Multivalent influenza nanoparticle vaccine

Abstract

The present invention discloses a multivalent nanoparticle vaccine composition suitable for influenza vaccine. The nanoparticles contain an effective amount of influenza glycoproteins, which provide an enhanced immune response compared to commercially available influenza vaccine compositions. The present disclosure also provides a vaccine formulation strategy that is cost-effective and convenient for clinical use. A method of administering the nanoparticle vaccine composition to a subject is also disclosed.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 644,623, filed on March 19, 2018, and U.S. Provisional Patent Application No. 62 / 787,980, filed on January 3, 2019, under 35 U.S.C. 119(e), the contents of each of which are incorporated herein by reference in their entirety for all purposes.

[0003] This application also incorporates by reference in their entirety the contents of U.S. Application No. 15 / 257,436, filed on September 6, 2016, and U.S. Application No. 15 / 819,962, filed on November 21, 2017, for all purposes. Technical Field

[0004] The present disclosure generally relates to influenza vaccine compositions that can be used to stimulate an immune response against influenza. Background Art

[0005] The heavy global burden of seasonal influenza is well documented. In the United States alone, approximately 140,000 to 10,000 hospitalizations and 12,000 to 56,000 deaths are attributable to influenza each year, with the elderly accounting for a disproportionate number of hospitalizations and 72% of deaths. Seasonal influenza vaccination has been a mainstay of prevention efforts and has been universally recommended in the United States since 2010.

[0006] However, recent developments, including the severe 2017-2018 A(H3N2) that dominated the U.S. flu season, suggest that vaccine efficacy is suboptimal in at least Australia, Canada, and the U.S. Other issues, such as antigenic mismatches associated with egg-based influenza vaccines and the ongoing challenge of antigenic drift, also suggest the need for more effective vaccination strategies.

[0007] Therefore, there is ongoing interest in producing vaccines against influenza viruses, and there remains a need for producing effective vaccines, particularly using methods that do not use recombinant egg proteins. Summary of the invention

[0008] The present disclosure provides a multivalent influenza composition. The composition stimulates an immune response against a variety of influenza strains. Advantageously, the immune response may include broadly neutralizing antibodies against variants of strains different from those used to prepare the composition. Influenza mutations lead to "drift strains", and the disclosed composition provides protection against "drift strains". In addition, the composition exhibits excellent stability, can be stored for a long time, and can be produced in pre-filled syringes ready for administration. In some aspects, a pre-filled syringe (PFS) may already contain an adjuvant and an influenza antigen and can be stored for a long time. The composition disclosed herein does not require refrigeration (2-8°C) and shows good stability at higher temperatures (e.g., room temperature, about 25°C). Therefore, the composition disclosed herein provides excellent convenience and excellent immune response.

[0009] In certain aspects, nanoparticles containing influenza antigens are co-formulated as vaccine compositions by mixing with an ISCOM matrix adjuvant (also referred to herein as a "matrix") for a period of time to form HaSMaN (hemagglutinin saponin matrix nanoparticles) before administration to a subject. Compositions containing HaSMaN exhibit good stability and immunogenicity, and are therefore well suited for packaging, for example, in prefilled syringes. In fact, HaSMaN is more stable than detergent core nanoparticles. The thermal stability of HaSMaN measured by differential scanning calorimetry is about 1 degree better than that of detergent core nanoparticles. Previously, methods using ISCOM matrix adjuvants included mixing the vaccine composition with the adjuvant at the bedside just before administration. In certain aspects, the compositions disclosed herein eliminate this requirement, thus providing an improved method for inducing an immune response. Surprisingly, it was found that the formation of HaSMaN varies depending on the type of influenza. HaSMaN is generally formed when the composition contains hemagglutinin (HA) protein from an influenza strain of type A, but not when the HA protein is from an influenza strain of type B.

[0010] In some embodiments, the ISCOM matrix adjuvant in the multivalent influenza composition is a matrix M, which is a combination of two types of matrices, the first type being a matrix A containing saponin fraction A (also referred to herein as a fraction A matrix), and the second type being a matrix C containing saponin fraction C (also referred to herein as a fraction C matrix). In certain aspects, the matrix M may comprise at least about 85% (w / w) fraction A matrix, with the remainder being fraction C matrix. Unless otherwise specified, the matrix M used herein (also referred to herein as matrix M1) comprises fraction A matrix and fraction C matrix in a ratio of 85:15 (w / w).

[0011] In some embodiments, each influenza HA protein in the quadrivalent nanoparticle influenza composition can be from a different influenza strain. In some embodiments, at least one strain can be a subtype A strain or a subtype B strain. In some embodiments, the subtype A strain can be complexed with the matrix M adjuvant, but the subtype B strain is not complexed with the matrix M adjuvant.

[0012] While the present disclosure contemplates multivalent compositions, particularly for seasonal vaccines, monovalent vaccine compositions may also be produced using HaSMaN for use, for example, in vaccination against emerging pandemic influenza strains.

[0013] Stability (especially room temperature stability) is a valuable property because it reduces or eliminates the need for cold chain storage, making distribution cheaper and easier to achieve. Advantageously, the multivalent compositions disclosed herein are stable for long periods of time, such as up to 3 months, up to 6 months, up to 12 months, and at room temperature (i.e., about 25° C.) during these periods. Excellent stability (especially when formulated with a matrix M adjuvant) means that the vaccine composition is suitable for delivery to a clinical setting in a ready-to-use form; e.g., a pre-filled syringe formulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Examples of transmission electron microscopy (TEM) images of influenza HA detergent core nanoparticles alone (left), matrix M alone (middle), and a combination of HA nanoparticles and matrix M, which form hemagglutinin saponin matrix nanoparticles (HaSMaN) (right), are shown.

[0015] Figure 2 Shown are reduced SDS-PAGE gel images of influenza antigens at 4°C for high and low dose quadrivalent bedside vial formulations (240 μg / mL / strain and 60 μg / mL / strain) and prefilled syringe (PFS) formulations (120 μg / mL / strain and 30 μg / mL / strain) with or without matrix M at T=0, 6 months and 12 months. The abbreviations in the figure are PFS: prefilled syringe; M1: matrix M.

[0016] Figure 3 Shown are non-reducing and reducing SDS-PAGE gel images of influenza antigens at time = 0 at 4°C for high dose bedside vial formulation (240 μg / mL / strain) and PFS formulation with or without matrix M (120 μg / mL / strain).

[0017] Figure 4Shown are non-reducing SDS-PAGE gel images of influenza antigens for high-dose and low-dose bedside vial formulations (240 μg / mL / strain and 60 μg / mL / strain) and PFS formulations with or without matrix M (120 μg / mL / strain and 30 μg / mL / strain) at 4°C and 25°C at the 3-month time point.

[0018] Figure 5 The results of single radial immunodiffusion assay (SRID) of A / Hong Kong, A / Michigan, B / Brisbane and B / Phuket strains at T=0, 2 weeks, 1 month, 3 months, 6 months, 9 months and 12 months at 2-8°C with or without PFS formulations of matrix M at a dose of 120 μg / mL / strain are shown. The abbreviations in the figure are MXM: matrix M; HK: Hong Kong; MI: Michigan; Bris: Brisbane; Phu: Phuket.

[0019] Figure 6 Shown are SRID results for A / Hong Kong, A / Michigan, B / Brisbane and B / Phuket strains at T=0, 2 weeks, 1 month, 3 months and 6 months at 25°C with or without matrix M at a dose of 120 μg / mL / strain of PFS formulation.

[0020] Figure 7 Hemagglutination inhibition antibody responses in mice administered with Quad-NIV co-formulations or bedside mixed formulations with or without matrix M are shown compared to administration of commercial influenza vaccine against A / Hong Kong / 4801 / 2014. Antibody responses are expressed as HAI titers. Abbreviations in the figure are HAI: hemagglutination inhibition; HA: hemagglutinin; Co-form: co-formulation; Quad-NIV: quadrivalent nanoparticle influenza vaccine; QIV: quadrivalent influenza vaccine.

[0021] Figure 8 Shown are hemagglutination inhibition antibody responses against A / Michigan / 45 / 2015 in mice administered Quad-NIV co-formulation or bedside mixed formulation with or without matrix M compared to administration of commercial influenza vaccine.

[0022] Fig. 9 Shown are hemagglutination inhibition antibody responses against B / Brisbane / 60 / 2008 in mice administered Quad-NIV co-formulation or bedside mixed formulation with or without matrix M compared to administration of a commercial influenza vaccine.

[0023] Fig.10Shown are hemagglutination inhibition antibody responses against B / Phuket / 3073 / 2013 in mice administered Quad-NIV co-formulation or bedside mixed formulation with or without matrix M compared to administration of commercial influenza vaccine.

[0024] Fig.11 Hemagglutination inhibition antibody responses in mice administered with Quad-NIV premixed formulations or bedside mixed formulations with matrix M against A / Hong Kong / 4801 / 2014, A / Michigan / 45 / 2015, B / Brisbane / 60 / 2008, and B / Phuket / 3073 / 2013 are shown. The PFS formulation was stored at 4°C for 3 months. For bedside mixed vaccines, viral antigens were stored at -60°C for 3 months and mixed with matrix M adjuvant just before administration. Antibody responses are expressed as HAI GMT titers. GMT results use 95% lower and 95% upper control limits. The abbreviations in the figure are HAI: hemagglutination inhibition; HA: hemagglutinin; GMT: geometric mean titer; LCL: lower control limit; UCL: upper control limit.

[0025] Fig.12 Shown are hemagglutination inhibition antibody responses against A / Michigan strain in mice administered Quad-NIV co-formulations or bedside mixed formulations with or without matrix M (100 μg / mL) at day 21. Premixed Quad-NIV co-formulations were stored at the indicated temperature for 6 months.

[0026] Fig.13 Shown are hemagglutination inhibition antibody responses against A / Michigan strain in mice administered Quad-NIV co-formulations or bedside mixed formulations with or without matrix M (100 μg / mL) at day 35. Premixed Quad-NIV co-formulations were stored at the indicated temperature for 6 months.

[0027] Fig.14 Shown are hemagglutination inhibition antibody responses against A / Hong Kong / 4801 / 2014 in mice administered Quad-NIV co-formulations or bedside mixed formulations with or without matrix M (100 μg / mL) at day 21. Premixed Quad-NIV co-formulations were stored at the indicated temperature for 6 months.

