBP may comprise a Q38K substitution, a Q38H substitution, a Q38F substitution, a Q38Y substitution, or a Q38W substitution.

The amino acid substitution of glutamic acid (E92) at amino acid 92 can be an E92K substitution. Other amino acids having positively charged or aromatic side chains, such as arginine, histidine, phenylalanine, tyrosine or tryptophan, may also be substituted at this position. Thus, for example, in some cases, a fHBP variant may comprise an E92R substitution, an E92H substitution, an E92F substitution, an E92Y substitution, or an E92W substitution.

The amino acid substitution of arginine (R130) at amino acid 130 may be an R130G substitution (G: glycine). Other amino acids having negatively charged or aromatic side chains, such as aspartic acid, glutamic acid, phenylalanine, tyrosine or tryptophan, may also be substituted at R130. Thus, for example, in some cases, a fHBP variant may comprise an R130D substitution, an R130E substitution, an R130F substitution, an R130Y substitution, or an R130W substitution.

The amino acid substitution of serine at amino acid 223 (S223) may be an S223R substitution (R: arginine). Other amino acids having positively charged or aromatic side chains, such as lysine, histidine, phenylalanine, tyrosine or tryptophan, may also be substituted at this position. Thus, for example, in some cases, a fHbp variant comprises an S223K substitution, an S223H substitution, an S223F substitution, an S223Y substitution, or an S223W substitution.

In one exemplary embodiment, the amino acid substitution of histidine (H248) at amino acid 248 may be an H248L substitution (L: leucine). Other amino acids having nonpolar, negatively charged or aromatic side chains, such as isoleucine, valine, aspartic acid, glutamic acid, phenylalanine, tyrosine or tryptophan, may also be substituted at H248. Thus, for example, in some cases, fHBP may comprise an H248I substitution, an H248V substitution, an H248D substitution, an H248E substitution, an H248F substitution, an H248Y substitution, or an H248W substitution.

In another embodiment, the non-naturally occurring or mutated fHBP B protein may comprise at least the amino acid substitution H248L. In another embodiment, the non-naturally occurring fHBP B protein may comprise only the amino acid substitution H248L based on the numbering of SEQ ID NO. 6.

In one embodiment, the non-naturally occurring (or mutated) fHBP B protein may be a non-lipidated mutant fHBP B protein comprising at least one amino acid substitution based on the numbering of SEQ ID NO:6 selected from the group consisting of: a) Amino acid substitution of glutamine (Q38) at amino acid 38; b) Amino acid substitution of glutamic acid (E92) at amino acid 92; c) Amino acid substitution of arginine (R130) at amino acid 130; d) Amino acid substitution of serine (S223) at amino acid 223; and e) an amino acid substitution of histidine (H248) at amino acid 248.

In another embodiment, the non-naturally occurring (or mutated) fHBP B protein may be a non-lipidated mutant fHBP B protein comprising at least one amino acid substitution based on the numbering of SEQ ID NO:6 selected from the group consisting of: Q38R substitution, Q38K substitution, Q38H substitution, Q38F substitution, Q38Y substitution, Q38W substitution, E92K substitution, E92R substitution, E92H substitution, E92F substitution, E92Y substitution, E92W substitution, R130G substitution, R130D substitution, R130E substitution, R130F substitution, R130Y substitution, R130W substitution, S223R substitution, S223K substitution, S223H substitution, S223F substitution, S223Y substitution, S223W substitution, H248L substitution, H248I substitution, H248V substitution, H248D substitution, H248E substitution, H248F substitution, H248Y substitution, or H248W substitution.

In another exemplary embodiment, the non-naturally occurring or mutant fHBP B protein may be a non-lipidated mutant fHBP B protein comprising at least the amino acid substitution H248L based on numbering of SEQ ID No. 6. In another exemplary embodiment, the non-lipidated mutant fHBP B protein may comprise only the amino acid substitution H248L based on the numbering of SEQ ID No. 6.

In one exemplary embodiment, the fHBP a protein may be a non-lipidated and mutated protein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID No. 3 and comprising at least the amino acid substitution H248L based on the numbering of SEQ ID No. 6. The non-lipidated mutant fHBP B protein may comprise only the amino acid substitution H248L based on SEQ ID NO. 6 numbering.

In one embodiment, the fHBP B protein can comprise at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% amino acid sequence identity to SEQ ID NO. 4.

In another embodiment, the fHBP B protein may comprise or consist of SEQ ID NO. 4.

Another embodiment relates to mRNA encoding a fHBP B protein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or at least about 99.5% amino acid sequence identity to SEQ ID NO. 4. The mRNA may encode a fHBP B protein comprising or consisting of SEQ ID NO. 3 or a fHBP A protein comprising or consisting of SEQ ID NO. 4.

Amino acid substitutions may be introduced into the fHBP B nucleotide sequence by any technique known in the art. For example, amino acid substitutions may be obtained as disclosed in WO 2016/014719 A1.

In one embodiment, fHBP B protein may be present in an immunogenic composition as disclosed herein in an amount of about 20 μg/dose to about 200 μg/dose, or about 25 μg/dose to about 180 μg/dose, or about 40 μg/dose to about 140 μg/dose, or about 50 μg/dose to about 120 μg/dose, or about 75 μg/dose to about 100 μg/dose. In one embodiment, fHBP a protein and/or fHBP B may be present in an amount of about 25 μg/dose, or about 50 μg/dose, or about 100 μg/dose.

NadA

Neisserial adhesin A (NadA, previously referred to as GNA 1994) is a surface-exposed trimeric protein forming oligomers, which is anchored to the outer membrane via a transmembrane domain and plays a critical role in adhesion and invasion to epithelial cells (Capecchi et al mol. Microbiol.2005;55 (687-98)). Sequences of NadA antigens from many strains have been published and the activity of this protein as neisseria adhesin has been well documented. nadA genes are present in approximately 50% of meningococcal isolates. NadA exhibits growth phase dependent expression with highest levels in the stable growth phase.

The NadA antigen is contained as gene NMB1994 (GenBank accession No. GI: 7227256) in the published genomic sequence of meningococcal serogroup B strain MC 58.

NadA polypeptides used according to the present disclosure may be wild-type polypeptides, or may be modified by amino acid substitutions, insertions or deletions, provided that the polypeptide can elicit an immune response against NadA.

In some embodiments, the NadA protein to be used may be an N-terminal and/or C-terminal truncated NadA or a NadA protein comprising an amino acid deletion or insertion, e.g. as disclosed in references WO 01/64920, WO 01/64922 or WO 03/020756.

In one embodiment, the recombinant NadA protein to be used in the immunogenic composition as disclosed herein may be a NadA1 variant. As shown in the examples, nadA1 was shown to induce a strong hSBA response.

NadA1 can be obtained from the NadA sequence of the MenB MC58 strain.

In one embodiment, the NadA protein may comprise or consist of the sequence SEQ ID No. 7.

In some embodiments, the NadA protein may comprise at least 190 consecutive amino acids from SEQ ID No.7, e.g., 200 or more, 210 or more, 220 or more, 230 or more, 240 or more, 250 or more consecutive amino acids from SEQ ID No.7, e.g., 260 or more, or 270 or more, or 280 or more, or 290 or more, or 300 or more, or 310 or more, or 320 or more, or 330 or more, or 340 or more, or 350 or more, or 360 or more amino acids from SEQ ID No. 7.

In some embodiments, the NadA protein may lack 5 to 10 amino acids, or 10 to 15, or 15 to 20, or 25, or 30 or 35, or 40 or 45, or 50 or 55 amino acids from, for example, the C-terminus and/or the N-terminus of SEQ ID No. 7. When the N-terminal residue is deleted, this deletion should not remove the ability of NadA to adhere to human epithelial cells.

In one embodiment, the NadA protein may lack a signal peptide at the N-terminus. For example, the NadA protein may lack 23 amino acids at the N-terminus of, for example, SEQ ID NO. 7.

In one embodiment, the NadA protein may lack a membrane anchoring peptide at the C-terminus. For example, the NadA protein may lack 55 amino acids at the C-terminus of, for example, SEQ ID NO. 7.

NadA may be used in monomeric or oligomeric form, for example in trimeric form.

For example, the NadA protein may have NO deletion of residues 308-362 of its C-terminal membrane anchor (e.g., strain MC58 (SEQ ID NO: 7)). Expression of NadA without its membrane anchoring domain in e.coli can result in secretion of the protein into the culture supernatant, while removing the 23 amino acid signal peptide (e.g. residues 2 to 24 of SEQ ID NO:7 are deleted leaving a 284 amino acid protein-SEQ ID NO: 5).

In one embodiment, the NadA protein may comprise at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% amino acid sequence identity to SEQ ID No. 5. In another embodiment, the NadA protein may comprise or consist of SEQ ID No. 5.

Another embodiment relates to mRNA encoding an NadA protein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or at least about 99.5% amino acid sequence identity to SEQ ID NO. 5. The mRNA can encode an NadA protein comprising or consisting of SEQ ID NO. 5.

The NadA protein for use in the immunogenic compositions disclosed herein may be obtained according to any recombinant technique known in the art, e.g., generally as disclosed above. NadA proteins can be obtained as recombinant proteins from recombinant expression vectors (or constructs) transfected into host cells (e.g., e.coli strains) for production. Recombinant NadA in purified form can be obtained from the culture by any purification method known in the art, as described for example in the examples section.

In one embodiment, nadA protein may be present in an amount ranging from about 20 μg/dose to about 200 μg/dose, or from about 25 μg/dose to about 180 μg/dose, or from about 40 μg/dose to about 140 μg/dose, or from about 50 μg/dose to about 120 μg/dose, or from about 75 μg/dose to about 100 μg/dose. In one embodiment, the NadA protein may be present in an amount of about 50 μg/dose.

dOMV

The immunogenic compositions as disclosed herein comprise detergent extracted outer membrane vesicles (dOMVs), also known as Outer Membrane Protein Complexes (OMPCs). Typically, detergent-extracted outer membrane vesicles are referred to as dOMVs or OMVs when used as antigens. When used as protein carriers, they are called OMPCs.

OMPC was used as a carrier protein platform for Phosphoribosyl Ribitol (PRP) conjugate vaccine PedvaxHIB (Haemophilus influenzae type b) (Einhorn et al, lancet (London, england) 1986;2 (8502): 299-302; moro et al, the Journal of pediatrics 2015;166 (4): 992-7) and VAXELIS (diphtheria, tetanus, pertussis, poliomyelitis, hepatitis B and Haemophilus influenzae type b vaccine) (Syed, PAEDIATRIC drugs 2017;19 (1): 69-80).

DOMVs are large proteolipid vesicles comprising integral outer membrane proteins found in the outer membrane of bacteria and residual lipo-oligosaccharides (LOS) (Helting, acta Pathol Microbiol Scand C,1981;89 (2): 69-78). Over 300 proteins can be identified in dOMVs. 75% of the total protein content of dOMVs is represented by the 10 most abundant proteins, including the outer membrane proteins porin A (PorA) and porin B (PorB), which represent up to 50% of the total protein.

Neisseria meningitidis porin (Por) is an antigenic determinant of serotyping variants. Two classes of porins, porA and PorB, were identified, as well as antigenically distinct variants in each class caused by sequence variations in the por gene Variable Region (VR) encoding surface exposed loops.

DOMVs suitable for use in the immunogenic compositions disclosed herein can be obtained from various MenB strains. dOMVs can be isolated from detergent extracts of MenB strains. Suitable MenB strains may be wild-type MenB strains or MenB strains engineered to overexpress porins (such as PorA or PorB proteins, and e.g. PorA proteins).

In one embodiment, the dOMVs may be obtained from a MenB strain that expresses PorA proteins.

In one embodiment, the dOMV may be obtained from a MenB strain expressing the PorA VR2 subtype. The PorA VR2 subtype may be PorA VR2 type P1.2, P1.4, P1.7, P1.10 or P1.13 protein.

In one embodiment, the dOMV may be obtained from a MenB strain expressing PorA VR2 type P1.2 protein.

In one embodiment, the dOMVs may be obtained from a MenB strain expressing PorA VR2 subtype and PorB P2.2a. dOMVs can be obtained from MenB strains expressing PorA VR 2P 1.2 and PorB P2.2 a.

In one embodiment, the dOMV may comprise a PorA VR2 subtype and PorB P2.2a. The dOMV may comprise PorA VR 2P 1.2 and PorB P2.2a.

In another embodiment, the dOMV may comprise PorA VR 2P 1.2 and PorB P2.2a and immune LOS L3,7.PorA and PorB may represent about 50% of the dcmv protein.

In some embodiments, the dOMVs may be obtained from a single MenB strain, or from different MenB strains. In the latter case, the MenB strain may express the same PorA protein subtype, or different PorA protein subtypes, or different types of porins, such as PorA and PorB proteins.

The available MenB strains of dOMVs exhibiting the porins sought can be identified, for example, from the PubMLST database (https:// pubmlst. Org /). For example, a suitable MenB strain may be obtained by selecting a MenB strain from an epidemic outbreak in such a database, and then selecting a MenB strain having a gene encoding a porin of interest, such as a PorA VR 2P 1.2 protein, in such a subset. The selected strain or strains may then be evaluated for efficient expression of the porin of interest using techniques known in the art.

As examples of MenB strains suitable for obtaining a dOMV according to the present disclosure, the following strains may be mentioned: NG H36, BZ 232, DK 353, B6116/77, BZ 163, 0085/00, NG P20, 0046/02, M1140123, M12 240069, N5/99, 99M or M07 240677.

In one exemplary embodiment, the MenB strain may be MenB strain 99M expressing a PorA VR 2P 1.2 protein subtype.

In one embodiment, the dOMV may comprise porin A (PorA) VR2 subtype P1.2.

In another embodiment, the dOMV may comprise outer membrane protein porin A (PorA) and/or outer membrane protein porin B (PorB). The PorA may be present in an amount ranging from about 3% to about 15%, or in an amount of about 5% to about 9% or 10%, relative to the total protein present in the dOMV. PorB may be present in an amount ranging from about 30% to about 70%, or about 35% to about 65%, or about 38% to about 58%, relative to the total protein present in the dOMV.

In one embodiment, the dOMVs may be obtained using a detergent extraction process using at least one deoxycholate treatment step.

Suitable methods for obtaining dOMVs may be as disclosed in Helting et al (Acta Pathol Microbiol Scand C.4, 1981; 89 (2): 69-78) or in example 2 of US 4,695,624. For example, the bacterial culture may be centrifuged to obtain a pellet, which is then extracted with a detergent, such as deoxycholate or Sodium Dodecyl Sulfate (SDS), under heat, e.g., from about 50 ℃ to about 60 ℃ or at about 56 ℃ for a period of time ranging from about 10 to about 20 minutes or about 15 minutes. The resulting material may then be centrifuged and the precipitate may be further suspended and purified according to any method known in the art.

In one embodiment, the dOMV may be present in an amount ranging from about 5 μg/dose to about 400 μg/dose, or from about 10 μg/dose to about 300 μg/dose, or from about 25 μg/dose to about 250 μg/dose, or from about 35 μg/dose to about 225 μg/dose, or from about 50 μg/dose to about 200 μg/dose, or from about 75 μg/dose to about 180 μg/dose, or from about 100 μg/dose to about 150 μg/dose, or from about 110 μg/dose to about 125 μg/dose. In one embodiment, the dOMVs may be present in an amount of about 25 μg/dose, or 50 μg/dose, or about 125 μg/dose.

Additional antigens

In one embodiment, an immunogenic composition as disclosed herein may comprise at least one additional antigen in addition to the combination of neisseria meningitidis antigens as disclosed herein.

In exemplary embodiments, the additional antigen may be a saccharide antigen from neisseria meningitidis serogroup A, C, W, Y, and/or X conjugated to a carrier protein. In one embodiment, the additional antigen may be a combination of MenA, menC, menW-135 and a conjugate of a MenY capsular polysaccharide and a carrier protein.

In exemplary embodiments, the additional antigen may be a saccharide antigen from neisseria meningitidis serogroup A, C, W and/or Y conjugated to a carrier protein. In one embodiment, the additional antigen may be a combination of MenA, menC, menW-135 and a conjugate of a MenY capsular polysaccharide and a carrier protein.

The carrier proteins of the different capsular polysaccharides may be different or the same. Carrier proteins may include inactivated bacterial toxins such as diphtheria toxoid, CRM197, tetanus toxoid, pertussis toxoid, escherichia coli LT, escherichia coli ST, and exotoxin a from pseudomonas aeruginosa. Bacterial outer membrane proteins such as porins, transferrin binding proteins, pneumonecropsy (pneumolysis), pneumococcal surface protein a (PspA) or pneumococcal adhesin protein (psa) may also be used. Other proteins, such as ovalbumin, keyhole Limpet Hemocyanin (KLH), bovine Serum Albumin (BSA), or purified protein derivatives of tuberculin (PPD), may also be used as carrier proteins. It may be CRM197 protein, tetanus or diphtheria toxoid. In one embodiment, it is tetanus toxoid.

The conjugate may be a population comprising molecules having a molecular weight in the range of 700kDa to 1400kDa or 800kDa to 1300 kDa.

The amount of individual saccharide antigens in each dose, measured as saccharide mass, may be between 1-50 μg. For example, a total of 40 μg of saccharide may be administered per dose. For example, 10 μg of each polysaccharide and about 55 μg of carrier protein (e.g., tetanus toxoid protein) may be administered.

In one embodiment, the additional antigen may be a combination of MenA, menC, menW-135 and MenY capsular polysaccharides each conjugated to a tetanus toxoid carrier protein, wherein the MenA polysaccharide is conjugated to the tetanus toxoid carrier via an adipic Acid Dihydrazide (ADH) linker, and the MenC, menW-135 and MenY polysaccharides are each conjugated directly to the tetanus toxoid carrier (TT).

In one embodiment, the additional antigen may be a combination of MenA, menC, menW-135 and a conjugate of a MenY capsular polysaccharide and a tetanus toxoid carrier protein. In exemplary embodiments, conjugated saccharide antigens from neisseria meningitidis serogroups A, C, W and/or Y may be as disclosed in WO 2018/045286 A1 or WO 2002/058737 A2.

In one embodiment, the additional antigen is a commercially available MenACYW-TT conjugate vaccineIs a target of the antigen (a).

Adjuvant

In one embodiment, the composition as disclosed herein may further comprise an adjuvant.

In one embodiment, the adjuvant may be an aluminum-based adjuvant (or aluminum salt). The aluminum-based adjuvant may be an aluminum hydroxide adjuvant (e.g., aluminum oxyhydroxide-AlOOH), an aluminum phosphate adjuvant (e.g., aluminum hydroxy phosphate-Al (OH) PO ₄ -or aluminum orthophosphate-AlPO ₄), an aluminum sulfate salt adjuvant, an aluminum hydroxy phosphate adjuvant, an aluminum potassium sulfate adjuvant, a combination of aluminum hydroxy carbonate, aluminum hydroxide, and magnesium hydroxide (commercially available asAlum), or mixtures thereof. In one embodiment, the aluminum-based adjuvant may be an aluminum hydroxide adjuvant (e.g., aluminum oxyhydroxide-AlOOH), an aluminum phosphate adjuvant (e.g., aluminum hydroxy phosphate-Al (OH) PO ₄ -or aluminum orthophosphate-AlPO ₄), or mixtures thereof. In one embodiment, the aluminum-based adjuvant may be an aluminum phosphate adjuvant, such as aluminum hydroxy phosphate (Al (OH) PO ₄) or aluminum orthophosphate- (AlPO ₄), which may take any suitable form, such as gel, crystalline, amorphous, and the like. In one embodiment, the aluminum-based adjuvant may be an amorphous aluminum hydroxy phosphate salt. In one embodiment, the aluminum-based adjuvant may be an aluminum orthophosphate. In another embodiment, the aluminum-based adjuvant may be a crystalline aluminum oxyhydroxide salt (diaspore).

Aluminum hydroxy phosphate or orthophosphate adjuvants can be obtained by precipitating aluminum hydroxy oxide in the presence of phosphate. The reaction conditions and reactant concentrations during the precipitation reaction affect the extent of hydroxyl groups in the phosphate substitution salt.

The aluminum hydroxyphosphate or orthophosphate adjuvant may have a molar ratio of PO/Al of between 0.3 and 0.99, and for example a ratio of between 0.8 and 0.95 (e.g., 0.88+0.05).

Aluminum hydroxy phosphate [ Al (OH) x (PO 4) y ] adjuvants, wherein the sum of the valences of each anion multiplied by its mole fraction of-3, can be distinguished from AlPO ₄ by the presence of a hydroxy group. For example, an IR band at 3146cm ^-1 (e.g., when heated to 200 ℃) indicates the presence of structural hydroxyl groups.

Aluminum oxyhydroxide [ AIO (OH) ] can be distinguished from Al (OH) 3 by IR spectroscopy, in particular by the absorption band at 1070cm ^-1 and the presence of a strong shoulder at 3090-3100cm ^-1.

Mixtures of different aluminium-based adjuvants may also be used.

In one embodiment, the adjuvant may be an aluminum phosphate adjuvant.

In exemplary embodiments, an immunogenic composition as disclosed herein may comprise a single aluminum-based adjuvant, i.e., it may represent more than 90%, more than 99% or even more than 99.9% of the aluminum adjuvant present in the composition. An aluminum-based adjuvant may be present such that the concentration of Al ³⁺ may be about 0.5mg/mL to about 1.5mg/mL of Al ³⁺, or about 0.8 to about 1.2mg/mL of Al ^{3 +}, or may have about 1.00mg/mL of Al ³⁺.

Aluminum-based adjuvants, such as aluminum phosphate adjuvants, may be present in the compositions disclosed herein in an amount ranging from about 100 μg/dose to about 1000 μg/dose, or from about 150 μg/dose to about 900 μg/dose, or from about 200 μg/dose to about 800 μg/dose, or from about 250 μg/dose to about 700 μg/dose, or from about 300 μg/dose to about 600 μg/dose, or from about 350 μg/dose to about 550 μg/dose, or from about 400 μg/dose to about 500 μg/dose, or from about 400 μg/dose to about 800 μg/dose, or about 400 μg/dose, or about 800 μg/dose.

In addition to (or instead of) the aluminum-based adjuvant, the composition may comprise other adjuvants.

Suitable adjuvants may include, but are not limited to: calcium phosphate adjuvants, MF59 (4.3% w/v squalene, 0.5% w/v Tween 80 ^TM, 0.5% w/v Span 85), cpG-containing nucleic acids (where cytosine is unmethylated), QS21, MPL, 3DMPL, extracts from Aquilla, ISCOMS, LT/CT mutants, poly (D, L-lactide-co-glycolide) (PLG) microparticles, quil A, interleukins, and the like. For experimental animals, freund's adjuvant (incomplete Freund's adjuvant; complete Freund's adjuvant), N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as norMDP), N-acetyl muramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (L ' -2' -dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) -ethylamine (CGP 19835A, referred to as MTP-PE) and RIBI (which contained three components extracted from bacteria in a 2% squalene/Tween 80 emulsion, namely monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS)) were used. The effectiveness of an adjuvant can be determined by measuring the amount of antibody directed against the immunogenic antigen or epitope thereof.

Other exemplary adjuvants that enhance the effectiveness of the composition may include, but are not limited to: (1) Oil-in-water emulsion formulations (with or without other specific immunostimulants, such as muramyl peptides (see below) or bacterial cell wall components), for example (a) MF59 (WO 90/14837) comprising 5% squalene, 0.5% Tween 80 and 0.5% Span 85 (optionally comprising MTP-PE), formulated into submicron particles using a microfluidizer, (b) SAF comprising 10% squalane, 0.4% Tween 80, 5% pluronic block polymer L121 and thr-MDP, microfluidization to submicron emulsions or vortexing to produce larger particle size emulsions, and (c) RIBI Adjuvant System (RAS) (RIBI Immunochem, hamilton, mont.) comprising 2% squalene, 0.2% Tween 80, and one or more bacterial cell wall components (such as monophosphoryl lipid a (MPL), trehalose Dimycolate (TDM), and Cell Wall Scaffold (CWS), e.g., mpl+cws (Detox); (2) A saponin adjuvant such as QS21 or Stimulon ^TM (Cambridge Bioscience, wood, ma) or particles produced therefrom such as ISCOMs (immunostimulatory complexes) which may be free of additional detergent, for example WO 00/07621; (3) Complete Freund's Adjuvant (CFA) or incomplete Freund's adjuvant (IF A); (4) Cytokines such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 (W099/44636), etc.), interferons (e.g., gamma interferon), macrophage colony stimulating factor (M-CSF), tumor Necrosis Factor (TNF), etc.; (5) Monophosphoryl lipid a (MPL) or 3-O-deacylated MPL (3 dMPL), such as GB-2220221, EP-a-0689454, optionally in the substantial absence of alum when used with pneumococcal saccharides, such as WO 00/56258; (6) Combinations of 3dMPL with, for example, QS21 and/or oil-in-water emulsions, for example EP-A-0835318, EP-A-0735898, EP-A-0761231; (7) An oligonucleotide comprising a CpG motif (see for example WO 98/52581), for example an oligonucleotide comprising at least one CG dinucleotide in which the cytosine is unmethylated; (8) Polyoxyethylene ethers or esters (see, e.g., WO 99/52549); (9) Polyoxyethylene sorbitan ester surfactants in combination with octoxynol (WO 01/21207) or polyoxyethylene alkyl ether or ester surfactants in combination with at least one additional nonionic surfactant such as octoxynol (WO 01/21152); (10) Saponins and immunostimulatory oligonucleotides (e.g., cpG oligonucleotides) (WO 00/62800); (11) Immunostimulants and metal salt particles, e.g. WO 00/23105; (12) saponins and oil-in-water emulsions, such as WO 99/11241; (13) Saponins (e.g. QS 21) +3dMPL+IM2 (optionally +sterols), e.g. WO 98/57659; (14) Other substances that act as immunostimulants to enhance the efficacy of the composition. Muramyl peptides include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-25 acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetyl muramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (L '-2' -dipalmitoyl-sn-glycerol-3-hydroxyphosphoryloxy) -ethylamine MTP-PE), and the like.

An adjuvant suitable for administration to humans may be an aluminium salt adjuvant (e.g. aluminium phosphate or aluminium hydroxide).

Immunogenic compositions

The immunogenic compositions disclosed herein can comprise a combination of meningococcal antigens comprising at least one factor H binding protein (fHBP) a protein, at least one fHBP B protein, at least one neisseria adhesion a (NadA) protein, and at least one detergent-extracted outer membrane vesicle (dOMV).

The immunogenic compositions disclosed herein may comprise a combination of neisseria meningitidis serogroup B antigens comprising at least one factor H binding protein (fHBP) a protein, at least one fHBP B protein, at least one neisseria adhesion a (NadA) protein, and at least one detergent-extracted outer membrane vesicle (dOMV). The fHBP a protein and/or the fHBP B protein may be non-lipidated.

The fHBP a protein may be a mutein comprising at least about 85% identity to SEQ ID No.1 and/or the fHBP B protein may be a mutein comprising at least about 85% identity to SEQ ID No. 3.

The fHBP a protein may comprise at least one amino acid substitution based on the numbering of SEQ ID No. 6 selected from at least one of: a) An amino acid substitution of asparagine (N115) at amino acid 115; b) Amino acid substitution of aspartic acid (D121) at amino acid 121; c) Amino acid substitution of serine at amino acid 128 (S128); d) Amino acid substitution of leucine (L130) at amino acid 130; e) Amino acid substitution of valine (V131) at position 131; f) Amino acid substitution of glycine (G133) at position 133; g) Amino acid substitution of lysine (K219) at position 219; and h) an amino acid substitution of glycine (G220) at position 220, or comprising or consisting of SEQ ID NO:2, and/or the fHBP B protein may comprise at least one amino acid substitution selected from at least one of the following based on the numbering of SEQ ID NO: 6: a) Amino acid substitution of glutamine (Q38) at amino acid 38; b) Amino acid substitution of glutamic acid (E92) at amino acid 92; c) Amino acid substitution of arginine (R130) at amino acid 130; d) An amino acid substitution of serine at amino acid 223 (S223); and e) an amino acid substitution of histidine at amino acid 248 (H248), or comprising or consisting of SEQ ID NO. 4.

The fHBP a protein and/or the fHBP B may be present in an amount ranging from about 20 μg/dose to about 200 μg/dose, or from about 25 μg/dose to about 180 μg/dose, or from about 40 μg/dose to about 140 μg/dose, or from about 50 μg/dose to about 120 μg/dose, or from about 75 μg/dose to about 100 μg/dose, or from about 25 μg/dose, or from about 50 μg/dose, or about 100 μg/dose.

The NadA protein may be a NadA1 protein or may comprise at least about 85% identity to SEQ ID No. 5 or comprise or consist of SEQ ID No. 5.

NadA protein may be present in an amount ranging from about 20 μg/dose to about 200 μg/dose, or from about 25 μg/dose to about 180 μg/dose, or from about 40 μg/dose to about 140 μg/dose, or from about 50 μg/dose to about 120 μg/dose, or from about 75 μg/dose to about 100 μg/dose, or about 50 μg/dose.

The dOMV may comprise porin A (PorA).

The dOMVs may be present in an amount ranging from about 5 μg/dose to about 400 μg/dose, or from about 10 μg/dose to about 300 μg/dose, or from about 25 μg/dose to about 250 μg/dose, or from about 35 μg/dose to about 225 μg/dose, or from about 50 μg/dose to about 200 μg/dose, or from about 75 μg/dose to about 180 μg/dose, or from about 100 μg/dose to about 150 μg/dose, or from about 110 μg/dose to about 125 μg/dose, or from about 25 μg/dose, or about 50 μg/dose, or about 125 μg/dose.

The composition may comprise an adjuvant, such as an aluminium-based adjuvant, for example an aluminium-based adjuvant selected from the group comprising: aluminum hydroxide adjuvants, aluminum phosphate adjuvants, aluminum sulfate adjuvants, aluminum hydroxy phosphate sulfate adjuvants, aluminum potassium sulfate adjuvants, aluminum hydroxy carbonate, combinations of aluminum hydroxide and magnesium hydroxide, and mixtures thereof, such as aluminum phosphate adjuvants.

The composition may comprise or consist of: about 25 to about 100 μg/dose of a non-lipidated fHBP a protein comprising or consisting of SEQ ID No. 2, about 25 to about 100 μg/dose of a non-lipidated fHBP B protein comprising or consisting of SEQ ID No. 4, about 25 to about 100 μg/dose of NadA protein comprising or consisting of SEQ ID No. 5, about 20 to about 150 μg/dose of dOMV from the MenB strain expressing PorA VR 2P 1.2, about 100 to about 600 μg/dose of aluminum phosphate adjuvant, 50mM acetate buffer and pH 6.0.

The composition may further comprise at least one conjugated capsular saccharide from one or more of neisseria meningitidis serogroups A, C, W, 135 and/or Y.

Vaccines comprising the compositions as described herein are also disclosed.

The compositions or vaccines as disclosed herein can be used to prevent meningococcal infection or can be used to induce an immune response against meningococcal bacteria.

Also disclosed is a composition comprising mRNA encoding a fHBP a protein comprising at least about 85%, at least about 90%, at least 95%, at least about 98%, at least about 99%, at least about 99.5%, or about 100% amino acid sequence identity to SEQ ID NO 2, an mRNA encoding a fHBP B protein comprising at least about 85%, at least about 90%, at least about 98%, at least about 99.5%, or about 100% amino acid sequence identity to SEQ ID NO 4, an mRNA encoding a NadA protein comprising at least about 85%, at least about 90%, at least about 99%, at least about 99.5%, or about 100% amino acid sequence identity to SEQ ID NO 5, and a dOMV from MenB expressing PorA VR 2P 1.2.

Formulation preparation

In another aspect, the present disclosure relates to a vaccine comprising a composition as disclosed herein.

The immunogenic or vaccine compositions as disclosed herein may be formulated as solid, semi-solid, liquid form preparations, such as tablets, capsules, powders, aerosols, solutions, suspensions or emulsions. Typical routes of administration of such compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, intranasal. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intradermal, intrasternal injection or infusion techniques. In some embodiments, vaccine compositions as disclosed herein may be administered by transdermal, subcutaneous, intradermal, or intramuscular routes. The compositions of the present disclosure are formulated based on the mode of delivery, including, for example, compositions formulated for delivery via parenteral delivery (e.g., intramuscular, intradermal, or subcutaneous injection).

The immunogenic compositions as disclosed herein can be administered via any suitable route, such as by mucosal administration (e.g., intranasal or sublingual), parenteral administration (e.g., intramuscular, subcutaneous, transdermal or intradermal route), or oral administration. As will be appreciated by those skilled in the art, the immunogenic composition may be suitably formulated to be compatible with the intended route of administration. In one embodiment, the immunogenic compositions as disclosed herein may be formulated for administration via an intramuscular or intradermal or subcutaneous route. In one embodiment, the immunogenic composition may be formulated for administration via the intramuscular route.