[0028] Fig.15 Shown are hemagglutination inhibition antibody responses against A / Hong Kong strain in mice administered Quad-NIV co-formulations or bedside mixed formulations with or without matrix M (100 μg / mL) at day 35. Premixed Quad-NIV co-formulations were stored at the indicated temperature for 6 months.

[0029] Fig.16 Shown are hemagglutination inhibition antibody responses against B / Brisbane strains in mice administered Quad-NIV co-formulations or bedside mixed formulations with or without matrix M (100 μg / mL) at day 21. Premixed Quad-NIV co-formulations were stored at the indicated temperature for 6 months.

[0030] Fig.17 Hemagglutination inhibition antibody responses against B / Brisbane strains in mice administered Quad-NIV co-formulations or bedside mixed formulations with or without matrix M (100 μg / mL) at 35 days are shown. Antibody responses are expressed as HAI titers. Premixed Quad-NIV co-formulations were stored at the specified temperature for 6 months.

[0031] Fig.18 Shown are hemagglutination inhibition antibody responses against B / Phuket strain in mice administered Quad-NIV co-formulations or bedside mixed formulations with or without matrix M (100 μg / mL) at day 21. Premixed Quad-NIV co-formulations were stored at the indicated temperature for 6 months.

[0032] Fig.19 Hemagglutination inhibition antibody responses against B / Phuket strains in mice administered Quad-NIV co-formulations or bedside mixed formulations with or without Matrix M (100 μg / mL) at 35 days are shown. Antibody responses are expressed as HAI titers. Premixed Quad-NIV co-formulations were stored at the specified temperature for 6 months.

[0033] Fig. 20 Shown are hemagglutination inhibition antibody responses against A / Hong Kong / 4801 / 2014 in mice administered Quad-NIV co-formulations or bedside mixed formulations with or without matrix M (100 μg / mL) at day 21. Premixed Quad-NIV co-formulations were stored at 4°C for 12 months.

[0034] Fig.21 Shown are hemagglutination inhibition antibody responses against A / Hong Kong strain in mice administered Quad-NIV co-formulations or bedside mixed formulations with or without matrix M (100 μg / mL) at day 35. Premixed Quad-NIV co-formulations were stored at 4°C for 12 months.

[0035] Fig. 22Shown are hemagglutination inhibition antibody responses against A / Michigan strain in mice administered Quad-NIV co-formulations or bedside mixed formulations with or without matrix M (100 μg / mL) at day 21. Premixed Quad-NIV co-formulations were stored at 4°C for 12 months.

[0036] Fig.23 Shown are hemagglutination inhibition antibody responses against A / Michigan strain in mice administered Quad-NIV co-formulations or bedside mixed formulations with or without matrix M (100 μg / mL) at day 35. Premixed Quad-NIV co-formulations were stored at 4°C for 12 months.

[0037] Fig.24 Shown are hemagglutination inhibition antibody responses against B / Brisbane strains in mice administered Quad-NIV co-formulations or bedside mixed formulations with or without matrix M (100 μg / mL) at day 21. Premixed Quad-NIV co-formulations were stored at 4°C for 12 months.

[0038] Fig.25 Hemagglutination inhibition antibody responses against B / Brisbane strains in mice administered Quad-NIV co-formulations or bedside mixed formulations with or without matrix M (100 μg / mL) at 35 days are shown. Antibody responses are expressed as HAI titers. Premixed Quad-NIV co-formulations were stored at 4°C for 12 months.

[0039] Fig.26 Shown are hemagglutination inhibition antibody responses against B / Phuket strain in mice administered Quad-NIV co-formulations or bedside mixed formulations with or without matrix M (100 μg / mL) at day 21. Premixed Quad-NIV co-formulations were stored at 4°C for 12 months.

[0040] Fig. 27 Hemagglutination inhibition antibody responses against B / Phuket strains in mice administered Quad-NIV co-formulations or bedside mixed formulations with or without matrix M (100 μg / mL) at 35 days are shown. Antibody responses are expressed as HAI titers. Premixed Quad-NIV co-formulations were stored at 4°C for 12 months.

[0041] Fig.28 AUC sedimentation curves for Quad-NIV PFS formulation (120 μg / mL) at 4°C at T=0, 3 months, 6 months, 9 months, or 12 months and Quad-NIV PFS formulation using 100 μg / mL matrix M1 at 4°C at T=3 months, 6 months, 9 months, or 12 months.

[0042] Fig.29 Analytical centrifugation (AUC) sedimentation curves of Quad-NIV PFS formulations with or without matrix M1 (100 μg / mL) at study initiation (T0), 1 month, 3 months, and 6 months are shown. Formulations were incubated at 25°C.

[0043] Fig.30 TEM images of high-dose Quad-NIV only (120 μg / mL / strain), matrix M only (100 μg / mL), high-dose Quad-NIV + matrix M, and low-dose Quad-NIV (30 μg / mL / strain) + matrix M (100 μg / mL) without incubation (T=0) are shown.

[0044] Fig.31 TEM images of high-dose Quad-NIV (120 μg / mL / strain) + Matrix M (100 μg / mL) at 4° C. or 25° C. and low-dose Quad-NIV (30 μg / mL / strain) + Matrix M (100 μg / mL) at 4° C. or 25° C. are shown. All samples were incubated for 4 hours.

[0045] Fig.32 TEM images of high dose Quad-NIV (120 μg / mL / strain) + Matrix M (100 μg / mL) at 4°C or 25°C and low dose Quad-NIV (30 μg / mL / strain) + Matrix M (100 μg / mL) at 4°C or 25°C are shown. All samples were incubated for 24 hours. Exemplary HaSMaN is circled.

[0046] Fig.33 TEM images of high-dose Quad-NIV (120 μg / mL / strain) + Matrix M (100 μg / mL) at 4°C or 25°C and low-dose Quad-NIV (30 μg / mL / strain) + Matrix M (100 μg / mL) at 4°C or 25°C are shown. All samples were incubated for 48 hours. Exemplary HaSMaN is circled.

[0047] Fig.34 TEM images of high-dose Quad-NIV (120 μg / mL / strain) + Matrix M (100 μg / mL) at 4°C or 25°C and low-dose Quad-NIV (30 μg / mL / strain) + Matrix M (100 μg / mL) at 4°C or 25°C are shown. All samples were incubated for 7 days. Exemplary HaSMaN is circled.

[0048] Fig.35TEM images of the low-dose PFS group (30 μg / mL / strain) with or without matrix M (100 μg / mL) incubated at various temperatures (2-8°C, 25°C, and 37°C) for 1 month are shown.

[0049] Fig.36 DSC curves of Quad-NIV formulations at a dose of 120 μg / mL / strain with or without matrix M1 (100 μg / mL) are shown. Molar heat capacity (Cp: kJ / mol.k) of the tetravalent vaccine antigens incubated at 4°C for 3, 6 and 12 months and at 25°C for 3 and 6 months are shown.

[0050] Fig.37 Shown is the formation of HaSMaN measured by the reduction of nHA (ie detergent core nanoparticles) using HA proteins from strain A, strain B, and C-terminally modified strain A fused with foldon. DETAILED DESCRIPTION

[0051] definition

[0052] 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 "a protein" may refer to one protein or a mixture of such proteins, and reference to "the method" includes reference to equivalent steps and / or methods known to those skilled in the art, and so forth.

[0053] As used herein, the term "adjuvant" refers to a compound that, when used in combination with an immunogen, augments or otherwise alters or modifies the immune response induced to the immunogen. Modification of the immune response may include enhancing or amplifying the specificity of either or both of the antibody and cellular immune response.

[0054] As used herein, the term "about" or "approximately" when preceding a numerical value indicates a range of plus or minus 10% of the numerical value. For example, "about 100" includes 90 and 110.

[0055] As used herein, the terms "immunogen," "antigen," and "epitope" refer to substances, such as proteins (including glycoproteins) and peptides, that are capable of eliciting an immune response.

[0056] As used herein, an "immunogenic composition" is a composition comprising an antigen, wherein administration of the composition to a subject results in the development of a humoral and / or cellular immune response to the antigen in the subject.

[0057] As used herein, "Quad-NIV," "QuadNIV," or "quadrivalent nanoparticle influenza vaccine" refers to an influenza vaccine formulation that includes antigens from four influenza strains.

[0058] As used herein, the terms "bedside mixture", "bedside preparation", "bedside vaccine composition", "bedside vial", "bedside vial preparation" refer to a vaccine preparation prepared just before administration. Such a vaccine preparation comprises a viral antigen and an adjuvant, which are stored in different containers and administered to a subject (e.g., two consecutive injections are administered, or the antigen and adjuvant are mixed into one injection before administration).

[0059] As used herein, the terms "co-formulation mixture", "co-formulation", "co-formulation vaccine composition", "pre-filled syringe", "premix" refer to vaccine formulations that are prepared for short- to long-term storage prior to the time of administration to a subject. Such vaccine formulations contain a combination of influenza antigens and ISCOM matrix adjuvants in the same container and are prepared under conditions sufficient to form HaSMaN (hemagglutinin saponin matrix nanoparticles).

[0060] As used herein, the terms "treat," "treatment," and "treating" refer to methods for obtaining beneficial or desired results (e.g., clinical results). For purposes of this disclosure, beneficial or desired results may include inhibiting or suppressing the initiation or progression of an infection or disease; ameliorating symptoms of an infection or disease or reducing its occurrence; or a combination thereof.

[0061] As used herein, "prevention" and "prophylaxis" are used interchangeably and may mean preventing an infection or disease completely, or preventing symptoms of such an infection or disease from occurring; delaying the onset of an infection or disease or its symptoms; or reducing the severity of a subsequent infection or disease or its symptoms.

[0062] As used herein, "effective dose" or "effective amount" refers to the amount of an immunogen sufficient to induce an immune response that reduces at least one symptom of a pathogen infection. An effective dose or effective amount can be determined, for example, by measuring the amount of neutralizing secretory antibodies and / or serum antibodies (e.g., by plaque neutralization, complement fixation, enzyme-linked immunosorbent assay (ELISA), microneutralization assay, and HAI titer).

[0063] As used herein, the term "vaccine" refers to an immunogenic composition, such as an immunogen derived from a pathogen, which is used to induce an immune response against the pathogen that provides protective immunity (e.g., immunity that protects the subject from infection with the pathogen and / or reduces the severity of a disease or condition caused by infection with the pathogen). The protective immune response may include the formation of antibodies and / or cell-mediated responses. Depending on the context, the term "vaccine" may also refer to a suspension or solution of an immunogen that is administered to a subject to produce protective immunity.