The compositions as disclosed herein are formulated such that the active ingredient contained therein is bioavailable when the composition is administered to a subject.

The actual methods of preparing such dosage forms are known or will be apparent to those skilled in the art; see, for example, remington, THE SCIENCE AND PRACTICE of Pharmacy, 20 th edition (PHILADELPHIA COLLEGE OF PHARMACY AND SCIENCE, 2000).

The immunogenic compositions as disclosed herein can be formulated with any pharmaceutically acceptable excipient. The composition may comprise at least one inert diluent or carrier. An exemplary pharmaceutically acceptable vehicle is physiological saline buffer. Other physiologically acceptable vehicles are known to those skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences (18 th edition), edit A.Gennaro,1990,Mack Publishing Company, oiston, pennsylvania. The immunogenic compositions as described herein may optionally comprise pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, human serum albumin, essential amino acids, non-essential amino acids, L-arginine hydrochloride, sucrose, D-trehalose dehydrate, sorbitol, tris (hydroxymethyl) aminomethane and/or urea. In addition, the vaccine composition may optionally comprise pharmaceutically acceptable additives including, for example, diluents, binders, stabilizers and preservatives.

In one embodiment, the composition may be in liquid form, such as a solution, emulsion, or suspension. The liquid may be for delivery by injection. The composition intended for administration by injection may comprise at least one of the following: surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers and isotonicity agents. The liquid composition as disclosed herein may comprise at least one of the following: sterile diluents such as water for injection, saline solutions such as physiological saline, ringer's solution, isotonic sodium chloride; fixed oils such as synthetic mono-or diglycerides, polyethylene glycols, glycerol, propylene glycol or other solvents useful as solvents or suspending media; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediamine tetraacetic acid; buffers such as acetate, citrate or phosphate; and agents for modulating tonicity, such as sodium chloride or dextrose; as cryoprotectant agents, for example sucrose or trehalose.

In another embodiment, the compositions as disclosed herein may have a pH in the range of about 4.0 to about 9.0.

The immunogenic compositions disclosed herein can have a pH ranging from about 4.5 to about 8.5, from about 4.8 to about 8.2, from about 5.0 to about 8.0, from about 5.2 to about 7.5, from about 5.4 to about 7.0, from about 5.5 to about 6.8, from about 5.7 to about 6.5, from about 5.8 to about 6.2. In one embodiment, the pH of a composition as disclosed herein may be about 6.0. The use of a buffer maintains a stable pH.

In one embodiment, the compositions as disclosed herein may further comprise a buffer. As possible buffers there may be mentioned Tris buffer, acetate buffer, citrate buffer, phosphate buffer, HEPES buffer or histidine buffer.

In exemplary embodiments, the compositions as disclosed herein may comprise sodium acetate buffer. The sodium acetate buffer may be present at a concentration ranging from about 10mM to about 300mM, or ranging from about 10mM to about 250mM, or ranging from about 20mM to about 150mM, or from about 20mM to about 130mM, or from about 30mM to about 120mM, or from about 40mM to about 100mM, or from about 50mM to about 80mM, or from about 50mM to about 60mM, or for example at a concentration of about 50 mM.

The immunogenic composition may be isotonic with respect to mammals such as humans.

The immunogenic composition may also comprise one or several additional salts, such as sodium, calcium or magnesium salts. In one embodiment, the sodium salt may be selected from the group comprising sodium chloride, sodium phosphate. In one embodiment, the sodium salt may be sodium chloride. The calcium salt may be a calcium chloride salt. The magnesium salt may be a magnesium chloride salt. In one embodiment, the sodium salt may be present at a concentration ranging from about 10mM to about 300mM, or about 30mM to about 280mM, or about 50mM to about 250mM, or about 60mM to about 220mM, or about 80mM to about 200mM, or about 100mM to about 180mM, or about 120mM to about 160mM, or may be, for example, at a concentration of about 150 mM. The calcium or magnesium may be present in an amount ranging from about 1mM to about 15mM, or from about 5mM to about 10 mM.

Parenteral formulations may be packaged in ampules, disposable syringes or multiple dose vials made of glass or plastic. The injectable composition is, for example, sterile.

The immunogenic compositions as disclosed herein may be sterilized by conventional sterilization techniques (e.g., with UV or gamma radiation), or may be sterile filtered. The compositions obtained by sterile filtration of the liquid immunogenic compositions as disclosed herein can be packaged and stored in liquid form or lyophilized. The lyophilized composition may be reconstituted with a sterile aqueous carrier prior to administration. The dry composition may comprise stabilizers such as mannitol, sucrose or dodecyl maltoside and mixtures thereof, e.g. lactose/sucrose mixtures, sucrose/mannitol mixtures and the like.

The compositions as disclosed herein are administered in a therapeutically effective amount, which will depend on a variety of factors, including the activity of the particular therapeutic agent used; metabolic stability and duration of action of the therapeutic agent; age, weight, general health, sex and diet of the patient; the mode and time of administration; excretion rate; a pharmaceutical combination; the severity of a particular disorder or condition; and a subject receiving the therapy.

In one embodiment, a composition as disclosed herein may comprise or consist of: a non-lipidated fHBP a protein comprising or consisting of SEQ ID No. 2, a non-lipidated fHBP B protein comprising or consisting of SEQ ID No. 4, a NadA protein comprising or consisting of SEQ ID No. 5, a dhmv from expression of PorA VR 2P 1.2. The composition may comprise an aluminium phosphate adjuvant. The composition may comprise 50mM acetate buffer and pH 6.0

In one embodiment, a composition as disclosed herein may comprise or consist of: non-lipidated fHBP a protein consisting of SEQ ID No. 2, non-lipidated fHBP B protein consisting of SEQ ID No. 4, nadA protein consisting of SEQ ID No. 5, dOMV from expression PorA VR 2P 1.2, aluminium phosphate adjuvant, 50mM acetate buffer and pH 6.0.

In another embodiment, a composition as disclosed herein may comprise or consist of: about 25 to about 100 μg/dose of non-lipidated fHBP a protein consisting of SEQ ID No. 2, about 25 to about 100 μg/dose of non-lipidated fHBP B protein consisting of SEQ ID No. 4, about 25 to about 100 μg/dose of NadA protein consisting of SEQ ID No. 5, about 20 to about 150 μg/dose of dOMV from the MenB strain expressing PorA VR 2P 1.2, about 100 to about 800 μg/dose of aluminium phosphate adjuvant, 50mM acetate buffer and pH 6.0.

In another embodiment, a composition as disclosed herein may comprise or consist of: about 25 μg/dose of non-lipidated fHBP a protein consisting of SEQ ID No. 2, about 25 μg/dose of non-lipidated fHBP B protein consisting of SEQ ID No. 4, about 25 μg/dose of NadA protein consisting of SEQ ID No. 5, about 25 μg/dose of dOMV from MenB strain expressing PorA VR 2P 1.2, about 100 μg/dose of aluminium phosphate adjuvant, 50mM acetate buffer and pH 6.0.

In another embodiment, a composition as disclosed herein may comprise or consist of: about 25 μg/dose of non-lipidated fHBP a protein consisting of SEQ ID No. 2, about 25 μg/dose of non-lipidated fHBP B protein consisting of SEQ ID No. 4, about 50 μg/dose of NadA protein consisting of SEQ ID No. 5, about 50 μg/dose of dOMV from MenB strain expressing PorA VR 2P 1.2, about 400 μg/dose of aluminium phosphate adjuvant, 50mM acetate buffer and pH 6.0.

In another embodiment, a composition as disclosed herein may comprise or consist of: about 50 μg/dose of non-lipidated fHBP A protein consisting of SEQ ID NO. 2, about 50 μg/dose of non-lipidated fHBP B protein consisting of SEQ ID NO. 4, about 50 μg/dose of NadA protein consisting of SEQ ID NO. 5, about 25 μg/dose of dOMV from MenB strain expressing PorA VR 2P 1.2, about 400 μg/dose of aluminium phosphate adjuvant, 50mM acetate buffer and pH 6.0.

In another embodiment, a composition as disclosed herein may comprise or consist of: about 50. Mu.g/dose of non-lipidated fHBP A protein consisting of SEQ ID NO. 2, about 50. Mu.g/dose of non-lipidated fHBP B protein consisting of SEQ ID NO. 4, about 50. Mu.g/dose of NadA protein consisting of SEQ ID NO. 5, about 50. Mu.g/dose of dOMV from MenB strain expressing PorA VR 2P 1.2, about 400. Mu.g/dose of aluminium phosphate adjuvant, 50mM acetate buffer and pH 6.0.

In another embodiment, a composition as disclosed herein may comprise or consist of: about 50 μg/dose of non-lipidated fHBP A protein consisting of SEQ ID NO. 2, about 50 μg/dose of non-lipidated fHBP B protein consisting of SEQ ID NO. 4, about 50 μg/dose of NadA protein consisting of SEQ ID NO. 5, about 125 μg/dose of dOMV from MenB strain expressing PorA VR 2P 1.2, about 400 μg/dose of aluminium phosphate adjuvant, 50mM acetate buffer and pH 6.0.

In another embodiment, a composition as disclosed herein may comprise or consist of: about 50. Mu.g/dose of non-lipidated fHBP A protein consisting of SEQ ID NO. 2, about 50. Mu.g/dose of non-lipidated fHBP B protein consisting of SEQ ID NO. 4, about 50. Mu.g/dose of NadA protein consisting of SEQ ID NO. 5, about 50. Mu.g/dose of dOMV from MenB strain expressing PorA VR 2P 1.2, about 200. Mu.g/dose of aluminium phosphate adjuvant, 50mM acetate buffer and pH 6.0.

In another embodiment, a composition as disclosed herein may comprise or consist of: about 75 μg/dose of non-lipidated fHBP A protein consisting of SEQ ID NO. 2, about 75 μg/dose of non-lipidated fHBP B protein consisting of SEQ ID NO. 4, about 75 μg/dose of NadA protein consisting of SEQ ID NO. 5, about 75 μg/dose of dOMV from MenB strain expressing PorA VR 2P 1.2, about 300 μg/dose of aluminium phosphate adjuvant, 50mM acetate buffer and pH 6.0.

In another embodiment, a composition as disclosed herein may comprise or consist of: about 100. Mu.g/dose of non-lipidated fHBP A protein consisting of SEQ ID NO. 2, about 100. Mu.g/dose of non-lipidated fHBP B protein consisting of SEQ ID NO.4, about 100. Mu.g/dose of NadA protein consisting of SEQ ID NO. 5, about 125. Mu.g/dose of dOMV from MenB strain expressing PorA VR 2P 1.2, about 400. Mu.g/dose of aluminium phosphate adjuvant, 50mM acetate buffer and pH 6.0.

In another embodiment, a composition as disclosed herein may comprise or consist of: about 100. Mu.g/dose of non-lipidated fHBP A protein consisting of SEQ ID NO. 2, about 100. Mu.g/dose of non-lipidated fHBP B protein consisting of SEQ ID NO. 4, about 50. Mu.g/dose of NadA protein consisting of SEQ ID NO. 5, about 50. Mu.g/dose of dOMV from MenB strain expressing PorA VR 2P 1.2, about 400. Mu.g/dose of aluminium phosphate adjuvant, 50mM acetate buffer and pH 6.0.

In another embodiment, a composition as disclosed herein may comprise or consist of: about 100. Mu.g/dose of non-lipidated fHBP A protein consisting of SEQ ID NO. 2, about 100. Mu.g/dose of non-lipidated fHBP B protein consisting of SEQ ID NO. 4, about 50. Mu.g/dose of NadA protein consisting of SEQ ID NO. 5, about 50. Mu.g/dose of dOMV from MenB strain expressing PorA VR 2P 1.2, about 800. Mu.g/dose of aluminium phosphate adjuvant, 50mM acetate buffer and pH 6.0.

In one embodiment, the present disclosure relates to a container comprising a composition as disclosed herein. The container may comprise an immunogenic composition comprising a combination of meningococcal antigens, the combination comprising at least one factor H binding protein (fHBP) a protein, at least one fHBP B protein, at least one neisseria adhesion a (NadA) protein, and at least one detergent-extracted outer membrane vesicle (dOMV).

Additionally, the container may comprise a composition comprising or consisting of: about 25 to 100 μg/dose of non-lipidated fHBP a protein consisting of SEQ ID No. 2, about 25 to 100 μg/dose of non-lipidated fHBP B protein consisting of SEQ ID No.4, about 25 to 100 μg/dose of NadA protein consisting of SEQ ID No. 5, about 20 to 150 μg/dose of dOMV from the MenB strain expressing PorA VR 2P 1.2, about 100 to 800 μg/dose of aluminium phosphate adjuvant, 50mM acetate buffer and pH 6.0.

The container may be a vial. The vials may be multi-dose vials or may be single-dose vials. Suitable vials may be small glass or plastic containers sealed with most suitable stoppers and seals.

Alternatively, the container may be a drug-loaded syringe. The drug-loaded syringe may comprise a syringe barrel storing a liquid composition as disclosed herein. The gasket and plunger are inserted into the syringe barrel. The gasket seals the syringe barrel in a fluid-tight manner to prevent leakage of the liquid drug, and the plunger slides the gasket. Various types of medicated syringes are known in the art, as described for example in US10,625,025 or WO 2013/046855.

In the case where the composition as disclosed herein is to be mixed and injected with another vaccine composition (e.g. as a tetravalent mencwy conjugated composition), both compositions may be packaged in one container as a single vial or drug carrier syringe or in a dual chamber syringe. Dual chamber syringes, also referred to as sequential or bypass syringes, may include a single barrel separated into proximal and distal compartments by a septum. Depression of the syringe plunger causes the two vaccine compositions to mix in the distal compartment. Various types of dual chamber syringes are known in the art, as described for example in US 10,695,505. The dual chamber syringe may also be used where the vaccine composition is formulated in a dry form, such as a lyophilized form, and stored with the liquid vehicle for reconstitution. In this case, the dried vaccine is stored in one chamber, while the liquid for reconstitution and injection is stored in a second chamber.

Method of manufacture

The compositions as disclosed herein may be prepared by methods well known in the pharmaceutical arts.

In one embodiment, the present disclosure relates to a method of preparing an immunogenic composition as disclosed herein or a vaccine as disclosed herein, the method comprising at least the step of mixing meningococcal antigen comprising at least one factor H binding protein (fHBP) a protein, at least one fHBP B protein, at least one neisseria adhesion a (NadA) protein, and at least one detergent-extracted outer membrane vesicle (dcmv), and optionally an aluminium salt.

In one embodiment, the step of mixing may include blending a first mixture of at least one factor H binding protein (fHBP) a protein optionally adsorbed on AlPO ₄ and at least one fHBP B protein optionally adsorbed on AlPO ₄ salt with a second mixture of at least one NadA protein optionally adsorbed on AlPO ₄ and at least one dOMV.

In one embodiment, the step of mixing may include blending a first mixture of at least one factor H binding protein (fHBP) a protein adsorbed on AlPO ₄ and at least one fHBP B protein adsorbed on AlPO ₄ salt with a second mixture of at least one NadA protein and at least one dOMV adsorbed on AlPO ₄.

The antigens fHBP a and B and NadA may be first adsorbed onto an aluminium-based adjuvant such as AlPO ₄ and then mixed together with the dOMV. The mixing may be performed in any order. Alternatively, fHBP a and B adsorbed on an aluminium-based adjuvant may be mixed together in a suitable buffer. NadA and dOMV adsorbed on an aluminium-based adjuvant can be mixed together in a suitable buffer. The two mixtures are then blended to obtain an immunogenic composition as disclosed herein.

Additional antigens, such as conjugated MenACWY polysaccharides, can then be mixed with the compositions as disclosed herein. Additional antigen compositions, such as conjugated MenACWY polysaccharide compositions, can be blended together with the immunogenic compositions as disclosed herein, just prior to administration to a patient, optionally via a dual chamber syringe that mixes the two compositions together prior to administration.

In another embodiment, an immunogenic composition or vaccine as disclosed herein is co-administered with a conjugated mencwy polysaccharide immunogenic composition or vaccine. Conjugated menac wy polysaccharide the immunogenic composition or vaccine that can be co-administered with the immunogenic composition or vaccine as disclosed herein can be MenA, menC, menW-135 and MenY capsular polysaccharides each conjugated to a tetanus toxoid carrier protein, optionally wherein the MenA polysaccharide is conjugated to the tetanus toxoid carrier via an adipic Acid Dihydrazide (ADH) linker, and the MenC, menW-135 and MenY polysaccharides are each directly conjugated to the tetanus toxoid carrier. The co-administered conjugated MenACWY polysaccharide immunogenic composition or vaccine may be as disclosed in WO 2018/045286 A1 or WO 2002/058737 A2. The co-administered conjugated mencwy polysaccharide immunogenic composition or vaccine may be a commercially available MenACYW-TT conjugate vaccine。

In another embodiment, the antigen and aluminum salt used in the preparation methods as disclosed herein may be in a buffer.

The compositions as disclosed herein may be manufactured in liquid or solid, e.g., lyophilized, form.

In one embodiment, the immunogenic composition as disclosed herein may be packaged and stored in a dry form, such as a lyophilized composition, or as pellets obtained by a granulation process as described in WO 2009/109550. In one embodiment, the different components of the composition (e.g., the MenB antigen) and possibly additional antigens may all be present in the same pellet. In another embodiment, the components of the immunogenic composition as disclosed herein may each be in a different pellet, i.e., one component per pellet; or all components or some of them (in which case the other component(s) may each be in a different pellet) may be combined in pairs or triplets (divalent or trivalent formulations) in the same pellet (fHBP a+b and nada+domv, fHBP a+nada and fHBP b+domv, fHBP b+nada and fHBP a+domv, fHBP a+nada+fhbp B and dOMV, or fHBP a+domv+fhbp B and NadA, etc.). In such embodiments, different pellets comprising different components alone or any combination of at least two different components may be mixed prior to administration to a subject. In one embodiment, they may be mixed prior to reconstitution in a liquid carrier. In another embodiment, they may be mixed by addition to a volume of liquid carrier as they are reconstituted in the liquid carrier. In another embodiment, they may be added separately to different volumes of liquid carrier first, and then the different volumes of liquid carrier may be mixed together to obtain the final liquid composition to be administered to the subject.

In one embodiment, the four antigens and AlPO ₄ are in the same container.

In another embodiment, the adjuvant and antigen may be prepared in at least two different compositions. The different compositions may then optionally be blended together just prior to administration to the patient via a dual chamber syringe that mixes the two compositions together prior to administration. In another embodiment, the different compositions may be administered separately, i.e., simultaneously (actually separated by only a few seconds or minutes, e.g., less than 5 minutes) but via at least two different administration sites (e.g., at least two different injection sites). In another embodiment, the different compositions may be administered sequentially, i.e., at least two different points in time, such as at least 5 minutes apart, or at most hours or 1 or 2 days apart. In such embodiments, the different compositions may be administered at the same administration site (e.g., the same injection site) or at different administration sites (e.g., different injection sites). In such embodiments, at least one of the different components of the compositions as disclosed herein may be provided separately as a multicomponent kit.

Multicomponent kit

In one embodiment, the disclosure relates to a multicomponent kit comprising a plurality of containers, wherein each of the containers comprises at least one meningococcal antigen or a combination of at least two meningococcal antigens selected from the group comprising: fHBP a protein, fHBP B protein, nadA protein and detergent-extracted outer membrane vesicles (dOMV).

In one embodiment, at least one of the antigens of the multicomponent kit may be in dry form. In one embodiment, at least one of the antigens may be in the form of lyophilized or dried pellets. In one embodiment, the kit may optionally comprise a container containing a physiologically injectable vehicle.

In one embodiment, the different antigens of the immunogenic compositions as disclosed herein can be prepared and stored in separate containers or vials. They may then be mixed at the time of administration to an individual.

In one embodiment, each of the antigens fHBP a, fHBP B, nadA and dOMV may be stored individually in one container. In another embodiment, the antigens may be combined in pairs or as triplets (bivalent or trivalent formulations). All types of combinations are conceivable: fhbpa+b and nada+domv, fhbpa+nada and fhbpb+domv, fhbpb+nada and fhbpa+domv, fhbpa+nada+dobpb and dOMV, or fhbpa+domv+fhbpb and NadA, etc.

In one embodiment, fHBP a and B antigens may be stored in a first container, nadA antigen may be stored in a second container, and dOMV antigen may be stored in a third container. In another embodiment, fHBP a and B antigens may be stored in a first container and both NadA antigen and dOMV antigen may be stored in a second container.

The antigen may be stored in liquid formulation or in dry form. When formulated in dry form, a refill container may be added to hold the injectable liquid carrier for re-suspending and mixing the different antigens. Suitable injectable liquid carriers may include buffers. Furthermore, it may comprise an adjuvant as indicated above. The adjuvant may be an aluminium-based adjuvant such as AlPO ₄.

Optionally, the kit of parts may comprise at least one additional container or a plurality of additional containers for at least one additional antigen or a plurality of additional antigens. In one embodiment, the additional antigen may be conjugated MenACWY polysaccharide.

In one embodiment, a kit of parts may comprise a first container comprising an immunogenic composition as disclosed herein and a second container comprising conjugated mencwy polysaccharide.

Use and method of treatment

In one embodiment, the present disclosure relates to a composition as disclosed herein for use as a medicament, in particular as a vaccine.

In another aspect, the present disclosure relates to a composition as disclosed herein for use in preventing meningococcal infection, and in one exemplary embodiment, for use in preventing neisseria meningitidis serogroup B infection.

In another aspect, the present disclosure relates to a composition as disclosed herein for inducing an immune response against meningococcal bacteria, and in one exemplary embodiment against neisseria meningitidis serogroup B bacteria.

In another aspect, the present disclosure relates to a composition as disclosed herein for inducing an immune response against neisseria meningitidis serogroup B bacteria from a ST-41/44, ST-32, ST-269, ST-213, ST-35, ST-461, ST-11, and/or ST-461 clone complex

In another aspect, the disclosure relates to a composition as disclosed herein for inducing an immune response against neisseria meningitidis serogroup B bacteria from an ST-11 clone complex.

In another aspect, the present disclosure relates to a method of protecting an individual from meningococcal infection and in one exemplary embodiment from neisseria meningitidis serogroup B infection, the method comprising at least the step of administering to the individual an immunogenic composition as disclosed herein or a vaccine as disclosed herein.

In another aspect, the present disclosure relates to a method for reducing the risk of developing an invasive meningococcal disease caused by a meningococcal infection in an individual, and in one exemplary embodiment by a neisseria meningitidis serogroup B infection, the method comprising at least the step of administering to the individual an immunogenic composition as disclosed herein or a vaccine as disclosed herein.

In another aspect, the present disclosure relates to a method of eliciting an immune response against neisseria meningitidis serogroup B bacteria in an individual, and in one exemplary embodiment, the method comprising at least the step of administering to the individual an immunogenic composition as disclosed herein or a vaccine as disclosed herein.

In another aspect, the present disclosure relates to a method of eliciting an immune response against neisseria meningitidis serogroup B bacteria from a ST-41/44, ST-32, ST-269, ST-213, ST-35, ST-461, ST-11 and/or ST-461 clone complex, the method comprising at least the step of administering to the individual an immunogenic composition as disclosed herein or a vaccine as disclosed herein.

In another aspect, the present disclosure relates to a method of eliciting an immune response against neisseria meningitidis serogroup B bacteria from an ST-11 clone complex, the method comprising at least the step of administering to the individual an immunogenic composition as disclosed herein or a vaccine as disclosed herein.

In one embodiment, an immunogenic composition as disclosed herein can be used to elicit an immune response in an individual against neisseria meningitidis serogroup B. The relevant individual may be a mammal, such as a human, and for example, infants, toddlers, children, adolescents, young adults, and elderly. In one embodiment, the individual may be from 6 weeks or more, 2 months or more, or 10 years or more. As an exemplary embodiment, the individual may be 6 weeks to 55 years old or older, such as 2 months to 55 years old or older, or such as 10 years old to 55 years old or older.

The methods generally involve administering to an individual in need thereof an effective amount of the subject immunogenic composition. The effective amount for therapeutic use will depend on, for example, the antigen composition, the mode of administration, the weight and general health of the patient, and the discretion of the prescribing physician. Depending on the dosage and frequency and route of administration desired and tolerated by the patient, single or multiple doses of the antigen composition may be administered.

The immunogenic compositions as disclosed herein can be administered in a2, 3, 2+1 or 3+1 dose regimen.

In one embodiment, the immunogenic compositions as disclosed herein may be administered in 2 or 3 doses. The follow-up agent may be administered about one month, about two months, about three months, about four months, about five months, about six months, about seven months, about eight months, about nine months, about ten months, about eleven months, about twelve months, about thirteen months, about fourteen months, about fifteen months, about sixteen months, about seventeen months, about eighteen months, about nineteen months, or about twenty months from the previous agent. In one embodiment, the follow-up agent may be administered about one month, about two months, about five months, about six months, about eight months, about ten months, about twelve months, about fourteen months, or about sixteen months from the previous agent. In one embodiment, the follow-up agent may be administered about one month, about two months, about five months, about six months, or about eight months from the previous agent. In one embodiment, the follow-up agent may be administered about 30 days, about 60 days, or about 180 days apart from the previous agent.

In a two dose regimen, the second dose may be administered about one month after the first dose, or about 2 months after the first dose, or 6 months after the first dose. Alternatively, in a two dose regimen, the second dose may be administered about 30 days after the first dose, or about 60 days after the first dose, or about 180 days after the first dose. This two dose regimen may be suitable for adults and/or teenagers.

In a two dose regimen, the second dose may be administered about 2 months after the first dose. Alternatively, in a two dose regimen, the second dose may be administered about 60 days after the first dose. This two dose regimen may be suitable for toddlers.

In a three dose regimen, the second dose may be administered about one month after the first dose, and the third dose may be administered about 6 months after the first dose. Alternatively, in a three dose regimen, the second dose may be administered about 30 days after the first dose, and the third dose may be administered about 180 days after the first dose. This three dose regimen may be suitable for adults and/or teenagers.

In a three dose regimen, the second dose may be administered about two months after the first dose, and the third dose may be administered about 10 months after the first dose. Alternatively, in a three dose regimen, the second dose may be administered about 60 days after the first dose, and the third dose may be administered at about 12 months of age. This three dose regimen may be suitable for infants.

In one embodiment, a third or fourth dose may be administered in addition to the 2 or 3 doses. The follow-up agent may be administered at least one year after the last of 2 or 3 doses, for example 16 months after the last dose. In this regimen, the first two or three doses may be characterized as priming agents, and the subsequent dose (+1) may be characterized as boosting agents.

In one embodiment, infants and toddlers, for example, from 6 weeks or 2 months of age to 2 years of age, may receive a 2+1 or 3+1 regimen. In another embodiment, a child, for example, from 2 years to 10 years of age, may receive a 2 dose regimen. In another embodiment, teenagers and adults, for example, from 10 to 55 years of age, may receive a 2+1 dose regimen.

The immunogenic compositions as disclosed herein may be administered by any suitable route. For example, administration by the intramuscular route may be considered.

The invention is further described in terms of the following clauses and embodiments.

According to embodiment 1, the present invention relates to an immunogenic composition comprising a combination of neisseria meningitidis serogroup B antigens, said combination comprising at least one factor H binding protein (fHBP) a protein, at least one fHBP B protein, at least one neisseria adhesion a (NadA) protein and at least one detergent-extracted outer membrane vesicle (dOMV).

According to embodiment 2, the present invention relates to a composition according to embodiment 1, wherein said fHBP a protein and/or said fHBP B protein is non-lipidated.

According to embodiment 3, the present invention relates to a composition according to embodiment 1 or 2, wherein said fHBP a protein is a mutein comprising at least about 85% identity to SEQ ID No.1 and/or wherein said fHBP B protein is a mutein comprising at least about 85% identity to SEQ ID No. 3.

According to embodiment 4, the present invention relates to a composition according to any one of embodiments 1 to 3, wherein the fHBP a protein comprises at least one amino acid substitution based on the numbering of SEQ ID NO:6 selected from at least one of: a) An amino acid substitution of asparagine (N115) at amino acid 115; b) Amino acid substitution of aspartic acid (D121) at amino acid 121; c) Amino acid substitution of serine at amino acid 128 (S128); d) Amino acid substitution of leucine (L130) at amino acid 130; e) Amino acid substitution of valine (V131) at position 131; f) Amino acid substitution of glycine (G133) at position 133; g) Amino acid substitution of lysine (K219) at position 219; and h) an amino acid substitution of glycine (G220) at position 220, or comprising or consisting of SEQ ID NO:2, and/or wherein the fHBP B protein comprises at least one amino acid substitution selected from at least one of the following based on the numbering of SEQ ID NO: 6: a) Amino acid substitution of glutamine (Q38) at amino acid 38; b) Amino acid substitution of glutamic acid (E92) at amino acid 92; c) Amino acid substitution of arginine (R130) at amino acid 130; d) An amino acid substitution of serine at amino acid 223 (S223); and e) an amino acid substitution of histidine at amino acid 248 (H248), or comprising or consisting of SEQ ID NO. 4.

According to embodiment 5, the present invention relates to a composition according to any one of embodiments 1 to 4, wherein said fHBP a protein and/or said fHBP B is present in an amount ranging from about 20 μg/dose to about 200 μg/dose, or from about 25 μg/dose to about 180 μg/dose, or from about 40 μg/dose to about 140 μg/dose, or from about 50 μg/dose to about 120 μg/dose, or from about 75 μg/dose to about 100 μg/dose, or about 25 μg/dose, or about 50 μg/dose, or about 100 μg/dose.

According to embodiment 6, the invention relates to a composition according to any one of embodiments 1 to 5, wherein the NadA protein is a NadA1 protein, or comprises at least about 85% identity to SEQ ID No. 5, or comprises or consists of SEQ ID No. 5.

According to embodiment 7, the invention relates to a composition according to any one of embodiments 1 to 6, wherein the NadA protein is present in an amount ranging from about 20 μg/dose to about 200 μg/dose, or from about 25 μg/dose to about 180 μg/dose, or from about 40 μg/dose to about 140 μg/dose, or from about 50 μg/dose to about 120 μg/dose, or from about 75 μg/dose to about 100 μg/dose, or about 50 μg/dose.

According to embodiment 8, the present invention relates to a composition according to any one of embodiments 1 to 7, wherein the dOMV comprises porin A (PorA).

According to embodiment 9, the present invention relates to a composition according to any one of embodiments 1 to 8, wherein the dOMV is present in an amount ranging from about 5 μg/dose to about 400 μg/dose, or from about 10 μg/dose to about 300 μg/dose, or from about 25 μg/dose to about 250 μg/dose, or from about 35 μg/dose to about 225 μg/dose, or from about 50 μg/dose to about 200 μg/dose, or from about 75 μg/dose to about 180 μg/dose, or from about 100 μg/dose to about 150 μg/dose, or from about 110 μg/dose to about 125 μg/dose, or about 25 μg/dose, or about 50 μg/dose, or about 125 μg/dose.

According to embodiment 10, the present invention relates to a composition according to any one of embodiments 1 to 9, further comprising an adjuvant, optionally an aluminium-based adjuvant, optionally selected from aluminium hydroxide adjuvants, aluminium phosphate adjuvants, aluminium sulphate adjuvants, aluminium hydroxy phosphate sulphate adjuvants, aluminium potassium sulphate adjuvants, combinations of aluminium hydroxy carbonate, aluminium hydroxide and magnesium hydroxide, and mixtures thereof.

According to embodiment 11, the present invention relates to a composition according to any one of embodiments 1 to 10, comprising or consisting of: 25 to 100 μg/dose of non-lipidated fHBP a protein consisting of SEQ ID No. 2, 25 to 100 μg/dose of non-lipidated fHBP B protein consisting of SEQ ID No.4, 25 to 100 μg/dose of NadA protein consisting of SEQ ID No. 5, 20 to 150 μg/dose of dOMV from the MenB strain expressing PorA VR 2P 1.2, 100 to 600 μg/dose of aluminium phosphate adjuvant, 50mM acetate buffer and pH 6.0.

According to embodiment 12, the present invention relates to a composition according to any one of embodiments 1 to 11, further comprising at least one conjugated capsular saccharide from one or more of neisseria meningitidis serogroups A, C, W and/or Y.

According to embodiment 13, the present invention relates to a vaccine comprising a composition according to any one of embodiments 1 to 13.

According to embodiment 14, the present invention relates to a composition according to any one of embodiments 1 to 12 or a vaccine according to embodiment 12 for preventing meningococcal infection or for inducing an immune response against meningococcal bacteria.

According to embodiment 15, the invention relates to a composition comprising mRNA encoding a fHBP a protein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, or about 100% amino acid sequence identity to SEQ ID NO 2, mRNA encoding a fHBP B protein comprising at least about 85%, at least about 90%, at least about 95%, at least about 99%, at least about 98%, at least about 99%, at least about 99.5%, or about 100% amino acid sequence identity to SEQ ID NO 4, and NadA protein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99.5%, or about 100% amino acid sequence identity to SEQ ID NO 5.