[0064] As used herein, the term "subject" includes humans and other animals. Typically, the subject is a human. For example, the subject can be an adult, teenager, child (2 to 14 years old), infant (1 month to 24 months) or newborn (up to 1 month). In some aspects, an adult is about 65 years old or older, or about 60 years old or older. In typical aspects, an adult is about 60 to about less than 75 years old, or at least about 75 years old. In some aspects, the subject is a pregnant woman or a woman who intends to become pregnant. In other aspects, the subject is not a human; for example, it is a non-human primate; for example, a baboon, a chimpanzee, a gorilla or a macaque. In some aspects, the subject can be a pet, such as a dog or a cat.

[0065] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the U.S. federal or state government or listed in the U.S. Pharmacopoeia, European Pharmacopoeia or other generally recognized pharmacopeia for use in animals and more particularly in humans. These compositions can be used as vaccines and / or antigenic compositions that induce a protective immune response in vertebrates.

[0066] Nanoparticles: structure and morphology

[0067] The compositions disclosed herein comprise two types of nanoparticles. The first type is referred to as detergent core nanoparticles, substantially as previously described. See U.S. Serial No. 15 / 257,436, which is incorporated herein by reference for all purposes. In short, these nanoparticles are produced in insect cells by expressing HA protein using a baculovirus expression system and then extracting the HA protein with a detergent. During the purification process, the first detergent is replaced with a second detergent (usually a nonionic detergent) to generate nanoparticles with a nonionic detergent core, in which the terminal portion of the HA protein is embedded in a trimer form. Figure 1 (Left) shows these structures as observed under an electron microscope.

[0068] The second type of nanoparticles is referred to herein as HaSMaN (hemagglutinin saponin matrix nanoparticles). Figure 1 (Right) shows these structures as observed under an electron microscope. HA glycoprotein modified matrix cage structure. The HaSMaN structure was formed by preparing HA nanoparticles and then incubating them with ISCOM matrix adjuvant particles for a period of time. ISCOM matrix particles such as Figure 1. Notably, HaSMaN is easily formed from influenza type A HA proteins, but not easily formed from influenza type B HA proteins. Thus, in certain aspects, a composition comprising nanoparticles having HA proteins from both type A and type B will contain both types of nanoparticles (i.e., detergent core nanoparticles and HaSMaN). In some aspects, the nanoparticles will transition between the two states, and thus the composition may comprise transitional nanoparticles.

[0069] In a specific embodiment, the detergent core nanoparticle is composed of multiple protein trimers around the nonionic detergent core. For example, each nanoparticle can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 15 trimers. In some embodiments, each nanoparticle includes 2 to 6 trimers. In a specific embodiment, each nanoparticle includes 2 to 9 trimers.

[0070] HA protein is associated with the nonionic detergent-containing core of the nanoparticle. Typically, detergents are selected from polysorbate 20 (PS20), polysorbate 40 (PS40), polysorbate 60 (PS60), polysorbate 65 (PS65) and polysorbate 80 (PS80). The presence of detergent promotes the formation of nanoparticles by forming a core that organizes and presents antigens. Therefore, in certain embodiments, the nanoparticles include antigens assembled into poly-oligosaccharide protein-PS80 protein-detergent nanoparticles, whose head regions protrude outward, and the hydrophobic region and the PS80 detergent form a central core surrounded by the antigen. In a specific embodiment, the nonionic detergent in the influenza vaccine composition is PS80. The Z-ave size range of detergent core nanoparticles is about 25nm to about 30nm. However, once the detergent core nanoparticles are mixed with the matrix M for a period of time, HaSMaN will be formed, and the formed HaSMaN is slightly larger than the detergent core nanoparticles, about 50nm to 60nm. Unless otherwise specified, particle size (Z-ave) was measured by dynamic light scattering (DLS) using a Malvern Zetasizer.

[0071] Nanoparticles: Production

[0072] The detergent core nanoparticles and HaSMaN of the present disclosure are non-naturally occurring products, the components of which do not occur together in nature.In general, the methods disclosed herein use a detergent exchange method, where a first detergent is used to isolate proteins, and then this first detergent is exchanged for a second detergent to form nanoparticles.

[0073] The glycoprotein antigen (usually HA) in the nanoparticle is usually produced by recombinant expression in a host cell. Usually, glycoproteins are expressed in insect host cells using a baculovirus system. In a preferred embodiment, the baculovirus is a cathepsin-L knockout baculovirus. In other preferred embodiments, the baculovirus is a chitinase knockout baculovirus. And in other preferred embodiments, the baculovirus is a double knockout type of both cathepsin-L and chitinase. High-level expression can be obtained in an insect cell expression system. Non-limiting examples of insect cells are Spodoptera frugiperda (Sf) cells (e.g., Sf9, Sf21), Trichoplusia ni cells (e.g., High Five cells), and Drosophila S2 cells. Preferably, the cell is an Sf9 cell or a derivative thereof.

[0074] Typical transfection and cell growth methods can be used to culture cells. Vectors, for example, vectors containing polynucleotides encoding fusion proteins can be transfected into host cells according to methods well known in the art. For example, nucleic acid introduction into eukaryotic cells can be achieved by calcium phosphate coprecipitation, electroporation, microinjection, lipofection, and transfection using polyamine transfection reagents.

[0075] Methods for growing host cells include, but are not limited to, batch, fed-batch, continuous, and perfusion cell culture techniques. Cell culture refers to the growth and propagation of cells in a bioreactor (fermentation chamber) where the cells propagate and express proteins (e.g., recombinant proteins) for purification and isolation. Typically, cell culture is performed in a bioreactor under sterile, controlled temperature and atmospheric conditions. A bioreactor is a chamber for culturing cells where environmental conditions such as temperature, atmosphere, agitation, and / or pH can be monitored. In one embodiment, the bioreactor is a stainless steel chamber. In another embodiment, the bioreactor is a pre-sterilized plastic bag (e.g., Wave Biotech, Bridgewater, NJ). In other embodiments, the pre-sterilized plastic bag is about 50 L to about 3500 L bag.

[0076] Detergent extraction and purification of nanoparticles

[0077] After the host cells are grown, detergents and purification schemes can be used to harvest proteins from the host cells. Once the host cells have grown for 48 to 96 hours, the cells are separated from the culture medium and a solution containing detergent is added to dissolve the cell membrane, thereby releasing the protein in the detergent extract. Triton X-100 and tergitol (also known as NP-9) are both preferred detergents for extraction. Detergents can be added to a final concentration of about 0.1% to about 1.0%. For example, the concentration can be about 0.1%, about 0.2%, about 0.3%, about 0.5%, about 0.7%, about 0.8% or about 1.0%. In certain embodiments, the range can be about 0.1% to about 0.3%. Preferably, the concentration is about 0.5%.

[0078] In other aspects, different first detergents can be used to separate proteins from host cells. For example, the first detergent can be bis(polyethylene glycol bis[imidazoylcarbonyl]), nonoxynol-9, bis(polyethylene glycol bis[imidazoylcarbonyl]), Decaglycone monododecyl ether, N-decanoyl-N-methylglucosamine, n-decyl α-D-glucopyranoside, decyl β-D-maltopyranoside, n-dodecanoyl-N-methylglucosamide, n-dodecyl α-D-maltoside, n-dodecyl β-D-maltoside, n-dodecyl β-D-maltoside, heptaglycone monodecyl ether, heptaglycone monododecyl ether, heptaglycone monotetradecyl ether, hexadecyl β-D-maltoside, hexaglycone monododecyl ether, hexaglycone monohexadecyl ether, hexaglycone monooctadecyl ether, hexaglycone monotetradecyl ether, Igepal CA-630, Igepal CA-630, methyl-6-0-(N-heptylcarbamoyl)-α-D-pyranoglucoside, nonaethylene glycol monododecyl ether, N-nonanoyl-N-methylglucamine, N-nonanoyl-N-methylglucamine, octaethylene glycol monodecyl ether, octaethylene glycol monododecyl ether, octaethylene glycol monohexadecyl ether, octaethylene glycol monooctadecyl ether, octaethylene glycol monotetradecyl ether, octyl-β-D-pyranoglucoside, pentaethylene glycol monodecyl ether, pentaethylene glycol monododecyl ether, pentaethylene glycol monohexadecyl ether, pentaethylene glycol monohexyl ether, pentaethylene glycol monooctadecyl ether, pentaethylene glycol monooctyl ether, polyethylene glycol diglycidyl ether, polyethylene glycol ether W-1, polyoxyethylene 10 tridecyl ether, polyoxyethylene 100 stearate, polyoxyethylene 20 isohexadecyl ether, polyoxyethylene 20 oleyl ether, polyoxyethylene 40 stearate, polyoxyethylene 50 stearate, polyoxyethylene 8 stearate, polyoxyethylene bis(imidazolylcarbonyl), polyoxyethylene 25 propylene glycol stearate, saponin from the bark of the genus Quillaja, 15-S-12 type Tergitol, 15-S-30 type Tergitol, 15-S-5 type Tergitol, 15-S-7 type Tergitol, 15-S-9 type Tergitol, NP-10 type Tergitol, NP-4 type Tergitol, NP-40 type Tergitol, NP-7 type Tergitol, NP-9 type Tergitol, TMN-10 type Tergitol, TMN-6 type Tergitol, Triton X-100 or a combination thereof.

[0079] Centrifugation can then be used to separate the nanoparticles from the cell debris. In some embodiments, gradient centrifugation can be used, such as using cesium chloride, sucrose, and iodixanol. Other techniques can be used as an alternative or supplement, such as standard purification techniques, including, for example, ion exchange chromatography, affinity chromatography, and gel filtration chromatography.

[0080] For example, the first column can be an ion exchange chromatography resin such as EMD TMAE (EMD Millipore), the second column can be a Lens culinaris lectin affinity resin, and the third column can be a cation exchange column such as EMD SO3 (EMD Millipore) resin. In other aspects, the cation exchange column can be an MMC column or a Nuvia C Prime column (Bio-Rad Laboratories, Inc). Preferably, the methods disclosed herein do not use a detergent extraction column; for example, a hydrophobic interaction column. Such columns are often used to remove detergents during purification, but may have a negative impact on the methods disclosed herein.