According to embodiment 16, the invention relates to an immunogenic composition comprising a combination of neisseria meningitidis serogroup B antigens, said combination comprising: at least one non-lipidated factor H binding protein (fHBP) a protein, at least one non-lipidated fHBP B protein, at least one neisseria adhesion protein a (NadA) protein and at least one detergent-extracted outer membrane vesicle (dOMV) containing at least about 85% identity with SEQ ID No. 1 and numbering based on SEQ ID No. 6 comprising at least the amino acid substitution G220S.

According to embodiment 17, the present invention relates to a composition according to embodiment 16, wherein the non-lipidated fHBP B protein is a mutein comprising at least about 85% identity with SEQ ID No. 3.

According to embodiment 18, the present invention relates to a composition according to embodiment 16 or 17, wherein the non-lipidated fHBP B protein comprises the amino acid substitution H248L based on SEQ ID No. 6 numbering.

According to embodiment 19, the present invention relates to a composition according to any one of embodiments 16 to 18, wherein said non-lipidated fHBP a protein comprises or consists of SEQ ID No. 4.

According to embodiment 20, the present invention relates to a composition according to any one of embodiments 16 to 19, wherein said non-lipidated fHBP a protein further comprises the amino acid substitutions L130R and G133D based on the numbering of SEQ ID No. 6.

According to embodiment 21, the present invention relates to a composition according to any one of embodiments 16 to 20, wherein the non-lipidated fHBP a protein comprises or consists of SEQ ID No. 2.

According to embodiment 22, the invention relates to a composition according to any one of embodiments 16 to 21, wherein the NadA protein is a NadA1 protein, or comprises at least about 85% identity to SEQ ID No. 5, or comprises or consists of SEQ ID No. 5.

According to embodiment 23, the present invention relates to a composition according to any one of embodiments 16 to 22, wherein the dOMV comprises a PorA VR2 subtype.

According to embodiment 24, the present invention relates to a composition according to any one of embodiments 16 to 23, wherein the dOMV comprises PorA VR 2P 1.2.

According to embodiment 25, the present invention relates to a composition according to any one of embodiments 16 to 24, further comprising an adjuvant, optionally an aluminium-based adjuvant, optionally selected from aluminium hydroxide adjuvants, aluminium phosphate adjuvants, aluminium sulphate adjuvants, aluminium hydroxy phosphate sulphate adjuvants, aluminium potassium sulphate adjuvants, combinations of aluminium hydroxy carbonate, aluminium hydroxide and magnesium hydroxide, and mixtures thereof.

According to embodiment 26, the present invention relates to a composition according to any one of embodiments 16 to 25, wherein said fHBP a protein and/or said fHBP B is present in an amount ranging from about 20 μg/dose to about 200 μg/dose, or from about 25 μg/dose to about 180 μg/dose, or from about 40 μg/dose to about 140 μg/dose, or from about 50 μg/dose to about 120 μg/dose, or from about 75 μg/dose to about 100 μg/dose, or about 25 μg/dose, or about 50 μg/dose, or about 100 μg/dose, the NadA protein is present in an amount ranging from about 20 μg/dose to about 200 μg/dose, or from about 25 μg/dose to about 180 μg/dose, or from about 40 μg/dose to about 140 μg/dose, or from about 50 μg/dose to about 120 μg/dose, or from about 75 μg/dose to about 100 μg/dose, or from about 50 μg/dose, and the dOMV is present in an amount ranging from about 5 μg/dose to about 400 μg/dose, or from about 10 μg/dose to about 300 μg/dose, or from about 25 μg/dose to about 250 μg/dose, or from about 35 μg/dose to about 225 μg/dose, or from about 50 μg/dose to about 200 μg/dose, or from about 75 μg/dose to about 180 μg/dose, or from about 100 μg/dose to about 150 μg/dose, or from about 110 μg/dose to about 125 μg/dose, or from about 25 μg/dose, or about 50 μg/dose or about 125 μg/dose.

According to embodiment 27, the present invention relates to a composition according to any one of embodiments 16 to 26, comprising or consisting of: 25 to 100 μg/dose of non-lipidated fHBP a protein consisting of SEQ ID No. 2, 25 to 100 μg/dose of non-lipidated fHBP B protein consisting of SEQ ID No. 4, 25 to 100 μg/dose of NadA protein consisting of SEQ ID No. 5, 20 to 150 μg/dose of dOMV from the MenB strain expressing PorA VR 2P 1.2, 100 to 600 μg/dose of aluminium phosphate adjuvant, 50mM acetate buffer and pH 6.0.

According to embodiment 28, the present invention relates to a composition according to any one of embodiments 16 to 27, further comprising at least one conjugated capsular saccharide from one or more of neisseria meningitidis serogroups A, C, W and/or Y.

According to embodiment 29, the present invention relates to a vaccine comprising a composition according to any one of embodiments 16 to 28.

According to embodiment 30, the invention relates to a method of treating a meningococcal infection or inducing an immune response against a meningococcal bacterium, the method comprising administering to a subject in need thereof a composition comprising or consisting of fHBP a protein, fHBP B protein, nadA protein, and dOMV, the fHBP a protein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5% or about 100% amino acid sequence identity to SEQ ID NO 2, the fHBP B protein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99.5% or about 100% amino acid sequence identity to SEQ ID NO 5, the NadA protein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5% or about 100% amino acid sequence identity to SEQ ID NO 2, the fHBP B protein comprising at least about 99.5% or about 100% amino acid sequence identity to SEQ ID NO 2.

It is to be understood that the present disclosure includes all variations, combinations and permutations in which at least one limitation, element, clause description, etc. from at least one of the listed claims is introduced into another claim that is dependent on the same base claim (or any other claim concerned), unless otherwise indicated or unless contradiction or inconsistency would occur to one of ordinary skill in the art. Where elements are presented as a list, for example in a markush group or the like, it is to be understood that each subgroup of the elements is also disclosed, and that one or more of any element may be removed from the group. It should be understood that, in general, where the present disclosure or aspects of the present disclosure are referred to as including particular elements, features, etc., embodiments of the present disclosure or aspects of the present disclosure also include embodiments consisting of or consisting essentially of such elements, features, etc. For the sake of simplicity, these embodiments are not explicitly set forth in each case in many of the words herein. It should also be understood that any embodiment or aspect of the present disclosure may be explicitly excluded from the claims, whether or not a particular exclusion is recited in this specification. Publications and other references cited herein, to describe the background of the disclosure and to provide additional details regarding the practice thereof, are hereby incorporated by reference.

The sequences disclosed in this specification are incorporated by reference. The same sequences are also presented in the sequence listing formatted according to standard requirements for patent transaction purposes. If any sequence difference exists between the sequence and the standard sequence table, the sequence described in the specification is subject to.

Without limiting the disclosure, various embodiments of the disclosure are described below for purposes of illustration.

Examples

Example 1: antigen preparation and immunogenic compositions

Preparation and purification of MenB antigen

1. Non-lipidated mutant fHBP A05 (A05 tmN)

To prepare non-lipidated A05tmN, three point mutations (G220S, L R and G133D, numbered relative to SEQ ID NO: 6) were introduced into the wild-type fHBP A05 sequence, and to obtain a non-lipidated protein, the first cysteine residue anchoring the lipid moiety at the N-terminus was replaced with methionine (non-lipidated A05tmN: SEQ ID NO: 2). The DNA sequence of a05tmN was synthesized and then used to prepare the plasmid construct. Briefly, the DNA sequence of Xba1 and Xho1 sites was added at both ends of the A05tmN sequence. To generate an expression plasmid, the Xba1/Xho 1-containing plasmid was digested and the resulting DNA fragment was ligated into Xba1/Xho 1-digested pET28a (+) and transformed into Top10 competent cells. Positive clones were confirmed by Xba l/Xho l digestion. The A05tmN plasmid was transformed into E.coli and the cell bank was prepared after three rounds of colony purification.

Coli strains transformed with A05tmN were amplified in semi-combinatorial medium at 37℃with stirring (pH 6.8-dissolved oxygen: 20%). Expression of the antigen was induced by addition of isopropyl β -D-1-thiogalactopyranoside (IPTG).

The culture was harvested as raw bulk material and the bacterial biomass was separated from the culture medium by centrifugation. The resulting cell pellet was resuspended in buffer (20 mM Tris-HCl, pH 8.5). The resuspended pellet was treated by a homogenizer to produce a cell homogenate. The homogenate is then centrifuged to collect the pellet. The homogenized pellet was resuspended in buffer (20 mM Tris-HCl, pH 8.5) and pH shock treated (pH 12, with mixing at room temperature for 1 hour). The pH was reduced to 8.5 with 85% phosphoric acid. After centrifugation, the supernatant fraction of the pH impact material was collected and then filtered to obtain a filtered supernatant.

The supernatant was adjusted to pH 8.5 and <5.0mS/cm conductivity and loaded onto a trapping column GigaCap Q-650M. The elution pool was adjusted to 0.9M ammonium sulfate (AmS) and then further purified using an intermediate chromatography, toyopearl Phenyl 600M. After hydrophobic interaction chromatography, the eluted pool was adjusted to pH 8.5 and <8.0mS/cm conductivity and further purified by Nuvia aPrime a chromatography. Followed by final ultrafiltration and diafiltration using a 5kDa regenerated cellulose Tangential Flow Filtration (TFF) membrane, and 0.2 μm filtration.

A05tmN was adsorbed onto AlPO ₄, defined as 1.00mg Al/mL AlPO 4 in acetate buffer (50 mM sodium acetate, 150mM NaCl,pH 6.0).

2. Non-lipidated mutant fHBP B01 (B01 smN)

To prepare non-lipidated B01smN, a single point mutation was introduced into the wild-type fHBP B01 sequence, and to obtain a non-lipidated protein, the first cysteine residue of the N-terminally anchored lipid moiety was replaced with methionine (non-lipidated B01smN: SEQ ID NO: 4). The DNA sequence of B01smN was synthesized and then used to prepare the plasmid construct. Briefly, the DNA sequence of Xba1 and Xho1 sites was added at both ends of the B01smN sequence. To generate an expression plasmid, the Xba1/Xho 1-containing plasmid was digested and the resulting DNA fragment was ligated into Xba1/Xho 1-digested pET28a (+) and transformed into Top10 competent cells. Positive clones were confirmed by Xba l/Xho l digestion. The B01smN plasmid was transformed into E.coli and the cell bank was prepared after three rounds of colony purification.

Coli strains transformed with B01smN were amplified in a semi-combinatorial medium at 37℃with stirring (pH 6.8-dissolved oxygen: 20%). Expression of the antigen was induced by addition of isopropyl β -D-1-thiogalactopyranoside (IPTG).

The culture was harvested as raw bulk material and the bacterial biomass was separated from the culture medium by centrifugation. The resulting cell pellet was resuspended in buffer (20 mM Tris-HCl, pH 8.5). The resuspended pellet was treated by a homogenizer to produce a cell homogenate. The homogenate is then centrifuged to collect the supernatant fraction. The supernatant fraction was then filtered.

The filtered supernatant was adjusted to pH 8.5 and <5.0mS/cm conductivity and loaded onto a chromatograph CaptoQ ImpRes and purified in binding and elution mode. The CaptoQ ImpRes elution pool was then adjusted to 1.8M AmS for loading onto the second chromatographic phenyl Sepharose HP. After elution, the material was concentrated and diafiltered into acetate buffer (50 mM sodium acetate, 150mM NaCl,pH 6.0) using a 5kDa Ultracel TFF membrane, followed by 0.2- μm filtration.

B01smN was adsorbed onto AlPO ₄, defined as 1.00mg Al/mL AlPO 4 in acetate buffer (50 mM sodium acetate, 150mM NaCl,pH 6.0).

3.NadA

Truncated forms of NadA were prepared from nada_mc 58. Truncated NadA removes the leader sequence (residues 1 to 23) and the anchor domain (residues 308 to 362) of NadA_MC58 (truncated NadA: SEQ ID NO: 5). In the truncated sequence of nada_mc58, the first amino acid after the leader sequence is alanine, which is replaced with methionine. The DNA sequence of NadA was synthesized and then used to prepare plasmid constructs. DNA sequences of Xba1 and Xho1 sites were added at both ends of the NadA sequence. To generate an expression plasmid, the Xba1/Xho 1-containing plasmid was digested and the resulting DNA fragment was ligated into Xba1/Xho 1-digested pET28a (+) and transformed into Top10 competent cells. Positive clones were confirmed by Xba l/Xho l digestion. The NadA plasmid was transformed into e.coli and the cell bank was prepared after three rounds of colony purification.

Coli strains transformed with NadA1 were amplified in half-combined medium at 37℃with stirring (pH 6.8-dissolved oxygen: 20%). Expression of the antigen was induced by addition of isopropyl β -D-1-thiogalactopyranoside (IPTG).

The culture was harvested as raw bulk material and the bacterial biomass was separated from the culture medium by centrifugation. The resulting cell pellet was resuspended in buffer (20 mM Tris-HCl, pH 8.5). The resuspended pellet was treated by a homogenizer to produce a cell homogenate. The homogenate is then centrifuged to collect the supernatant fraction. The supernatant fraction was then filtered.

The supernatant fraction was loaded onto Capto DEAE column. The Capto DEAE elution fraction was adjusted with powdered AmS until a concentration of 500mM AmS was reached. The conditioned Capto DEAE-eluted fraction was loaded onto a Toyopearl Butyl-650M column. The Toyopearl Butyl-650M elution fraction was loaded onto a CHT Type I40 μm column and the CHT elution fraction was concentrated using a 30kDa regenerated cellulose TFF membrane followed by diafiltration into 50mM sodium acetate, 150mM NaCl (pH 6.0). After TFF, the product was filtered through 0.2- μm to produce NadA antigen.

NadA was adsorbed onto AlPO ₄, defined as 1.00mg Al/mL AlPO 4 in acetate buffer (50 mM sodium acetate, 150mM nacl, ph 6.0).

4.dOMV

DOMVs were purified from wild type Neisseria meningitidis serotype B strain 99M, which was supplied by Woltt's Rehd army institute (WALTER REED ARMY Institute of Research, WRAIR).

Nm B99M was cultured in the presence of 1g/L yeast extract and Hepes 1M in chemically defined medium as described in Fu et al (Biotechnology (N Y), 1995, month 2; 13 (2): 170-4) and US 5,494,808 at 37℃under CO ₂%.

Culture harvest was performed using low-speed centrifugation (55 ℃ for 2 hours) of the heat-treated suspension to recover wet bacterial pellet. Two consecutive detergent-mediated extraction steps (15 min at 56 ℃) were performed with an extraction buffer composed of detergent (sodium deoxycholate) to extract dOMVs from the bacterial outer membrane and deplete lipooligosaccharides (as disclosed Helting et al, acta Pathol Microbiol Scand C.4 month 1981; 89 (2): 69-78). Sodium deoxycholate and EDTA solubilize the bacterial adventitia, and then they reorganize themselves into domvs (vesicles and microparticles). The re-suspension is done by homogenizing the particles suspended in the extraction buffer using an Ultra-Turrax (rotor-stator device). dOMV supernatants were pooled followed by treatment with omnipotent nuclease in the presence of MgCl 2 (37℃for 15 min).

After the dOMV extraction, concentration of dOMV was performed in 300kDa modified polyethersulfone (mPES) using hollow fiber. Several ultracentrifugation steps are used to separate the dOMVs from the "soluble" contents (e.g., nucleic acids, cytoplasmic proteins, extracted lipopolysaccharide, or buffer components). The resulting pellet was then resuspended in extraction buffer using an Ultra-Turrax (rotor-stator apparatus) at minimum speed for a few seconds. After the initial resuspension, the dOMV was completely resuspended in extraction buffer using high pressure homogenization and the accessibility of the detergent to the dOMV surface was increased.

Centrifugation was then performed, followed by final filtration of the supernatant with a 0.45/0.2- μm cellulose acetate filter.

5. Combination of antigens

The MenB antigens in the multicomponent immunogenic composition are purified non-lipidated mutant a05 fHBP (a 05 tmN), non-lipidated mutant fHBP (B01 smN), nadA and dOMV. The a05tmN, B01smN and NadA antigens were adsorbed onto aluminium phosphate (AlPO ₄).

The vehicle consisted of acetate buffer (50 mM sodium acetate, 150mM NaCl,pH 6.0) and AlPO ₄(1.00mg AL³⁺/mL).