[0081] Detergent exchange

[0082] In order to form detergent core nanoparticles, the first detergent for extracting protein from the host cell is substantially replaced by the second detergent to form a nanoparticle structure. NP-9 is a preferred extraction detergent. Usually, when measured by HPLC, the nanoparticles do not contain detectable NP-9. The second detergent is usually selected from the group consisting of PS20, PS40, PS60, PS65 and PS80. Preferably, the second detergent is PS80.

[0083] In certain aspects, affinity chromatography is used to bind glycoproteins via their sugar moieties for detergent exchange. For example, affinity chromatography can use a leguminous lectin column. Leguminous lectins are proteins originally identified in plants and found to interact specifically and reversibly with sugar residues. See, e.g., Sharon and Lis, "Legumelectins--a large family of homologous proteins," FASEB J. 1990 Nov; 4(14): 3198-208; Liener, "The Lectins: Properties, Functions, and Applications in Biology and Medicine," Elsevier, 2012. Suitable lectins include concanavalin A (conA), pea lectin, sainfoin, and lentil lectin. Lentil lectin is a preferred column for detergent exchange due to its binding properties. Lectin columns are commercially available; for example, Capto LentilLectin, available from GE Healthcare. In certain aspects, the lentil lectin column can use recombinant lectin. At the molecular level, it is believed that the sugar moiety binds to the lentil lectin, releasing the amino acids of the protein to associate around the detergent, thereby forming a detergent core that provides nanoparticles with multiple copies of the antigen, such as glycoprotein oligomers, which can be dimers, trimers or tetramers anchored in the detergent.

[0084] When detergent is incubated with protein to form nanoparticles during a detergent exchange process, detergent may be present up to about 0.1% (w / v) in early purification steps.

[0085] Typically, influenza detergent core nanoparticles are prepared separately as monovalent single strain products. For example, a nonionic detergent (e.g., PS80) can be about 0.03% to about 0.5% nonionic detergent. In particular aspects, the monovalent drug substance may include about 1.5 mg / mL of protein measured by A280 and about 0.12% PS80, which provides a molar ratio of about 40: 1. In a specific quadrivalent composition, the protein concentration can be about 0.48 mg / mL as measured by a single radial immunodiffusion assay (SRID), and the amount of PS80 calculated is about 0.04%.

[0086] Detergent swaps can be performed with purified protein as discussed above and the protein purified, stored frozen, and then thawed for detergent swap.

[0087] Formation of HaSMaN

[0088] The HaSMaN disclosed herein are produced by incubating detergent core nanoparticles with an ISCOM matrix adjuvant comprising a saponin fraction, cholesterol, and phospholipids.

[0089] Typically, formation requires incubation at 4°C or 25°C for about 24 to 48 hours. Higher temperatures promote the formation of HaSMaN. See, e.g., Fig.33 and Fig.35 Thus, in certain aspects, HaSMaNs are formed by incubation at about 25°C for at least 24 hours. Mixing the detergent core nanoparticles with the ISCOM matrix adjuvant shortly before administration to a subject - i.e., bedside mixing - does not produce HaSMaNs. Longer incubation periods do not negatively affect the formation of HaSMaNs.

[0090] Nanoparticle influenza HA protein

[0091] The HA glycoprotein used as an influenza antigen can be from any influenza virus strain. Human influenza viruses type A and B cause seasonal epidemics almost every winter in the United States. Influenza A viruses are further divided into subtypes based on two proteins on the surface of the virus: hemagglutinin (HA) and neuraminidase (NA).

[0092] The HA protein can be selected from subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17 and H18. Phylogenetically, influenza is divided into several groups. For HA, group 1 contains H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17 and H18, while group 2 contains H3, H4, H7, H10, H14 and H15.

[0093] In certain aspects, the influenza nanoparticles are trypsin-resistant nanoparticles produced using neutral pH purification. During the purification and formulation of the HA nanoparticles, trypsin resistance is achieved by a neutral pH range of 6.9 or more to 8.5. Trypsin-resistant influenza glycoproteins and trypsin-resistant influenza nanoparticles; and methods for making them are described in detail in U.S. Application No. 15 / 819,962, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0094] Modified Antigen

[0095] Typically, the antigen is a full-length wild-type sequence, however, the antigen may be a variant or mutant of a wild-type antigen. In certain aspects, the antigen may share identity with the disclosed antigen; for example, the identity percentage may be at least 80%, at least 90%, at least 95%, at least 97%, or at least 98%. The identity percentage may be calculated using the alignment program ClustalW2, which is available at www.ebi.ac.uk / Tools / msa / clustalw2 / . The following default parameters may be used for paired alignments: protein weight matrix = Gonnet; gap opening = 10; gap extension = 0.1.

[0096] Other variants may be used. HA is a homotrimer in which each monomer consists of approximately 550 amino acid residues. Each monomer of HA has been conceptually divided into three domains: an extracellular domain of approximately 515 residues constitutes the extraviral portion of the molecule; a single segment of 27 residues defines the transmembrane (TM) domain; and approximately 10 residues constitute the cytoplasmic tail (CT). Although there may be some variation in the antigen, the formation of both detergent core nanoparticles and HaSMaN requires a complete transmembrane (TM) domain. Thus, in a particular example, the modified HA protein sequence has 100% identity (i.e., is wild type) in the TM and CT domains, while having some flexibility in the remaining extracellular domain portion, where the identity can be at least 90% or at least 95%. The domains can be modified by conformation with Melikyan et al. (Mol Biol Cell. 1999 Jun; 10(6): 1821-1836) Figure 1 The HA domains were identified based on the homology of the amino acid sequences of the TM domain and CT of Japan / 305 / 57 HA shown in , but it should be noted that the boundaries between the ectodomain, TM, and CT domains may vary from HA protein to HA protein by up to three amino acids.

[0097] Vaccine stability

[0098] Advantageously, the disclosed nanoparticle influenza vaccine formulations are stable and suitable for long-term storage. The formulations provided herein may be particularly suitable for co-formulation strategies (i.e., wherein the antigen and matrix adjuvant are fully mixed before administration; e.g., in a pre-filled syringe). As disclosed herein, the co-formulation influenza vaccine strategy may provide advantages for clinical practice and may serve as a cost-effective alternative to the currently used bedside mixed formulations.

[0099] In some embodiments, the influenza antigens in the co-formulated vaccine composition are stable when the composition is stored for up to 12 months.While the vaccine compositions can be stored at 2-8°C, they exhibit excellent stability at 25°C.

[0100] The stability of the premixed (co-formulated) formulation can be evaluated by methods and protocols known in the art. Stability can be measured by the level of degradation of the vaccine antigens and the immunogenicity of the vaccine. Stability can be tested by evaluating the thermal stability of the viral antigens in the vaccine.

[0101] Long-term stability of vaccine formulations at room temperature (25°C) facilitates cost-effective vaccine strategies, especially for regions where cold chain distribution and storage may be limited. As stated in the WHO guidance on assessing vaccine stability, controlling the temperature chain during manufacturing, distribution and storage is very important to ensure vaccine efficacy (“Guidelines for stability assessment of vaccines used in controlled temperature chains; available at www.who.int / biologicals / WHO_CTC_first_draft_22_Dec_2014_amended.pdf).

[0102] Without being bound by theory, it is believed that the HaSMaN structure may contribute to the stability of the co-formulated vaccine formulation. (See Fig.36 , which showed better thermal stability in the presence of a matrix adjuvant; that is, the HaSMaN particles were shown to have better stability than formulations using only a detergent core). The detergent core nanoparticles were mixed with a matrix M to provide HaSMaN to provide a vaccine composition that had a temperature increase of about 0.5°C to about 1.0°C (measured by differential scanning calorimetry) compared to a vaccine composition containing only detergent core nanoparticles.

[0103] This improved thermal stability helps to extend the shelf life of the vaccine. In particular aspects, the vaccine formulation can be stable for up to 6 months or up to 12 months. If after storage at 2-8°C for 12 months, there is no statistical difference in the HAI titer of the test sample from the HAI titer of the freshly prepared sample; and if after storage at 2-8°C for 12 months, the value obtained by the single radial immunodiffusion (SRID) assay is at least about 70%, then in the context of the present disclosure, the test sample is considered to be "stable". The statistical significance analysis of the HAI titer measurement was performed as follows. The geometric mean titer (GMT) and the associated 95% confidence interval (CI) were calculated by group. Using Tukey HSD analysis with JMP13 software, the mean value of the log10 transformed titer measurements of HAI between the groups was compared. P value <0.05 indicates a statistically significant difference between the two comparison groups.

[0104] Vaccine composition

[0105] The compositions disclosed herein can be used prophylactically or therapeutically. The present disclosure includes methods for preventing influenza virus infection. The method comprises administering to a subject a therapeutic or prophylactic amount of an immunogenic composition of the present disclosure. Preferably, the pharmaceutical composition is a vaccine composition that provides a protective effect. In some aspects, the protective effect may include improving symptoms associated with infection in a certain proportion of exposed people. For example, compared to untreated subjects, the composition can prevent or reduce one or more influenza symptoms selected from the following: fever fatigue, muscle pain, headache and sore throat.

[0106] The vaccine composition may include various excipients, pharmaceutically acceptable buffers, and the like. For example, the pharmaceutically acceptable buffer in the vaccine composition may include one or more of sodium phosphate, sodium chloride, histidine, arginine hydrochloride, and trehalose. Sodium phosphate may be present at about 10 mM to about 50 mM, about 15 mM to about 30 mM. In certain cases, there is about 25 mM sodium phosphate. Histidine may be present in a range of about 0.1% (w / v) to about 2.5% (w / v); for example, histidine may be present at about 0.1% (w / v), about 0.5% (w / v), about 0.7% (w / v), about 1% (w / v), about 1.5% (w / v), about 2% (w / v), or about 2.5% (w / v).

[0107] Sodium chloride may range from about 50 mM to about 300 mM. Typically, when present in the composition, sodium chloride is about 150 mM.

[0108] Arginine hydrochloride may be present at about 50 mM to about 200 mM, about 80 mM to about 150 mM, or about 100 mM to about 180 mM. In particular instances, arginine hydrochloride is present at about 100 mM.

[0109] Trehalose may be present in the range of about 1% to about 10%; for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%.

[0110] The pH range of the influenza vaccine composition is generally close to neutral and is maintained above pH 6.9 during purification and in the composition. For example, the pH of the buffer and composition can be about pH 7.2 to about pH 7.8, more preferably about 7.2 to about 7.5. In a specific aspect, the pH is about pH 7.5. Avoid pH values ​​below 6.9 because they can negatively affect the stability of the HA structure.