To formulate the final immunogenic composition formulation, alPO ₄, B01smN adsorbed on AlPO ₄, A05tmN adsorbed on AlPO ₄, and acetate buffer (50 mM sodium acetate, 150mM NaCl, pH 6.0) were blended together to obtain the antigen of interest and aluminum concentration (250 μg/mL for B01smN, 250 μg/mL for A05tmN, and 1.00mg Al/mL of AlPO ₄). Different NadA-dOMV compositions were formulated by blending dOMV with acetate buffer (50 mM sodium acetate, 150mM NaCl,pH 6.0) and NadA adsorbed on AlPO ₄ to obtain different antigen concentrations for the different formulations (F1 to F5) tested below. The fHBP-only formulation was diluted in a carrier to obtain formulation F6.

2.

Is a MenB bivalent recombinant lipidated fHBP (rLP 2086) composition consisting of 60 μg/mL/subfamily fHBP subfamily A (A05) and B (B01) proteins formulated in 10mM histidine buffer pH 6.0, sodium chloride (NaCl) and 0.5mg/mL aluminum as aluminum phosphate, alPO ₄) and polysorbate 80 (PS 80) in sterile liquid suspension.

3.

The method comprises the following steps: three recombinant proteins, rp287-953 (NHBA chimera) at a dose of 50 μg/0.5mL, rp936-741 (fHBP chimera with non-lipidated B24 at a dose of 50 μg/0.5 mL) and rp961c (NadA) at a dose of 50 μg/0.5 mL; 25 μg/0.5mL dose of dOMV and 1.5mg/0.5mL dose of AlOOH (e.g., 0.5mg of Al) in a buffer containing 0.776mg/0.5mL dose of histidine, 3.125 μg/0.5mL dose of sodium chloride, 10mg/0.5mL dose of sucrose and water.

4.

Is a commercially available vaccine comprising an ACWY polysaccharide antigen obtained as disclosed in WO 2018/045286 A1 and conjugated to Tetanus Toxoid (TT). The formulation comprises neisseria meningitidis capsular polysaccharides from serogroups A, C, Y and W135, which are conjugated to tetanus toxoid protein alone. The target active ingredient concentration was 10 μg of each polysaccharide and approximately 55 μg of tetanus toxoid protein per 0.5mL dose. The antigen was formulated in a sterile aqueous solution containing 30mM sodium acetate buffer (1.23 mg/dose) and sodium chloride (0.67%, 3.35 mg/dose).

5. Test compositions

Six formulations of the MenB multicomponent vaccine as disclosed herein (F1-F5, and F1 co-administered with MENQUADFI) were tested in the following study and compared to reference composition BEXSERO, TRUMENBA and comparative bivalent (fHBP a and fHBP B) compositions (F6). The different amounts of the different antigens of the test compositions are summarized in table 1 below. F1, F2, F4-F6 and F1 co-administered with MENQUADFI contained 0.4mg AlPO ₄ per dose, whereas F3 contained 0.8mg AlPO ₄ per dose.

Table 1: formulation of the immunogenic compositions tested

The following volumes of each formulation were used for the administration in example 2:

F1：400μL

F2：400μL

F3：800μL

F4：400μL

F5：400μL

F6：400μL

f1+ MENQUADFI: animals received 400 μ L F1 at D0, D28 and D56. On day 0, they also received 500 μ l MENQUADFI on the contralateral thigh.

TRUMENBA：500μL

BEXSERO：500μL

Example 2: preclinical evaluation of MenB vaccine immunogenicity: rabbit immunogenicity studies

1. Materials and methods

1. Study design

Seven groups of rabbits (new zealand KBL-female-8 weeks old) received three immunizations by the IM route at D0, D28 and D56, each of seven formulations-designated F1 to F6 and f1+ MENQUADFI-in table 1 of example 1 or licensed reference vaccines TRUMENBA and BEXSERO (see example 1). Formulation F1 co-administered with MENQUADFI was also tested. The first injection is at the right thigh, the second injection is at the left thigh, and the last injection is at the right thigh, the composition being administered via the IM route. For the group receiving f1+ MENQUADFI, animals were co-administered MENQUADFI given by IM route at the contralateral thigh at D0.

Clinical signs and body temperature were measured 4, 24 and 48 hours post immunization. Two weeks after D0 and the second immunization (at D42) and at D63, blood samples were collected in tubes containing clot activators and serum separators (BD Vacutainer SST II 8.5.5 mL, reference 366468 a). The tube was centrifuged at 3500rpm for 15min to separate serum from blood cells. Serum was transferred to 4.5mL NUNC tubes and heat inactivated at +56 ℃ for 30min. Serum was stored at-20 ℃ until used in ELISA, purification and bacterial killing assays.

The plateau of the antibody response has been reached after dose 2, so immunogenicity was analyzed after dose 2.

The immunogenicity of the test compositions was measured at D42 by evaluating antigen (Ag) -specific IgG responses measured by ELISA and functional serum bactericidal antibody activity (hSBA) against a panel of seven or eighteen MenB strains. Responders showed at least a 4-fold increase in hSBA between D0 and D42.

The positive threshold for a positive response is defined as follows:

front (D0): hSBA < 8-post (D42): serum is positive if hSBA >8

Front (D0): hSBA is more than or equal to 8-and is (D42): serum is positive if hSBA is increased by 4 times or more compared to D0.

The number of immunized animals with serum capable of inducing specific hSBA was greater than 70% of the responders observed (i.e., 5/7 animals per formulation).

2. Determination of IgG antibody titers by ELISA

Specific IgG responses generated against each antigen were measured from rabbit serum at D0 and D42. ELISA analysis was performed on individual sera collected from each immune group.

Briefly, 96-well microplates were coated with 100. Mu.L of 1. Mu.g/mL specific antigen in a carbonate buffer coating solution per well and kept overnight at +4℃. The coating solution was removed by plate inversion, followed by patting the plate on paper towels. The empty sites were blocked with 150. Mu.L buffer 1 (PBS/Tween 20 0.05%/skim milk 1%) and after incubation at +37℃for a period of 60min, the plates were emptied. Serum was serially diluted (12 times) in buffer 1 in microplates at a volume of 100 μl. Plates were incubated for 90min at +37℃, then washed with buffer 2 (PBS/Tween 20.05%). Then, 100. Mu.L of diluted anti-rabbit IgG was added to each well. After incubation for 90min at +37℃, the plates were washed with buffer 2. The reaction was developed by adding 100. Mu.L of tetramethylbenzidine substrate solution to each well. The reaction was chemically terminated after 30min of treatment with HCl (1N) at room temperature and the absorbance at 450-650nm was measured on a spectrophotometer (Versamax reader p248, molecular Devices). Results were expressed in arbitrary ELISA units/mL by reciprocal of the dilution corresponding to od=1 using CODUNIT procedure of robotic ELISA.

3. Rabbit serum IgG purification for hSBA test

To avoid non-specific bactericidal killing induced by rabbit serum at D0, D42, it is necessary to purify IgG. Purification of rabbit serum was performed using RPROTEIN A GRAVTITRAP ^TM columns (GE HEALTHCARE GE-9852-54) and Ab buffer kit GE HEALTHYCARE reference number 28-9030-59. After the first step of equilibrating the column with binding buffer (sodium phosphate 20mM ph=7), serum (2 mL) adjusted to neutral pH with binding buffer (V/V) was added to the column for IgG binding. The column was washed with binding buffer and eluted with elution buffer (glycine HCl 0.1m pH 2.7) to collect IgG. To maintain IgG activity, a neutralization buffer (Tris-HCl 1m, pH 9.0) was added to the eluted fraction to obtain a final near neutral pH. Each sample was dialyzed using Slide-A-Lyser G2 dialysis cartridge (Thermo Scientific 87730) to convert the sample into PBS buffer. Quantification of IgG concentration was performed by NANODROP.

HSBA test

Measurement of serum bactericidal antibodies using human complement as a source of complement is widely accepted as an alternative marker for protection against meningococcal disease (Borrow et al, vaccine.2005;23 (17-18): 2222-7; narrow et al, vaccine.2006;24 (24): 5093-107; frasch et al, vaccine.2009;27 journal 2: b112-6; et al, J Exp med.1969;129 (6): 1307-26).

SBA assays measure the ability of antibodies to lyse and kill bacteria in the presence of complement. The source of complement is human complement (Pel Freez IgG/IgM depleted lot number 13441). Briefly, serum was heat-inactivated at +56 ℃ for 30min, followed by IgG purification. Purified IgG was then serially diluted twice (9 times) in Dulbecco PBS buffer (dilution buffer) containing ca++ and mg++) plus 0.2% gelatin in 96-well microplates.

Bacteria were pre-incubated in 5% CO ₂ at +37℃onMueller Hinton agar (Petri dish) for 18h to obtain confluent bacterial growth. Thereafter, the bacteria were grown in BHI suspension with an initial OD of about 0.20 at λ600nm and incubated for 2h 30 with shaking (100 rpm) at +37℃. After incubation, the bacteria were diluted 5-fold to obtain 1.4.104CFU/mL. mu.L of working bacterial suspension, 50. Mu.L of pre-diluted serum and 25. Mu.L of diluted human complement (15% final concentration) were placed in 96-well microplates and incubated for 1 hour with shaking (100 rpm) at +37℃. The Zephyr robot application automatically places 40 μl per well on a square plate with Mueller Hinton agar (40 x 40). Agar plates were incubated for 10 hours at +37℃and 5% CO 2. After incubation, the colony count per well was calculated using Cybele software from Microvision corporation.

The bactericidal titer is defined as the serum dilution that results in a 50% decrease in Colony Forming Units (CFU) per mL of bacteria compared to complement control wells. SBA titers were calculated by modeling 4 parameter curves in Soft Max Pro v6.5.1gxp by a user-defined scheme that adapts to the plate plan. If the modeling does not conform to the 4 parameter curve, applying a trend function around K50 and manual readings; the bactericidal titer is defined as the reciprocal of the final serum dilution that resulted in at least a 50% reduction.

The positive threshold for a positive response is defined as follows:

front (D0): hSBA < 8-post (D42): serum is positive if hSBA >8

Front (D0): hSBA is more than or equal to 8-and is (D42): serum is positive if hSBA is increased by 4 times or more compared to D0.

The number of immunized animals with serum capable of inducing specific hSBA was greater than 70% of the responders observed (i.e., 5/7 animals per formulation).

MenB Strain

A panel of 18 wild-type serogroup B neisseria meningitidis isolates were selected, which were isolated from geographically diverse locations on different separation dates (most recently clinical isolates), and represented different MLST clone complexes. The MenB strain was selected to evaluate the immunogenicity of each component of the immunogenic composition and to evaluate coverage breadth. Immunogenicity is measured using the MenB strain with SBA, which is selectively recognized by antibodies against one of the antigens of the composition but not any other antigen of the composition. In addition, selection was performed to ensure that some selected strains represented a well-described sequence diversity of fHBP and distribution between the a and B subfamilies. Coverage breadth was assessed using MenB strains that represent epidemiological characteristics such as Clonal Complex (CC) distribution, geographical origin, antigen popularity and diversity.

The main characteristics of the selected strains are listed in table 2.

Table 2: strains selected for measuring antigen-specific SBA response to vaccine components

18 Strains were selected based on epidemiological characteristics such as clone complex distribution, popularity and supervirulence of clone complexes, geographical origin, antigen popularity and diversity, and the following were characterized for antigen distribution:

11 strains expressed fHBP subfamily A (2 near fHBP A05 antigen-n.degree.1 and 8,9 far from fHBP A05 antigen-n.degree.2, 5, 7, 9, 10, 11, 12, 13, 14) and

7 Strains expressed fHBP subfamily B (1 near fHBP B01 antigen-n°3,6 far away fHBP B01 antigen-n°4, 6, 15, 16, 17, 18).

14 Strains did not express NadA (no gene present or frameshift NadA4/5 lost), and 4 strains expressed homologous NadA1 variants (3 strains) or heterologous NadA2/3 variants (1 strain); and

2 Strains expressing dOMVs, matched PorA VR 2P 1.2, expressing low levels of fHBP A, one not expressing the NadA protein (n.degree.5), and one expressing a heterologous NadA protein (n.degree.13).

2. Results

ELISA results

Total IgG antibodies from pooled serum (grey) were quantified by ELISA at D0 and total IgG antibodies from individual sera collected from rabbits immunized with various formulations (F), TRUMENBA or BEXSERO (blue) at D42.

The results within each group were homogeneous regardless of ELISA reaction (all p values. Gtoreq.0.061). No abnormal animals were detected.

ELISA results showed that all F1-F5 formulations and F1 formulations co-administered with MENQUADFI elicited antibody responses, with Ag specific IgG titers ranging from 4.05 to 4.99Log 10, as plotted for fHBP specific IgG responses in FIG. 1A and FIG. 1B, and for nadA specific IgG responses and dOMV specific IgG responses in FIGS. 2A and 2B.

In addition, as depicted in fig. 1A and 1B and fig. 2A and 2B, injection of each of the four MenB antigens formulated in combination with AlPO ₄ resulted in the production of antibodies with medium to high titers against fHBP a05tmN, fHBP B01smN, nadA proteins and dOMV in 100% of animals, regardless of the formulation used. Formulations prepared with 50 μ g A05tmN and B01smN fHBP Ag (F1 +/-meridafi, F4, F5 and F6) induced fHBP-specific IgG responses with average titers ranging from 4.28log10 to 4.59log10 and 4.05log10 to 4.52log10, respectively. Formulations prepared with 50 μg NadA (F1 to F5) induced NadA specific IgG responses with average titers ranging from 4.42log10 to 4.81log10. Formulations prepared with 50 μg dOMVs (F1 to F3) induced dOMV specific IgG responses with average titers ranging from 4.77Log10 to 4.99Log10. Finally, similar average titers were measured for all doses for either fHBP protein (25. Mu.g for F2 and 100. Mu.g for F3) or dOMV (25. Mu.g for F4 and 125. Mu.g for F5) for the various doses.

TRUMENBA is capable of inducing specific IgG against fHBP a05 tmN, fHBP B01 smN antigens with average titers of 5.16 and 5.15log10 respectively.

BEXSERO was able to induce specific IgG against NadA variant 1 antigen with an average titer of 4.34log10.

2. HSBA results obtained with different compositions F1 to F6 or F1+ MENQUADFI, TRUMENBA and BEXSERO for 7 MenB strains (n.degree.1 to 6 and 18)

In the first set of experiments, different compositions F1 to F6 or f1+ MENQUADFI, TRUMENBA and BEXSERO were determined for 7 MenB strains (n°1 to 6 and 18).

The hSBA results show that the six formulations F1-F5, together with MENQUADFI co-administered F1, were immunogenic in rabbits. The percentage of responders was the percentage of animals in serum with a 4-fold increase in hSBA GMT (a surrogate marker confirming protection) measured after immunization compared to before immunization.

As depicted in fig. 3, regardless of the immunization dose used (25 μ g F2, 50 μ g F1, F4, F5, F6, and 100 μ g F), the mutant non-lipidated fHBP B01smN in combination with the mutant non-lipidated fHBP a tmN +nada+domv was able to induce bactericidal activity against closely related fHBP B44 variant strains in D42 in 100% animals, GMT ranging from 443 to 1147.

TRUMENBA and BEXSERO are capable of inducing bactericidal activity against MenB strains expressing closely related fHBP B44 variants in 100% of animals, GMT 5773 and 646, respectively.

As depicted in fig. 4 and 5, mutant non-lipidated fHBP B01 smN in combination with mutant non-lipidated fHBP a tmN +nada+domv was able to induce bactericidal activity against the first MenB strain expressing the variant fHBP B24 in 43% to 86% of animals (fig. 4) and the second variant fHBPB variant strain tested in 86% to 100% of animals (fig. 5), GMT ranges from 5 to 18 and 28 to 219, respectively.

Notably, for the first heterologous B24 variant strain, only the mutant non-lipidated fHBP B01smN in formulation F3 induced an hSBA response in 86% of animals, GMT was 18.

TRUMENBA are capable of inducing bactericidal activity against two B24 strains in 100% of animals, GMT 44 and 143, respectively. BEXSERO are capable of inducing bactericidal activity against two B24 strains in 100% of animals, GMT 2274 and 2587 respectively.

In addition, no significant dose effect was shown between the 25 to 100 μ g B01 smN fHBP doses (all p values ≡0.056).

Finally, no significant differences were shown with respect to the mutant non-lipidated B01 smN response when MENQUADFI was co-administered with formulation F1 (50 μg for all MenB Ag, all p values ∈0.339).