[0111] Adjuvant

[0112] In certain embodiments, the compositions disclosed herein can be combined with one or more adjuvants to enhance the immune response. In other embodiments, the compositions are prepared without an adjuvant and can therefore be administered as a non-adjuvanted composition.

[0113] The immunogenicity of a particular composition can be enhanced by using a non-specific stimulator of the immune response (called an adjuvant). An effective amount of an adjuvant has long been used to promote a general increase in immunity to an antigen (e.g., U.S. Patent No. 4,877,611). Immunization protocols have been using adjuvants to stimulate reactions for many years, and therefore, adjuvants are well known to those of ordinary skill in the art. Some adjuvants affect the way antigens are presented. For example, when protein antigens are precipitated with alum, immune responses are increased. Emulsification of antigens also prolongs the duration of antigen presentation. It is contemplated that any adjuvant described in "A Compendium of Vaccine Adjuvants and Excipients (Second Edition)" contained in Vogel et al. is within the scope of the present disclosure, and the reference is incorporated herein by reference in its entirety for all purposes.

[0114] Although various adjuvants may be included, HaSMaN is produced using an ISCOM matrix adjuvant that retains effective adjuvant activity after administration to a subject. Thus, in some aspects, no additional adjuvant may be added to the formulation. As described above, this formulation method allows for the preparation of a pre-filled syringe containing the vaccine without the need for mixing the antigen with the adjuvant at the bedside.

[0115] Saponin adjuvant for matrix

[0116] ISCOM matrices are prepared using saponin fractions. Saponins are glycosides derived from the bark of the Quillaja saponaria Molina tree. Typically, saponins are prepared using a multi-step purification process, thereby producing multiple fractions. As used herein, the term "saponin fraction from Quillaja saponaria Molina" is generally used to describe a semi-purified or defined saponin fraction of Quillaja saponaria or a substantially pure fraction thereof.

[0117] Saponin fraction

[0118] There are several methods suitable for producing saponin fractions. Fractions A, B and C are described in U.S. Patent No. 6,352,697, and the fractions can be prepared as follows. The lipophilic fraction from Quil A (a crude aqueous extract of Quillaja Molina) is separated by chromatography and eluted with 70% acetonitrile in water to recover the lipophilic fraction. This lipophilic fraction is then separated by semi-preparative HPLC, where a gradient of 25% to 60% acetonitrile in acidic water is used for elution. The fraction referred to herein as "fraction A" or "QH-A" is or corresponds to a fraction eluted with about 39% acetonitrile. The fraction referred to herein as "fraction B" or "QH-B" is or corresponds to a fraction eluted with about 47% acetonitrile. The fraction referred to herein as "fraction C" or "QH-C" is or corresponds to a fraction eluted with about 49% acetonitrile. Additional information about fraction purification can be found in U.S. Patent No. 5,057,540. When prepared as described herein, fractions A, B and C of Molina each represent a group or family of chemically closely related molecules with definable properties. Under the chromatographic conditions under which the fractions were obtained, batch-to-batch reproducibility was highly consistent in terms of elution profiles and biological activity.

[0119] Other saponin fractions have been described. Fractions B3, B4 and B4b are described in EP 0436620. Fractions QA1-QA22 are described in EP 03632279 B2, Q-VAC (Nor-Feed, AS Denmark), Quillaja saponaria Molina Spikoside (lsconova AB, Ultunaallén 2B, 75651 Uppsala, Sweden). Fractions QA-1, QA-2, QA-3, QA-4, QA-5, QA-6, QA-7, QA-8, QA-9, QA-10, QA-11, QA-12, QA-13, QA-14, QA-15, QA-16, QA-17, QA-18, QA-19, QA-20, QA-21 and QA-22, especially QA-7, QA-17, QA-18 and QA-21 of EP 03632279B2 can be used. The fractions are obtained as described in EP 03632279B2, in particular on page 6 and in Example 1 on pages 8 and 9.

[0120] The saponin fractions described herein and used to form adjuvants are generally substantially pure fractions; that is, the fractions are substantially free of contamination by other substances. In particular aspects, substantially pure saponin fractions may contain up to 40% by weight, up to 30% by weight, up to 25% by weight, up to 20% by weight, up to 15% by weight, up to 10% by weight, up to 7% by weight, up to 5% by weight, up to 2% by weight, up to 1% by weight, up to 0.5% by weight, or up to 0.1% by weight of other compounds, such as other saponins or other adjuvant substances.

[0121] Other saponin fractions, such as QS-7 and QS-21 fractions, are produced and used as described in U.S. Pat. No. 5,057,540; U.S. Pat. No. 6,231,859; U.S. Pat. No. 6,352,697; U.S. Pat. No. 6,524,584; U.S. Pat. No. 6,846,489; U.S. Pat. No. 7,776,343 and U.S. Pat. No. 8,173,141. These fractions can be used in the methods and compositions disclosed herein.

[0122] Matrix particles

[0123] The matrix particle adjuvants disclosed herein can be used to produce HaSMaN or as discrete particles due to their adjuvant properties. The ISCOM matrix comprises a lipid and at least one saponin fraction. The lipid is at least a sterol, such as cholesterol. In a particular aspect, the lipid is a phospholipid. The ISCOM matrix complex may also comprise one or more other immunomodulatory (adjuvant active) substances (not necessarily glycosides) and may be produced as described in EP0436620B1.

[0124] The saponin fraction may be Quillaja saponaria fraction A, fraction B or fraction C, a semi-purified preparation of Quillaja saponaria, a purified preparation of Quillaja saponaria or any purified sub-fraction, for example QA 1-21.

[0125] The matrix particles may comprise a mixture of saponin fractions, or the particles may be formed using only one saponin fraction. The compositions disclosed herein may comprise a plurality of particles, wherein each particle comprises only one saponin fraction. That is, certain compositions may comprise one or more different types of ISCOM matrices, wherein each individual particle comprises a saponin fraction from Molina, wherein the saponin fraction in one particle is different from the saponin fraction in the other composite particles.

[0126] In certain aspects, one type of saponin fraction or crude saponin fraction can be incorporated into one ISCOM matrix particle, while another type of substantially pure saponin fraction or crude saponin fraction can be incorporated into another ISCOM matrix particle. The composition or vaccine can comprise at least two types of complexes or particles, each type having one type of saponin incorporated into physically distinct particles.

[0127] In the composition, a mixture of ISCOM base particles may be used, wherein one saponin fraction Quillaja Molina and another saponin fraction Quillaja Molina are separately incorporated into different ISCOM base complex particles and / or ISCOM complex particles.

[0128] ISCOM matrices each having a saponin fraction may be present in the composition in any combination of wt%. In particular aspects, the composition may comprise 0.1% to 99.9% by weight, 5% to 95% by weight, 10% to 90% by weight, 15% to 85% by weight, 20% to 80% by weight, 25% to 75% by weight, 30% to 70% by weight, 35% to 65% by weight, 40% to 60% by weight, 45% to 55% by weight, 40 to 60% by weight, or 50% by weight of an ISCOM matrix containing a first saponin fraction, the remainder being composed of an ISCOM matrix containing a different saponin fraction. In some aspects, the remainder is one or more ISCOM matrix particles comprising only one saponin fraction. In other aspects, the ISCOM matrix particles may comprise more than one saponin fraction.

[0129] In a preferred composition, the saponin fraction in the first ISCOM matrix is ​​Fraction A ("Fraction A Matrix"), and the saponin fraction in the second ISCOM matrix or ISCOM composite particle is Fraction C ("Fraction C Matrix"). Thus, a preferred composition comprises a Fraction A matrix adjuvant and a Fraction C matrix adjuvant as adjuvants. The amount of each matrix in the composition may vary. For example, the amount of Fraction A matrix may be about 80% (w / w), about 85% (w / w), about 90% (w / w), about 92% (w / w), or about 95% (w / w), with the remainder being Fraction C matrix. An example of a suitable 85:15 combination of Fraction A matrix and Fraction C matrix (referred to herein as Matrix M or Matrix M1) may be provided as Matrix-M. TM Available from Novavax AB, Uppsala, Sweden.

[0130] In some aspects, matrix M can be used as an adjuvant in the compositions provided herein. In some aspects, matrix M can be used as the sole adjuvant in the nanoparticle influenza vaccine compositions provided herein.

[0131] In some embodiments, the amount of matrix M in each administered dose can be in the range of about 20 μg to about 140 μg; for example, about 20 μg, about 30 μg, about 40 μg, about 50 μg, about 60 μg, about 70 μg, about 75 μg, about 80 μg, about 90 μg, about 100 μg, about 110 μg, about 120 μg, about 130 μg or about 140 μg. In a specific aspect, the adjuvant can be present at about 50 μg to about 75 μg.

[0132] Other adjuvants

[0133] Exemplary adjuvants include complete Freund's adjuvant (containing nonspecific stimulators of the immune response that kill Mycobacterium tuberculosis), incomplete Freund's adjuvant, and aluminum hydroxide adjuvant. Other adjuvants include GMCSP, BCG, MDP compounds (such as thur-MDP and nor-MDP), CGP (MTP-PE), lipid A and monophosphoryl lipid A (MPL), MF-59, RIBI, which are in 2% squalene / The 80 emulsion contains three components extracted from bacteria, namely MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS). In other preferred aspects, alum is used, such as 2% Alhydrogel (Al(OH) 3 ). In some aspects, the adjuvant can be a paucilamellar lipid vesicle; for example, Novasomes are paucilamellar, non-phospholipid vesicles ranging from about 100 nm to about 500 nm. They include Brij 72, cholesterol, oleic acid, and squalene. Novasomes have been shown to be effective adjuvants (see U.S. Pat. Nos. 5,629,021, 6,387,373, and 4,911,928.

[0134] Administration and Dosage of Nanoparticle Vaccine Compositions

[0135] The compositions disclosed herein can be administered by systemic routes or mucosal routes or transdermal routes or directly administered to specific tissues. As used herein, the term "systemic administration" includes parenteral routes of administration. In particular, parenteral administration includes subcutaneous, intraperitoneal, intravenous, intramuscular or intrasternal injection. Typically, the composition is administered by intramuscular injection. In particular aspects, the composition can be administered by mucosal administration. As used herein, the term "mucosal administration" includes oral, intranasal, intravaginal, rectal and intratracheal.

[0136] The composition may be administered to a subject, typically a human, in need thereof.

[0137] Composition can be used in single dose regimen or multiple dose regimen.Multiple doses can be used in primary immunization regimen or in booster immunization regimen.In multiple dose regimen, various dosages can be given by the same or different approaches, for example, parenteral for the first time and mucosal reinforcement, mucosal for the first time and parenteral reinforcement, etc. In some aspects, subsequent booster doses are used in about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks or about 6 weeks after the previous dose.

[0138] In some embodiments, at least one of the four strains of the quadrivalent influenza vaccine composition is a type A strain. For example, a quadrivalent influenza vaccine may include three type A strains and one type B strain.

[0139] The total amount of influenza HA in the vaccine composition may be in the range of about 25 μg to about 200 μg, about 30 μg to about 150 μg, about 50 μg to about 100 μg, about 45 μg to about 180 μg, about 60 μg to about 190 μg, or about 100 μg to about 200 μg. In certain embodiments, the amount of influenza HA protein in the vaccine composition may be in the range of about 5 μg per strain to about 80 μg per strain, about 10 μg per strain to about 75 μg per strain, about 15 μg per strain to about 70 μg per strain, about 20 μg per strain to about 65 μg per strain, about 25 μg per strain to about 60 μg per strain, about 30 μg per strain to about 55 μg per strain, about 35 μg per strain to about 50 μg per strain, about 15 μg per strain to about 60 μg per strain. Advantageously, the composition exhibits stability for up to 9 to 12 months such that the remaining amount measured by SRID is a large percentage of the initial amount; for example, at least about 70%, at least about 75%, or at least about 80% of the initial amount.

[0140] In some embodiments, the disclosure provides a co-formulation (i.e., pre-filled syringe or pre-mix) strategy for nanoparticle influenza vaccine compositions. The typical influenza vaccine administration strategy currently utilized is a bedside mixed formulation. That is, the vaccine composition and adjuvant are stored separately and mixed before administration. Due to concerns about the stability of influenza antigens and their subsequent immunogenicity, pre-mixture, co-formulation or pre-filled syringe strategies for influenza vaccines are less common. The disclosure provides nanoparticle influenza vaccine compositions that can be pre-mixed and stored in advance. The disclosed vaccination strategies and formulations can improve vaccination efficiency, and can reduce the risk of bedside mixing errors while maintaining overall safety and immunogenicity.

[0141] Immunogenicity of nanoparticle influenza vaccines

[0142] The present disclosure provides methods for preventing influenza infection. The immunogenicity of the nanoparticle influenza vaccine disclosed herein can be determined using a suitable method, including performing an HAI assay or by measuring neutralizing antibodies. In some embodiments, the immunogenicity of the nanoparticle influenza vaccine can be compared with a commercially available influenza vaccine composition. As used herein, a "commercially available influenza vaccine composition" can be any influenza vaccine composition that can be used for medical purposes. For example, a commercially available influenza vaccine composition can be formulated into a trivalent or quadrivalent injection. In some aspects, the preparation for injection can include an inactivated form of the virus. In another example, a commercially available influenza vaccine composition can be formulated into a nasal spray. In some aspects, the preparation for the nasal spray can include an attenuated or weakened form of the virus.

[0143] The compositions disclosed herein provide a non-inferior immune response compared to other vaccines, particularly commercially available vaccines including Quadrivalent Afluria, Quadrivalent Fluarix, Quadrivalent FluLaval, Quadrivalent Fluzone, Quadrivalent Flucelvax, Intradermal Quadrivalent Fluzone, Afluria, Fluvirin, Fluad, High Dose Fluzone, Quadrivalent Flublok, Flublok, and Quadrivalent FluMist.

[0144] Advantageously, the compositions disclosed herein induce neutralizing antibodies that bind to strains that have drifted (i.e., have undergone slight mutations) relative to the sequences used by viruses within the same influenza subtype. In particular aspects, one, two, three, four, or all of the strains used in the composition induce neutralizing antibodies against one drift strain, against two drift strains, against three drift strains, against four drift strains, or against five drift strains. Without being bound by theory, it is believed that the presence of a matrix adjuvant in the composition promotes exposure of additional antigens, thereby providing expanded protection against drift strains. Similarly, the formation of HaSMaN after incubation of a matrix adjuvant with the HA protein of subtype A is believed to contribute to this process.

[0145] Importantly, HaSMaN is at least as immunogenic as detergent core nanoparticles. For example, as shown in Table 6 below, at day 35, at 25°C, the HAI titer of 1.5 μg premixed formulation against A / Michigan was about 538, while at 25°C, the HAI titer of 1.5 μg bedside mixed formulation against A / Michigan was about 453. In another example shown in Table 8, at day 35, at 25°C, the HAI titer of 1.5 μg premixed formulation against A / Hong Kong was about 538, while at -60°C, the HAI titer of 1.5 μg bedside mixed formulation against A / Michigan was about 494. The similar immunogenicity between the premixed formulation and the bedside mixed formulation further indicates that the premixed formulation is stable at room temperature. In addition, surprisingly, the premixed formulation of the present invention stored at room temperature can have similar immunogenicity compared to the bedside mixed formulation incubated at -60°C.

[0146] container

[0147] Various containers can be used to store and transport premixed preparations, including syringes and plastic ampoules for single administration. In some cases, blow-fill-seal manufacturing techniques or methods can be used to manufacture plastic ampoules. Typically, blow-fill-seal (BFS) manufacturing methods include extruding a plastic material (e.g., a resin) to form a parison, which is then placed in a mold and cut to size. The plastic is then inflated using a filling needle or mandrel, which in turn produces a hollow ampoule that substantially conforms to the shape of the mold. Once inflated, the desired volume of liquid can be injected into the ampoule, the filling needle or mandrel can be removed, and the ampoule can be sealed. Therefore, BFS can be an automated process that can be performed in a sterile environment without direct human intervention.

[0148] In some cases, the ability to aseptically manufacture sterile ampoules containing a desired liquid can make BFS-made ampoules particularly suitable for use in the pharmaceutical industry. However, BFS technology is not compatible with all pharmaceutical liquids, products, etc. For example, some known BFS manufacturing methods involve delivering the liquid or product into the ampoule while the plastic is still relatively hot, which may have an adverse effect on temperature-sensitive liquids and / or products such as vaccines, biologics, etc. However, advances in cooling BFS technology have increased the variety of applicable products, liquids, etc., thereby allowing some vaccines, biologics, and / or other temperature-sensitive drugs to be included in BFS ampoules.

[0149] In some cases, the size, shape, and / or configuration of a BFS ampoule may be based at least in part on the desired use and / or the desired drug liquid or dose that the ampoule is configured to contain. For example, some known BFS ampoules may include a pierced top, a twist-off top, a top with a male or female Luer, etc. Some known BFS ampoules may be sized and / or shaped based on the volume of the liquid or dose that is configured to be placed therein. In addition, some known BFS ampoules may be manufactured as a strip of multiple temporarily connected ampoules, which may improve manufacturing, packaging, and / or storage efficiency, etc.

[0150] All patents, patent applications, references, and journal articles cited herein are expressly incorporated by reference in their entirety for all purposes.

[0151] Example

[0152] Example 1: Purification of HA nanoparticles

[0153] HA protein from a single strain was expressed in Sf9 cells by baculovirus infection and grown for 48-96 hours before harvesting. HA protein was then harvested by detergent extraction and converted into detergent core nanoparticles during purification. In short, the TMAE column was pre-equilibrated with a buffer consisting of 25mM Tris, pH 8.0, 1.5M sodium chloride, 0.02% NP9. The sample was loaded at ≤90cm / h (residence time 24 minutes) and then washed with EQ buffer (25mM Tris, pH 8.0, 50mM sodium chloride or 81mM sodium chloride (for A strain, B strain, respectively), 0.02% NP-9). The purified sample was then eluted using 1.5CV EQ buffer.

[0154] For strain A, the product from the TMAE column was nanofiltered and then applied to a lentil lectin affinity chromatography column pre-equilibrated with 3 CV of buffer consisting of 25 mM Tris, 50 mM sodium chloride and 107 mM sodium chloride (for strain A and strain B, respectively), 0.02% (w / v) NP-9, pH 8.0 (flow rate: 150 cm / h). The sample was loaded at a residence time of 4 minutes. After loading, washing was performed with 3 CV of lentil lectin equilibration buffer. 2 CV of product was collected at 75 cm / h and at a residence time of 8 minutes and eluted with 25 mM sodium phosphate, pH 7.5, 200 mM sodium chloride, 500 mM methyl-α-D-mannopyranoside, 0.01% (w / v) PS80, pH 7.5.

[0155] For B strain, the TMAE column product was further purified using a Capto Blue column. The column was pre-equilibrated with 25 mM Tris, pH 8.0, 107 mM sodium chloride, 0.02% (w / v) NP-9, and then the TMAE product was loaded at a flow rate of 225 cm / h at a 4-minute residence time and collected with 2 CV of equilibrium buffer. The product from the Capto Blue column was nanofiltered and then applied to a lentil lectin affinity chromatography column, which was pre-equilibrated with 3 CV of buffer consisting of 25 mM Tris, 50 mM sodium chloride and 107 mM sodium chloride (for A strain and B strain, respectively), 0.02% (w / v) NP-9, pH 8.0 (flow rate: 150 cm / h). The sample was loaded at a 4-minute residence time. After loading, it was washed with 3 CV of lentil lectin equilibrium buffer. The product was collected for 2 CV at 75 cm / h and 8 min residence time, eluting with 25 mM sodium phosphate, pH 7.5, 200 mM sodium chloride, 500 mM methyl-a-D-mannopyranoside, 0.01% (w / v) PS80, pH 7.5.

[0156] The lentil lectin products of both strain A and strain B were concentrated to the target HA concentration and then buffer exchanged into the final drug substance formulation buffer. Concentration and buffer exchange were performed by ultrafiltration and diafiltration.

[0157] Example 2 - SDS PAGE analysis

[0158] To evaluate the stability of co-formulated Quad-NIV and ISCOM matrix adjuvant (matrix M) in pre-filled syringes and to understand the physical, chemical and biological properties of this vaccine, formulations were prepared as shown in Table 1 below. The vaccine compositions tested were distributed into three groups: (1) glass bedside vials containing HA antigen only; (2) pre-filled syringes (PFS) containing HA vaccine antigen co-formulated with matrix M1; and (3) PFS containing HA vaccine antigen. Glass vials for bedside mixtures and PFS were commercially available (Schott).