As depicted in fig. 6 and 7, regardless of which immunizing dose (25 μ g F2, 50 μ g F1, F4, F5, F6, and 100 μ g F3) was used, the mutant non-lipidated fHBP a05tmN in combination with the mutant non-lipidated fHBP b01smN +nada+domv was able to induce bactericidal activity against the closely related fHBP a56 variant-expressing strain (fig. 6) and against the variant fHBP a22 variant-expressing strain (fig. 7) in 100% animals, with a Geometric Mean Titer (GMT) ranging from 226 to 382 and 45 to 108, respectively.

TRUMENBA are capable of inducing bactericidal activity against fHBP a56 and fHBP a22 variant-expressing strains in 100% of animals, GMTs 2058 and 463, respectively. BEXSERO were able to induce bactericidal activity against fHBP a56 variant-expressing strains in 57% of animals and against fHBP a22 variant-expressing strains in 100% of animals, GMTs 7 and 331, respectively.

In addition, no significant dose effect was shown between the 25 to 100 μg doses of mutant non-lipidated a05 tmN fHBP (all p values ≡0.066).

Finally, when MENQUADFI was co-administered with formulation F1, no significant differences were shown with respect to the mutant non-lipidated a05 tmN response against the a56 strain (p-value = 0.281). For hSBA against fHBP a22 expressing strain, the titers obtained with f1+ MENQUADFI were significantly lower than those obtained with F1 alone (p=0.020), but a 2.4-fold decrease was not biologically relevant.

As depicted in fig. 8, formulations prepared with 50 μg NadA (F1 to F5) were able to induce bactericidal activity against homologous NadA variant 1 strains in 100% animals, GMT ranging from 217 to 696.

TRUMENBA and BEXSERO were able to induce bactericidal activity against homologous NadA variant 1 strains in 86% and 100% of animals, GMT was 118 and 1191, respectively.

Finally, when MENQUADFI was co-administered with the multicomponent MenB vaccine, no significant differences were shown with respect to NadA response (p-value=0.688).

As depicted in fig. 9, either of the formulations prepared with the dcmv at 25 μ g F4, 50 μ g F1, F2, F3 and 125 μ g F5, the dcmv was able to induce bactericidal activity against the homologous pora_1.2 strain in 100% of animals, GMT ranging from 350 to 1324.

TRUMENBA and BEXSERO are incapable of inducing bactericidal activity against such dOMV strains.

In addition, no significant dose effect of dOMVs was shown (all p values. Gtoreq.0.637).

No significant effect of NadA and dOMV antigens on fHBP a or B responses was observed (all p values ∈0.208).

The only significant effect of the dose of fHBP antigen combined with NadA/dOMV antigen was on NadA-specific hSBA response between F1 (50 μg) and F3 (100 μg) formulations. As depicted in fig. 8, the titers obtained with the F3 formulation were higher than those obtained with the F1 formulation (p=0.004, 3.2 fold increase). No significant differences were shown between all formulations for porA/dOMV specific hSBA responses.

This study demonstrates the immunogenicity of each of the following four selected MenB antigens combined together formulated with AlPO ₄, mutant non-lipidated fHBP a05 tmL and B01 smL, nadA and dOMV.

All antigens contained in the new vaccine formulation were able to generate Ag-specific bactericidal antibody responses measured by hSBA against Ag-specific MenB strains. In the rabbit model, no significant dose effect was observed for fHBP. Similarly, no significant dose effect was observed for dOMV, and dOMV at the lowest dose (25 μg) provided an effective hSBA response. Of note, formulation F3, a formulation containing 100 μg fHBP, 50 μg NadA, and 50 μg/dose of dOMV, tended to provide the highest hSBA response and induced positive responses against all test strains in more than 71% of animals. Furthermore, no effect of MenQuadfi co-administration on MenB-specific hSBA response was observed. In all formulations tested, fHBP a05tmN vaccine Ag induced hSBA responses in 100% of animals against closely related a56 variant strains and heterologous a22 variant strains, with Geometric Mean Titers (GMT) ranging from 226 to 382 and 45 to 108, respectively. In all formulations tested, fHBP B01smN vaccine Ag induced hSBA responses in 100% of animals against closely related B44 variant strains, GMT ranging from 443 to 1147. In testing the hSBA response against the first heterologous H44/76B24 variant strain, fHBP B01smN in formulation F3 alone induced a positive hSBA response in 86% of animals, GMT was 18, while all other tested formulations induced an hSBA response in only 29% to 57% of animals, GMT ranged from 5 to 10. In contrast, fHBP B01smN induced hSBA in 86% to 100% of animals, GMT ranging from 28 to 219, when the second heterologous B24 strain, strain 03S-0291, was used in all formulations tested.

In all formulations tested, nadA vaccine Ag induced hSBA against NadA1 variant strains in 100% of animals, GMT ranging from 217 to 696.

In all formulations tested, the dOMV induced hSBA against the PorA subtype VR 2P 1.2 strain in 100% of animals, GMT ranging from 350 to 1324.

Overall, the immunogenicity results show that six formulations of the immunogenic compositions disclosed herein are immunogenic in rabbits. All antigens contained in the new vaccine were able to generate Ag-specific bactericidal antibody responses measured by hSBA against Ag-specific MenB strains. No significant dose effect was observed for fHBP in the rabbit model, even formulations prepared with 100 μg fHBP tended to provide the highest hSBA response, and responses to all test strains were also induced in more than 70% of animals. Similarly, no significant dose effect was observed for dOMV, and the lowest dOMV dose (25 μg) was sufficient to induce an effective hSBA response. Notably, no effect of MENQUADFI co-administration on MenB-specific hSBA response was observed.

Notably, no temperature rise and significant findings (such as erythema … …) were observed in the animals throughout the study.

In summary, the immunogenicity results show that the six formulations of the disclosed immunogenic compositions are immunogenic in rabbits. All antigens contained in the MenB multicomponent vaccine were able to generate Ag-specific bactericidal antibody responses as measured by hSBA against Ag-specific MenB strains.

3. HSBA results obtained with compositions F1, F3, TRUMENBA and BEXSERO for 17 MenB strains (n°1 to 17)

In the second set of experiments, the serum obtained after immunization with compositions F1, F3, TRUMENBA and BEXSERO in the previous experiments was determined for 11 additional MenB strains (total of 17 MenB strains including the 6 strains tested above).

The overall results are summarized in table 3 below, representing the efficacy of the different test compositions against the selected MenB strain group.

Table 3: testing the efficacy of the compositions against the MenB Strain group

* : Percentage of respondents

The results showed that strains n°4 and 5 were either poorly covered (< 71% responders) or uncovered (0% responders) by TRUMENBA. While these strains are covered to a greater extent or completely by the F1 and F3 formulations.

Furthermore, the results show that strains n°1, 5, 7, 8, 9 and 11 are poorly covered or uncovered by BEXSERO, whereas they tend to be well or completely covered by F1 and F3 formulations.

Notably, strain n°5 was not covered at all by TRUMENBA or BEXSERO.

Table 3 shows that 16 of the 17 MenB strains and 17 of the 17 MenB strains were covered by the F1 and F3 compositions. In contrast TRUMENBA appeared to cover 15 of the 17 MenB strains, while BEXSERO covered only 11 of the 17 MenB strains.

As shown in FIG. 19, formulation F3 tested was able to induce protection against all the different MenB strains from the different cloning complexes ST-41/44, ST-32, ST-269, ST-213, ST-35, ST-461, ST-11 and ST-461. In contrast, the coverage induced by TRUMENBA and BEXSERO is somewhat notched.

Summarizing the data, it can be noted that the MenB multicomponent immunogenic compositions of the present disclosure provide good strain coverage even for fHBP low expressing strains and distinct strains. The F3 formulation covered the MenB strain of 17/17, respondent >71%, and the F1 formulation covered the MenB strain of 16/17, respondent >71%.

TRUMENBA covers the MenB strain of 15/17. Uncovered strains (0% responders) did not express fHBP a10 and belonged to the ST-11 clone complex. The poorly covered strain (71% responders) was the B24 expressing strain.

BEXSERO covers the MenB strain of 11/17. The 6 strains, poorly covered or uncovered (0-57% responders), expressed fHBP a variants. They can represent up to 29% of circulating MenB strains and belong to a circulating or super virulent clonal complex: ST-213; ST-11; ST-35; and ST-461.

Newly developed vaccine compositions provide increased protective breadth against 18 strains representing current MenB molecular epidemiology; the strains are those from the most prevalent and avirulent Clone Complexes (CC) and the most prevalent antigen variants.

Example 3: simulated PTE assay

1. Materials and methods

1. Test compositions and experimental design

In the first study, commercially available vaccines BEXSERO and TRUMENBA were compared in simulated adult and neonatal Peripheral Tissue Equivalent (PTE) cultures (see below) each using 20 donors. TRUMENBA and BEXSERO vaccine compositions are as disclosed in example 1.

In a second study, simulated PTE cultures (see below) were treated with five vaccine candidate formulations F1, F2, F3, F4 and F5 (formulations as indicated in table 1 of example 1) in a dose range curve (based on 10 fold dilutions of human dose) of 1:100 to 1:1000000. F1 is considered a standard formulation because it contains 50 μg/dose of each component and a standard dose of AlPO4 (0.4 mg/dose); f2 formulation was a combination of low dose fHBP (25 μg/dose) and standard dose NadA and dOMV (50 μg/dose) in 0.4 mg/dose AlPO ₄; f3 formulations were a combination of high dose fHBP (100 μg/dose), standard dose NadA and dOMV (50 μg/dose) and high dose AlPO4 (0.8 mg/dose); f4 formulations combined standard doses of fHBP and NadA (50 μg/dose each), low dose dOMV (25 μg/dose) and standard dose of AlPO4; and the F5 formulation contained a combination of standard doses of fHBP and NadA (50 μg/dose), high dose dOMV (125 μg/dose) and standard doses of AlPO ₄. In this study, a dmenb vaccine BEXSERO based on dwmv was used as a baseline reference control.

The simulated or control composition is a no-treatment control containing only serum-free medium.

2. Adult and neonate simulated PTE assay

The simulated PTE constructs were assembled on a robotic line using the method taught by Ma et al (Immunology, 2010, 130:374-87) (Higbee et al, altern Lab Anim.2009, 9; 37 journal 1:19-27).

Briefly, endothelial cells grew to confluence on a collagen matrix (Advanced Biomatrix, san diego, california). Thereafter, donor PBMCs (adult simulated PTE) or donor umbilical cord blood (neonatal simulated PTE) prepared from frozen stock were applied to the assay wells. After incubation for 90 minutes (adult form) or 3 hours (umbilical cord blood form), non-migrating cells were washed away, after which various treatments (table 1 of example 1) were added in the final incubation step for 48 hours. A mixture of 100ng/mL LPS (from Pseudomonas aeruginosa, catalog number L8643, millipore Sigma, berlington, mass.) and 10 μg/mL R848 (catalog number TLRL-R848, invivoGen, san Diego, calif.) was used as a positive control (assay control) in these assays. The counter-migrating cells were harvested after a 48 hour treatment period and phenotyped for cell viability using flow cytometry, whereas culture supernatants harvested at the same time point were analyzed for cytokines/chemokines by multiplex assays.

In a first series of experiments BEXSERO and TRUMENBA were compared together. In a second series of experiments, the immunogenic compositions of F1, F2, F3, F4 and F5 of the present disclosure were compared to BEXSERO.

Culture supernatants were harvested after a 48 hour treatment period and analyzed for cytokines/chemokines by multiplex assays. Cells harvested at the same time point were phenotyped for cell viability using flow cytometry.

3. Cytokine/chemokine analysis

The mock culture supernatants were analyzed using a Milliplex human 12-way multi-cytokine detection system (Millipore). The kit comprises IL-1 beta, IL-6, MIP-1 beta and TNF alpha. Analyte concentrations were calculated based on the relevant standard curve using Bio-Plex manager software (Luna et al PloS One, volume 13,6e0197478.2018, 6 th month).

For the run acceptance criteria, a lower limit of quantification (LLOQ) and an upper limit of quantification (ULOQ) for each analyte were established based on percent recovery (observed/expected) per point based on a5 parameter logic (5 PL) curve fit for standard values. The percent recovery of 80% -120% is considered acceptable, so values falling within this range define the lower and upper limits of the standard curve. Bead counts of the original data file were reviewed; data points are considered valid when at least 35 beads per region are counted.

4. Flow cytometry

Will bePTE-derived cells were washed with PBS and stained with Live-read Aqua (InvitroGen, calif.) on ice for 20min to assess cell viability. Data analysis was performed using FlowJo software (Tree Star, ashland, oregon). For flow gating, a singlet state is selected, followed by a viable cell.

5. Data analysis and mapping

The data is exported to GRAPHPAD PRISM (GraphPad Software, san Diego, calif., U.S.) for graphics fabrication. Cytokine data was exported into an excel database. An out-of-range high (> OOR) value (a value above the highest point of the curve) is replaced with ULOQ; out-of-range low (< OOR) values are replaced with 1/2LLOQ (lower limit of quantification).

6. Statistical analysis

Statistical analysis was performed using a non-bad efficacy model. Parameters including cell viability, CD86, IL-6, IL-1b, TNF-a and MIP-1b were used as endpoints for this analysis. Comparing different antigen formulationsGeometric Mean (GM) of the vaccine and δ=2/3 was used as a measure of non-inferior efficacy. The analysis described here was done using the SA program PROC TTEST and is represented in a dot plot showing GM and 95% CI ranges, as recommended in the published literature.

2. Results

1. BEXSERO and TRUMENBA in adult and neonatal analog PTE

Culture supernatants from untreated and treated adult and neonatal mock PTE cultures were harvested 48 hours later and analyzed for cytokine/chemokine secretion using Millipore custom multiple arrays. The innate chemokines/cytokines IL-6, TNFa, MIP-1 beta and IL-1 beta are included in this assay because they are critical for innate immune activity and can also drive immune cytotoxicity.

The results obtained (fig. 10) show that TRUMENBA exhibited a stronger pro-inflammatory cytokine profile than BEXSERO in adult simulated PTE. Furthermore TRUMENBA exhibited a stronger pro-inflammatory cytokine profile than BEXSERO in neonatal mock PTE (figure 11).

Statistical analysis as presented in fig. 12 confirmed the observation that TRUMENBA produced a stronger response than BEXSERO in both adult and neonatal platforms for most cytokines. In fact, TRUMENBA is better than BEXSERO as shown by the geometric mean forest map ratio (with 95% confidence interval) in the case of BEXSERO triggered cytokine secretion compared to TRUMENBA in adult and neonatal PTEs.

2. F1, F2, F3, F4, F5 and BEXSERO in adult and neonatal simulated PTEs

Culture supernatants from untreated and treated adult and neonatal mock PTE cultures were harvested 48 hours later and analyzed for cytokine/chemokine secretion using Millipore custom multiple arrays. The innate chemokines/cytokines IL-6, TNFa, MIP-1 beta and IL-1 beta are included in this assay because they are critical for innate immune activity and can also drive immune cytotoxicity.

In an adult simulated PTE system (see fig. 13), at the highest treatment dose (1:100, 1:1000, 1:10000), all test formulations produced about one log higher cytokine secretion than the simulated control (i.e., without antigen). In almost all cases, the formulation follows a path equal to or slightly below BEXSERO.

In fig. 14A, 14B and 14C, statistical analysis showed that F1, F2, F4 and F5 formulations produced fewer inflammatory responses in adult simulated PTEs than BEXSERO for all cytokines reported. In the adult-simulated PTE system, F3 formulations induced similar (TNF-. Alpha.or IL-1β) or less (IL 6 or MIP-1 b) pro-inflammatory cytokine secretion compared to Bexsero.

In addition, in the adult PTE model, all formulations exhibited immune cytotoxicity in a dose-dependent manner and within a range similar to BEXSERO. In fact, as shown in fig. 15, in some of the test conditions, at the lowest treatment doses (1:100000 and 1:1000000 dilutions) cell viability was hardly affected, while at the highest dose (1:100 dilutions) cell viability was reduced by up to about 50%.

Furthermore, in neonatal simulated PTEs, F1, F2, F3, F4, and F5 formulations exhibited pro-inflammatory cytokine profiles consistent with BEXSERO (fig. 16). All formulations produced about 4-fold to one log higher cytokine secretion relative to the simulated conditions. The F1, F2, F3 and F4 formulations induced similar or less secretion of IL-6 and TNF- α compared to BEXSERO. The F5 formulation induced slightly higher pro-inflammatory cytokine secretion compared to BEXSERO.