[0159] Table 1. Comparative study of bedside vial formulation and PFS formulation

[0160]

[0161] Figure 2Shown are images of reducing SDS-PAGE gels showing protein bands from bedside vials stored at 4°C; or prefilled syringes (PFS) with or without matrix M1 stored at 4°C. The banding patterns of proteins from the PFS were similar to those from the bedside vials at all time points tested, indicating that the proteins in the PFS were at least as stable as those in the bedside vials. Neither the presence nor the absence of matrix M altered the banding patterns between the PFS groups, nor after 6 or even 12 months of storage. The LD bedside vial group and the LD PFS group also had consistently similar banding patterns at 4°C, although the LD group had weaker banding patterns compared to its HD cohort at the same time points.

[0162] Figure 3 The banding patterns of proteins from PFS or bedside vials stored at 4° C. detected on non-reducing SDS-PAGE gels and reducing SDS-PAGE gels are shown. The results show that the protein banding patterns of all groups are similar even on non-reducing gels, further indicating that the proteins in PFS are similar to those in bedside vials at time zero.

[0163] In addition, non-reducing SDS-PAGE assay ( Figure 4 ) detected the protein stability of bedside vial groups stored at 4°C or 25°C for 3 months and PFS groups with or without matrix M. Some of the groups stored at higher temperatures (25°C) tended to have higher molecular weight bands compared to those stored at lower temperatures (e.g., 4°C). Stronger expression of proteins was also observed in the groups stored at lower temperatures. The results showed that even after long-term storage (such as storage for 3 months), the proteins in the PFS were at least as stable as those in the bedside vials.

[0164] These data show that the PFS co-formulation vaccine strategy is feasible in terms of stability. PFS co-formulation vaccines are feasible when stored at both 4°C and 25°C. The presence of matrix M1 and the duration of storage time (e.g., 3 months) do not negatively affect the stability of the vaccine formulation, confirming that the formation of HaSMaN does not interfere with the stability of the protein over a long period of time.

[0165] Example 3 - SRID Analysis

[0166] The stability of the protein was tested by SRID assay on various influenza A strains and influenza B strains formulated in the PFS group. The SRID assay was performed at 2-8°C (e.g., 4°C) at T=0, 2 weeks, 1 month, 3 months, 6 months, 9 months, and 12 months; and at 25°C at T=0, 2 weeks, 1 month, 3 months, and 6 months. Figure 5 and Figure 6It shows that at 2-8℃( Figure 5 ) and 25℃( Figure 6 ) for 120 μg / mL PFS formulations containing A / Hong Kong, A / Michigan, B / Brisbane, or B / Phuket with or without 100 μg / mL matrix M.

[0167] The SRID immunoreactivity results of PFS with matrix M were similar to or better than those of PFS without matrix M. The data showed that the overall trend of immunoreactivity over time when stored at 25°C was not significantly different from the results observed at 4°C. The absence of significant changes in vaccine immunoreactivity indicates that the Quad-NIV PFS formulation is stable at various temperatures for at least 6 months. The stability of the Quad-NIV PFS formulation at room temperature (25°C) advantageously provides a cost-effective vaccine strategy because the PFS formulation does not require long-term refrigeration or is limited to low-temperature processing. These data further show that it is a feasible method to mass-produce stable premixed vaccines (containing influenza antigens and matrix M) long before vaccination is performed in the clinic.

[0168] Example 4 - Comparative Immunogenicity Study of Quad-NIV and Commercial Influenza Vaccines in Ferrets

[0169] Ferret studies were conducted to compare the immunogenicity of various test vaccine strategies (bedside and co-formulation) with commercially available influenza vaccines.

[0170] Table 2. Comparative immunogenicity study of Quad-NIV and commercial vaccines in ferrets

[0171]

[0172] * - Each B strain dose is 90 μg; and each A strain dose is 60 μg.

[0173] # - Figure 7-10 The results depicted in correspond to day 42.

[0174] For the Quad-NIV preparation, A / Michigan / 45 / 2015-H1N1 strain, A / Hong Kong / 4801 / 2014-H3N2 strain, B / Brisbane / 60 / 2008 strain, B / Phuk et / 3073 / 2013 strain were used. For Fluzone HD (TIV; Sanofi Pasteur), A / Michigan / 45 / 2015-H1N1, A / HongKong / 4801 / 2014-H3N2 and B / Brisban e / 60 / 2008 were used. For Fluzone HD (QIV; Sanofi Pasteur), A / Michigan / 45 / 2015-H1N1, A / HongKong / 4801 / 2014-H3N2, B / Brisbane / 60 / 2008, and B / Phuket / 3073 / 2013 were used. For Fluad (TIV; Seqirus), A / Singapore / GP1908 / 2015 (similar to A / Michigan / 45 / 2015-H1N1), A / HongKong / 4801 / 2014-H3N2, and B / Brisbane / 60 / 2008 were used. For Flublok (QIV; Protein Sciences), A / Michigan / 45 / 2015-H1N1, A / Hong Kong / 4801 / 2014-H3N2, B / Brisbane / 60 / 2008, and B / Phuket / 3073 / 2013 were used. For Flucelvax (QIV; Seqirus), A / Singapore / GP1908 / 2015 (similar to A / Michigan / 45 / 2015-H1N1), A / Singapore / GP2050 / 2015 (similar to A / Hong Kong / 4801 / 2014-H3N2), B / Hong Kong / 259 / 2010 (similar to B / Brisbane / 60 / 08), and B / Utah / 9 / 2014 (similar to B / Phuket / 3073 / 2013) were used.

[0175] Ferrets were immunized on day 0 and day 21 of the study. Blood was drawn one day before the start of the study, on day 21 and day 42 of the study. HAI assays were performed using human red blood cells to assess the immunogenicity of the test formulations in ferrets. The experimental protocol is known in the art and has been discussed above. The following HAI reagents were used in the assay as reference influenza antigens in VLP form: (1) A / Michigan 45 / 15 (BV#001), (2) A / HongKong / 4801 / 14 (BV#1808), (3) B / Bris / 60 / 08 (BV#714), (4) B / Phuket / 3073 / 13 (BV#1659), (5) A / Switzerland / 9715293 / 13 (BV#1660), (6) A / Singapore / 2016 (BV#2165), (7) A / Texas / 50 / 2012 (BV#1324), (8) A / Victoria / 36 / 11 (BV#1577) and (9) A / Perth / 16 / 09. Methods of making influenza antigen VLPs are disclosed, for example, in US Patent Application No. 15 / 901,000, which is incorporated herein by reference in its entirety for all purposes.

[0176] All commercially available influenza vaccines had lower HAI titers compared to all Quad-NIV formulations in the presence of matrix M, regardless of dosing regimen or formulation type (i.e., bedside and co-formulation). Figure 7 (A / Hong Kong / 4801-2014), Figure 8 (A / Michiga n / 45 / 2015)、 Fig. 9 (B / Brisbane / 60 / 2008) and Fig.10 (B / Phuket / 3073 / 2013).

[0177] In summary, compared with commercial vaccines, for all tested influenza strains disclosed herein, Quad-NIV formulations can induce stronger immunogenic effects in ferrets, and the difference is more significant with the presence of matrix M in the formulation. Quad-NIV co-formulations (premixed) containing HaSMaN have similar immunogenicity compared to Quad-NIV bedside formulations. Among the co-formulation groups, the formulations stored at 4°C and at 25°C have very similar immunogenicity overall.

[0178] Example 5 - Evaluation of the immunogenicity of quadrivalent nanoparticle influenza vaccines stored at 4°C

[0179] Table 3 shows the mouse study design used to test the immunogenicity and stability of the premixed PFS vaccine formulation and the bedside mixed vaccine. The PFS formulation was stored at 4°C for 3 months. For the bedside mixed vaccine, the viral antigen was stored at -60°C for 3 months and mixed with the matrix M adjuvant just before administration.

[0180] Table 3. Quad-NIV formulations used for immunogenicity assessment

[0181]

[0182] Immunization was administered intramuscularly on days 0 and 21. Blood samples were collected for immunogenicity analysis on the day before the start of the study and again on day 42 of the study (or day 21 after the second immunization). Immunogenicity was determined based on the hemagglutination inhibition (HAI) reaction of the following influenza A and B strains: (1) A / Hong Kong / 4801 / 2014; (2) A / Michigan / 45 / 2015; (3) B / Brisbane / 60 / 2008 and (4) B / Phuket / 3073 / 2013. HAI was measured as described in Manual of Laboratory Diagnosis and Virological Surveillance of Influenza (World Health Organization 2011, accessed on 02 / 15 / 2018, at: www.who.int / influenza / gisrs_laboratory / manual_diagnosis_surveillance_influenza / en / , the disclosures of which are incorporated by reference).

[0183] Fig.11 The HAI titers of the formulations listed in Table 3 against four influenza strains for all HD groups at 3 months are shown. The results show that the premixed formulations stored at 4°C for 3 months have similar immunogenicity compared to the bedside mixed formulations stored at -60°C. The results also show that matrix M does not change the immunogenicity of the tested Quad-NIV formulations even when the premixed formulations are stored for a long time (such as 3 months).

[0184] Example 6 - Immunogenicity and long-term stability of premixed HA nanoparticles at bedside with matrix M at 25°C

[0185] Groups 1 to 14 were prepared as shown in Table 4 below. For the premixed group, HA nanoparticles and matrix M (85:15 w / w of fraction A matrix and fraction C matrix) were mixed and stored at 4°C for 6 months or 12 months, or at 25°C for 6 months before administration. For the bedside mixed group, HA was stored at 25°C or frozen at -60°C for 6 months, or frozen at -60°C for 12 months, and then mixed with matrix M just before administration to mice. HA protein nanoparticles contained HA from the following strains: A / Michigan H1N1, A / Hong Kong-H3N2 B / Brisbane, and B / Phuket.

[0186] Table 4. 6 and 12 months 3 Stability analysis of formulations

[0187]

[0188]

[0189] 1 High dose: 120 μg / mL / strain (480 μg HA total) + 100 μg / mL matrix.

[0190] 2 Low dose: 30 μg / mL / strain HA (120 μg HA total) + 100 μg / mL matrix.

[0191] 3 The 12-month stability study did not include samples stored at 25°C.

[0192] We measured HAI titers for each of the four strains. Figure 12-Figure 19 and Tables 5-12 show the HAI titers of the HD and LD groups against A / Hong Kong, A / Michigan, B / Brisbane, and B / Phuket strains at 6 months.