In fig. 17A and 17B, statistical analysis showed that overall, the inflammatory response generated by F1, F2 and F3 formulations in neonatal simulated PTEs was comparable to BEXSERO. F5 formulations tended to produce a slightly higher inflammatory response in neonatal simulated PTE compared to BEXSERO, while F4 formulations tended to produce a weaker response in neonatal simulated PTE compared to BEXSERO.

Similar to the adult simulated PTE system, all formulations exhibited immune cytotoxicity characteristics similar to BEXSERO. In fact, as shown in figure 18, in some test conditions, cell viability was hardly affected in simulated neonatal PTEs at the lowest treatment dose (1:100000 and 1000000 dilutions), whereas at the highest dose (1:100 dilutions) cell viability was reduced by up to about 50%.

3. Conclusion(s)

In the adult and neonatal forms of simulated PTE, TRUMENBA induced a stronger pro-inflammatory cytokine response than BEXSERO.

In contrast to BEXSERO, the F1, F2, F3, F4, and F5 formulations produced similar or less proinflammatory cytokine secretion in adult-simulated PTE constructs.

Overall, the F5 formulation induced more pro-inflammatory cytokines in neonatal mock PTE than BEXSERO, while the F1, F2, F3 and F4 formulations induced similar or lower cytokine responses.

F1, F2, F3, F4 and F5 formulations had little effect on cell viability (except that at 1:100 dilutions, some formulations induced a decrease in cell viability of about 40%) and the trend was similar to BEXSERO.

Example 4: general conclusion

From the results presented above, it can be concluded that the disclosed immunogenic composition comprising a combination of meningococcal antigens comprising at least one factor H binding protein (fHBP) a protein, at least one fHBP B protein, at least one neisseria adhesion a (NadA) protein and at least one detergent-extracted outer membrane vesicle (dcmv) is capable of inducing an immunogenic protective response, as demonstrated by hSBA results. This confirms the utility of these compositions as vaccines and to induce an immunogenic protective response against meningococcal infection.

In addition, the data show that the epidemic and virulent MenB strain covered a greater breadth than TRUMENBA and BEXSERO and that it can fill some of the gaps left by both vaccines.

Finally, the data show that these compositions exhibit good safety, exhibit reactivity characteristics similar to BEXSERO or lower, while BEXSERO has a reactivity lower than TRUMENBA.

In summary, the present disclosure relates specifically to the following:

1. An immunogenic composition comprising a combination of neisseria meningitidis serogroup B antigens, the combination comprising at least one factor H binding protein (fHBP) a protein, at least one fHBP B protein, at least one neisseria adhesion a (NadA) protein, and at least one detergent-extracted outer membrane vesicle (dOMV).

2. The composition of item 1, wherein the fHBP a protein and/or the fHBP B protein is non-lipidated.

3. The composition of item 1 or 2, wherein the fHBP a protein is a mutein comprising at least about 85% identity to SEQ ID No. 1, and/or wherein the fHBP B protein is a mutein comprising at least about 85% identity to SEQ ID No. 3.

4. The composition according to any one of items 1 to 3, wherein the fHBP a protein comprises at least one amino acid substitution based on the numbering of SEQ ID NO:6 selected from at least one of: a) An amino acid substitution of asparagine (N115) at amino acid 115; b) Amino acid substitution of aspartic acid (D121) at amino acid 121; c) Amino acid substitution of serine at amino acid 128 (S128); d) Amino acid substitution of leucine (L130) at amino acid 130; e) Amino acid substitution of valine (V131) at position 131; f) Amino acid substitution of glycine (G133) at position 133; g) Amino acid substitution of lysine (K219) at position 219; and h) an amino acid substitution of glycine (G220) at position 220, or comprising or consisting of SEQ ID NO:2, and/or wherein the fHBP B protein comprises at least one amino acid substitution selected from at least one of the following based on the numbering of SEQ ID NO: 6: a) Amino acid substitution of glutamine (Q38) at amino acid 38; b) Amino acid substitution of glutamic acid (E92) at amino acid 92; c) Amino acid substitution of arginine (R130) at amino acid 130; d) An amino acid substitution of serine at amino acid 223 (S223); and e) an amino acid substitution of histidine at amino acid 248 (H248), or comprising or consisting of SEQ ID NO. 4.

5. The composition of any one of claims 1 to 4, wherein the fHBP a protein and/or the fHBP B is present in an amount ranging from about 20 μg/dose to about 200 μg/dose, or from about 25 μg/dose to about 180 μg/dose, or from about 40 μg/dose to about 140 μg/dose, or from about 50 μg/dose to about 120 μg/dose, or from about 75 μg/dose to about 100 μg/dose, or about 25 μg/dose, or about 50 μg/dose, or about 100 μg/dose.

6. The composition of any one of claims 1 to 5, wherein the NadA protein is a NadA1 protein, or comprises at least about 85% identity to SEQ ID No. 5, or comprises or consists of SEQ ID No. 5.

7. The composition of any one of claims 1 to 6, wherein the NadA protein is present in an amount ranging from about 20 μg/dose to about 200 μg/dose, or from about 25 μg/dose to about 180 μg/dose, or from about 40 μg/dose to about 140 μg/dose, or from about 50 μg/dose to about 120 μg/dose, or from about 75 μg/dose to about 100 μg/dose, or about 50 μg/dose.

8. The composition of any one of claims 1 to 7, wherein the dOMV comprises porin a (PorA).

9. The composition of any one of claims 1 to 8, wherein the dOMV is present in an amount ranging from about 5 μg/dose to about 400 μg/dose, or from about 10 μg/dose to about 300 μg/dose, or from about 25 μg/dose to about 250 μg/dose, or from about 35 μg/dose to about 225 μg/dose, or from about 50 μg/dose to about 200 μg/dose, or from about 75 μg/dose to about 180 μg/dose, or from about 100 μg/dose to about 150 μg/dose, or from about 110 μg/dose to about 125 μg/dose, or about 25 μg/dose, or about 50 μg/dose, or about 125 μg/dose.

10. The composition according to any one of claims 1 to 9, further comprising an adjuvant, in particular an aluminium-based adjuvant selected from the group comprising: aluminum hydroxide adjuvants, aluminum phosphate adjuvants, aluminum sulfate adjuvants, aluminum hydroxy phosphate sulfate adjuvants, aluminum potassium sulfate adjuvants, aluminum hydroxy carbonate, combinations of aluminum hydroxide and magnesium hydroxide, and mixtures thereof, particularly aluminum phosphate adjuvants.

11. The composition according to any one of claims 1 to 10, comprising or consisting of: 25 to 100 μg/dose of non-lipidated fHBP a protein consisting of SEQ ID No. 2, 25 to 100 μg/dose of non-lipidated fHBP B protein consisting of SEQ ID No. 4, 25 to 100 μg/dose of NadA protein consisting of SEQ ID No. 5, 20 to 150 μg/dose of dOMV from the MenB strain expressing PorA VR 2P 1.2, 100 to 600 μg/dose of aluminium phosphate adjuvant, 50mM acetate buffer and pH 6.0.

12. The composition of any one of claims 1 to 11, further comprising at least one conjugated capsular saccharide from one or more of neisseria meningitidis serogroups A, C, W and/or Y.

13. A vaccine comprising the composition of any one of claims 1 to 12.

14. The composition according to any one of items 1 to 12 or the vaccine according to item 13 for use in preventing meningococcal infection or for inducing an immune response against meningococcal bacteria.

15. A composition comprising or consisting of mRNA encoding a fHBP a protein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, or about 100% amino acid sequence identity to SEQ ID NO:4, a NadA protein comprising at least about 85%, at least about 90%, at least about 95%, at least about 99.5%, or about 100% amino acid sequence identity to SEQ ID NO:4, a dOMV encoding a fHBP B protein comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99.5%, or about 100% amino acid sequence identity to SEQ ID NO:4, a NadA protein comprising at least about 98%, at least about 99%, at least about 99.5%, or about 100% amino acid sequence identity to SEQ ID NO:5, and a dOMV encoding a NadA VR2P1.2.

16. An immunogenic composition comprising a combination of neisseria meningitidis serogroup B antigens, the combination comprising at least one non-lipidated H factor binding protein (fHBP) a protein, at least one non-lipidated fHBP B protein, at least one neisseria adhesion a (NadA) protein, and at least one detergent-extracted outer membrane vesicle (dOMV), the fHBP a protein comprising at least about 85% identity with SEQ ID NO:1 and comprising at least an amino acid substitution G220S based on the numbering of SEQ ID NO: 6.

17. The composition of item 16, wherein the non-lipidated fHBP B protein is a mutein comprising at least about 85% identity to SEQ ID No. 3.

18. The composition of item 17, wherein the non-lipidated fHBP B protein comprises the amino acid substitution H248L based on the numbering of SEQ ID No. 6.

19. The composition of item 18, wherein the non-lipidated fHBP a protein comprises or consists of SEQ ID No. 4.

20. The composition of item 16, wherein the non-lipidated fHBP a protein further comprises the amino acid substitutions L130R and G133D based on the numbering of SEQ ID No. 6.

21. The composition of item 20, wherein the non-lipidated fHBP a protein comprises or consists of SEQ ID No. 2.

22. The composition of item 16, wherein the NadA protein is a NadA1 protein or comprises at least about 85% identity to SEQ ID No. 5 or comprises or consists of SEQ ID No. 5.

23. The composition of item 16, wherein the dOMV comprises a PorA VR2 subtype.

24. The composition of item 23, wherein the dOMV comprises PorA VR 2P 1.2.

25. The composition according to item 16, further comprising an adjuvant, in particular an aluminium-based adjuvant selected from the group comprising: aluminum hydroxide adjuvants, aluminum phosphate adjuvants, aluminum sulfate adjuvants, aluminum hydroxy phosphate sulfate adjuvants, aluminum potassium sulfate adjuvants, aluminum hydroxy carbonate, combinations of aluminum hydroxide and magnesium hydroxide, and mixtures thereof, particularly aluminum phosphate adjuvants.

26. The composition of item 16, wherein the fHBP A protein and/or the fHBP B protein is present in an amount ranging from about 20 μg/dose to about 200 μg/dose, or from about 25 μg/dose to about 180 μg/dose, or from about 40 μg/dose to about 140 μg/dose, or from about 50 μg/dose to about 120 μg/dose, or from about 75 μg/dose to about 100 μg/dose, or from about 25 μg/dose, or from about 50 μg/dose, or from about 100 μg/dose, and the NadA protein is present in an amount ranging from about 20 μg/dose to about 200 μg/dose, or from about 25 μg/dose to about 180 μg/dose, or from about 40 μg/dose to about 140 μg/dose, or from about 50 μg/dose to about 120 μg/dose, or from about 75 μg/dose to about 100 μg/dose, or about 50 μg/dose, the dOMV is administered in a range of about 5 to about 400, or about 10 to about 300, or about 25 to about 250, or about 35 to about 225, or about 50 to about 200, or about 75 to about 180, or about 100 to about 150, or about 110 to about 125, or about 25, or about 50, or about 125 μg/dose.

27. The composition of item 16, comprising or consisting of: 25 to 100 μg/dose of non-lipidated fHBP a protein consisting of SEQ ID No. 2, 25 to 100 μg/dose of non-lipidated fHBP B protein consisting of SEQ ID No. 4, 25 to 100 μg/dose of NadA protein consisting of SEQ ID No. 5, 20 to 150 μg/dose of dOMV from the MenB strain expressing PorA VR 2P 1.2, 100 to 600 μg/dose of aluminium phosphate adjuvant, 50mM acetate buffer and pH 6.0.

28. The composition of item 16, further comprising at least one conjugated capsular saccharide from one or more of neisseria meningitidis serogroup A, C, W and/or Y.

29. A vaccine comprising the composition of item 16.

30. A vaccine comprising the composition of item 27.

31. A therapeutic method for preventing a meningococcal infection or for inducing an immune response against a meningococcal bacterium, the method comprising administering to an individual in need thereof a composition comprising or consisting of mRNA encoding a fHBP a protein comprising an amino acid sequence identity of at least about 85%, at least about 95%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 99.5% or about 100% with SEQ ID NO 4, mRNA encoding a fHBP B protein comprising an amino acid sequence identity of at least about 85%, at least about 95%, at least about 98%, at least about 99.5% or about 100% with SEQ ID NO 2, mRNA encoding a fHBP B protein comprising an mRNA encoding a NadA protein comprising an amino acid sequence identity of at least about 85%, at least about 90%, at least about 95%, at least about 5% or about 100% with a MenB expressing a PorA VR 2P 1.2, and a dOMV.

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Claims (10)

1. An immunogenic composition comprising a combination of neisseria meningitidis serogroup B antigens, the combination comprising at least one factor H binding protein (fHBP) a protein, at least one fHBP B protein, at least one neisseria adhesion a (NadA) protein, and at least one detergent-extracted outer membrane vesicle (dOMV).

2. The composition of claim 1, wherein the fHBP a protein and/or the fHBP B protein is non-lipidated.

3. The composition of claim 1 or 2, wherein the fHBP a protein is a mutein comprising at least about 85% identity to SEQ ID No. 1, and/or wherein the fHBP B protein is a mutein comprising at least about 85% identity to SEQ ID No. 3.

4. A composition according to any one of claims 1 to 3, wherein the fHBP a protein comprises at least one amino acid substitution based on the numbering of SEQ ID NO:6 selected from at least one of: a) An amino acid substitution of asparagine (N115) at amino acid 115; b) Amino acid substitution of aspartic acid (D121) at amino acid 121; c) Amino acid substitution of serine at amino acid 128 (S128); d) Amino acid substitution of leucine (L130) at amino acid 130; e) Amino acid substitution of valine (V131) at position 131; f) Amino acid substitution of glycine (G133) at position 133; g) Amino acid substitution of lysine (K219) at position 219; and h) an amino acid substitution of glycine (G220) at position 220, or comprising or consisting of SEQ ID NO:2, and/or wherein the fHBP B protein comprises at least one amino acid substitution selected from at least one of the following based on the numbering of SEQ ID NO: 6: a) Amino acid substitution of glutamine (Q38) at amino acid 38; b) Amino acid substitution of glutamic acid (E92) at amino acid 92; c) Amino acid substitution of arginine (R130) at amino acid 130; d) An amino acid substitution of serine at amino acid 223 (S223); and e) an amino acid substitution of histidine at amino acid 248 (H248), or comprising or consisting of SEQ ID NO. 4.

5. The composition of any one of claims 1 to 4, wherein the fHBP a protein and/or the fHBP B is present in an amount ranging from about 20 μg/dose to about 200 μg/dose, or from about 25 μg/dose to about 180 μg/dose, or from about 40 μg/dose to about 140 μg/dose, or from about 50 μg/dose to about 120 μg/dose, or from about 75 μg/dose to about 100 μg/dose, or from about 25 μg/dose, or from about 50 μg/dose, or about 100 μg/dose.

6. The composition of any one of claims 1 to 5, wherein the NadA protein is a NadA1 protein, or comprises at least about 85% identity to SEQ ID No. 5, or comprises or consists of SEQ ID No. 5.

7. The composition of any one of claims 1 to 6, wherein the NadA protein is present in an amount ranging from about 20 μg/dose to about 200 μg/dose, or from about 25 μg/dose to about 180 μg/dose, or from about 40 μg/dose to about 140 μg/dose, or from about 50 μg/dose to about 120 μg/dose, or from about 75 μg/dose to about 100 μg/dose, or about 50 μg/dose.

8. The composition of any one of claims 1 to 7, wherein the dOMV comprises porin a (PorA).

9. The composition of any one of claims 1 to 8, wherein the dOMV is present in an amount ranging from about 5 μg/dose to about 400 μg/dose, or from about 10 μg/dose to about 300 μg/dose, or from about 25 μg/dose to about 250 μg/dose, or from about 35 μg/dose to about 225 μg/dose, or from about 50 μg/dose to about 200 μg/dose, or from about 75 μg/dose to about 180 μg/dose, or from about 100 μg/dose to about 150 μg/dose, or from about 110 μg/dose to about 125 μg/dose, or about 25 μg/dose, or about 50 μg/dose, or about 125 μg/dose.

10. Composition according to any one of claims 1 to 9, further comprising an adjuvant, in particular an aluminium-based adjuvant selected from the group comprising: aluminum hydroxide adjuvants, aluminum phosphate adjuvants, aluminum sulfate adjuvants, aluminum hydroxy phosphate sulfate adjuvants, aluminum potassium sulfate adjuvants, aluminum hydroxy carbonate, combinations of aluminum hydroxide and magnesium hydroxide, and mixtures thereof, particularly aluminum phosphate adjuvants.