[0193] Table 5. A / Michigan H1N1-HAI titers on day 21

[0194]

[0195] Table 6. A / Michigan H1N1-HAI titers on day 35

[0196]

[0197]

[0198] Table 7. A / Hong Kong-H3N2 HAI titers on day 21

[0199]

[0200] Table 8. A / Hong Kong-H3N2 HAI titers on day 35

[0201]

[0202] Table 9. B / Brisbane HAI titers on day 21

[0203]

[0204] Table 10. B / Brisbane HAI titers on day 35

[0205]

[0206] Table 11. B / Phuket HAI titers on day 21

[0207]

[0208]

[0209] Table 12. B / Phuket HAI titers on day 35

[0210]

[0211] Figure 20-Figure 27 Shown are HAI titers against A / Hong Kong strain, A / Michigan strain, B / Brisbane strain, and B / Phuket strain for the high dose (HD) group and the low dose (LD) group at 12 months.

[0212] The data showed that bedside mixed vaccine and premixed vaccine produced similar immunogenicity in mice, as measured by HAI titers, even after 6 or 12 months of storage. The study also confirmed that the presence of matrix M favored a greater immune response. Matrix M did not alter the immunogenicity of the tested Quad-NIV formulations, even after long-term storage.

[0213] The premixed formulation at 25°C had similar immunogenicity compared to the bedside mixed formulation at 25°C. For example, on day 21, the 1.5 μg premix group stored at 4°C or 25°C elicited similar HAI titer responses against the A / Michigan strain (Table 5; GMT: 57 vs. 67, respectively). Combined with the stability of the premixed formulation stored for a long time at 25°C, the vaccine is particularly suitable for environments where cold chain storage may be limited or lacking.

[0214] Figure 12-Figure 27 The data shown in show that the high-dose groups, especially the premixed formulations, had higher HAI titer responses compared to the low-dose groups for all strains on day 21. Compared to the HAI titer responses observed on day 21, the HAI titer responses on day 35 were higher in all groups for all strains (e.g., A / Hong Kong HAI titer of 1.5 μg premixed formulation at 25°C: 22 vs. 538 on day 21 vs. day 35), while minimal differences were observed between the high-dose and low-dose groups on day 35.

[0215] The data confirmed that HaSMaN particles formed from type A HA proteins were at least as effective as HA nanoparticles with a detergent core in a non-HaSMaN format in inducing an immune response. The data showed that the interaction of HA nanoparticles with the matrix to form HaSMaN did not negatively affect the adjuvant effect of the matrix or the immunogenicity of the HA protein itself. Although HA nanoparticles appear to preserve the stability of the HA protein by being inserted into the detergent core, the data therefore suggest that HASMaN particles can provide some protection for the HA protein structure and can also have a positive impact on presentation to the immune system without the HA protein being embedded in a detergent core.

[0216] Example 7 - Sedimentation Coefficient Analysis

[0217] The tetravalent compositions previously tested for stability in mice in Example 6 were analyzed by measuring sedimentation velocity (SV) by analytical ultracentrifugation (AUC) to determine particle size. Fig.28 Results are shown for Quad-NIV alone at 120 μg / mL / strain (QIV alone) at 0, 3, 6, 9, and 12 months at 4° C. or QIV + 100 μg / mL M1 at 3, 6, 9, and 12 months at 4° C. The data show that the amount of rapidly settling species (i.e., structures that appear at higher values) increases over time, indicating that HA particles form HaSMaN over time. Fig.29 Results are shown for Quad-NIV alone or with 100 μg / mL M1 at 0, 3, or 6 months at 25°C and demonstrate that formation of HaSMaN is faster at higher temperatures.

[0218] Example 8 - Transmission Electron Microscopy (TEM) Analysis of HaSMaN Formation

[0219] As mentioned earlier, Figure 1Different structures of HA nanoparticles (Flu), matrix M and HaSMaN (Flu-matrix interaction) observed using TEM are shown. As expected, HA nanoparticles are free of HaSMaN formation. Specifically, HA nanoparticles exhibit a detergent core structure with HA protein surrounding the core. Matrix M exhibits a cage-like structure that contains a saponin fraction (either fraction A or fraction C, but not both), phospholipids and cholesterol. In the HaSMaN structure, the HA glycoprotein is attached to the cage-like matrix particles with the HA head portion extending outward.

[0220] Next, we observed the time course of the various samples described below under TEM to visually assess the formation of HaSMaN under different conditions. Table 13 below shows the sample conditions and time points for testing the nanoparticle structure of the premixed Quad-NIV formulations (high dose HD and low dose LD) by using TEM. The mark "X" indicates the sample conditions available at each time point. In addition, the LD formulation ( Fig.35 ).

[0221] Figure 30-Figure 34 Representative images of HaSMaN structures are shown. The data show that the formation of HaSMaN is visible at about 4 hours of incubation; and there is no evidence of HaSMaN formation before 4 hours ( Fig.30 and Fig.31 By 24 hours, HaSMaN was formed at both low temperature (4°C) and high temperature (25°C); however, no HaSMaN formation was observed in high-dose Quad-NIV at 4°C. High-dose Quad-NIV showed that by 48 hours ( Fig.33 ) and on day 7 ( Fig.34 ) formed HaSMaN. The data also showed that by 1 month, the HaSMaN structure was still retained in the LD group and was more prominent at higher temperatures, especially at 37°C ( Fig.35 ).

[0222] Table 13. TEM images: study design

[0223]

[0224]

[0225] Matrix M concentration: 100 μg / mL in all samples

[0226] These data show that the formation of HaSMaN requires incubation of HA nanoparticles with the matrix M1 for more than about 4 hours. All conditions showed the formation of HASMaN within 48 hours. The formed HaSMaN particles are stable in the long term. Under the conditions of higher temperature and lower dose of Quad-NIV, the formation of HaSMaN developed earlier.

[0227] This suggests that pre-filled syringe formulations containing HaSMaN can be stored at room temperature or higher (eg, 37°C), which would avoid the costs of cold storage and shipping, and prevent potential inconsistencies in mixing and preparing vaccines just before vaccination in the clinic.

[0228] Example 9

[0229] Differential Scanning Calorimetry (DSC) Curve of Quad-NIV

[0230] The stability of the Quad-NIV formulations described in Examples 6 and 7 was tested by DSC assay. 120 μg / mL / strain Quad-NIV with 100 μg / mL matrix M1 was prepared in a buffer containing 25 mM NaPi, 150 mM NaCl, 100 mM arginine, 5% trehalose, 0.03% PS80, pH 7.5. All Quad-NIV samples were incubated at 4°C for 3 months, 6 months, or 12 months; or at 25°C for 3 months or 6 months. DSC scans were performed from 4°C to 120°C at 1°C per minute, and the molar heat capacity (Cp: kJ / mol.k) of Quad-NIV was measured.

[0231] Fig.36 The results showed that the preparations incubated at 25°C showed a significant decrease in melting point temperature (T m ) has a smaller shift and a wider peak width. Table 14 shows the T values ​​of 120 μg / mL / strain Quad-NIV+matrix M1 formulations incubated at 25°C m This is lower than at 4°C at 3 months (59.6°C vs. 60.4°C) and 6 months (59.0°C vs. 60.4°C). Table 14 also shows that the change in Hcal required to reach peak temperature for the formulation at 25°C is less than that at 4°C, further indicating that this formulation has relatively less HaSMaN complexes.

[0232] DSC curve and T m The results showed that the formation of HaSMaN increased the T of HA protein. m The increase was about 0.5°C to about 1°C, thereby improving the thermal stability of the HA protein in HaSMaN compared to the detergent core nanoparticles.

[0233] Table 14. DSC curves of high-dose and low-dose Quad-NIV formulations

[0234]

[0235] Example 10 - Type A strains instead of Type B strains for HaSMaN

[0236] We investigated the ability of HA glycoproteins from different influenza strains to form HaSMaNs. Detergent core nanoparticles were prepared and purified as described in Example 1 for the following strains: Type A: A / Hunan, A / Guangdong (both H7N9 subtypes) and A / Panama and A / Hong Kong / 4801 / 2014 (both H3N2 subtypes) and Type B: B / Brisbane / 60 / 2008.

[0237] The nanoparticles were mixed with matrix M (85:15 w / w of fraction A matrix and fraction C matrix) for up to 4 weeks. The final concentration of HA was 120 μg / mL (60 μg of each A strain + 60 μg of each B strain in 0.5 mL). Free nHA was measured. The results are shown in Fig.37 As shown in . The reduction of free nHA species corresponds to the formation of HaSMaN.

[0238] These data indicate that the type A strain HA glycoprotein forms HaSMaN, while the type B strain HA glycoprotein does not form HaSMaN. We also tested HA glycoprotein fusion proteins with foldon at the C-terminus. The presence of foldon prevented the formation of HaSMaN, indicating that the C-terminus must be free to allow the HA glycoprotein to form HaSMaN.

Claims

1. A multivalent immunogenic influenza composition comprising (a) a detergent core nanoparticle, wherein the detergent core nanoparticle comprises a recombinant influenza hemagglutinin (HA) glycoprotein from an influenza B strain; and (b) hemagglutinin saponin matrix nanoparticles (HaSMaN), wherein the HaSMaN are formed by incubating the detergent core nanoparticles with an ISCOM matrix adjuvant for at least 4 hours; and (c) a pharmaceutically acceptable buffer, wherein the ISCOM matrix adjuvant comprises a fraction A matrix and a fraction C matrix, and The detergent is selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65 and polysorbate 80.

2. The multivalent immunogenic influenza composition of claim 1, further comprising one or more detergent core nanoparticles and one or more HaSMaNs, wherein each detergent core nanoparticle comprises a type B strain HA glycoprotein, and each HaSMaN comprises a type A strain HA glycoprotein, and wherein each HA glycoprotein is from a different influenza strain.

3. The multivalent immunogenic influenza composition of claim 1, wherein each of the nanoparticles is a trypsin-resistant nanoparticle.

4. The multivalent immunogenic influenza composition of claim 1, wherein the Fraction A matrix and the Fraction C matrix are present in a ratio of 85:15; w / w.

5. The multivalent immunogenic influenza composition of claim 1, wherein the detergent is PS80.

6. The multivalent immunogenic influenza composition of claim 5, wherein the PS80 detergent is present at 0.03% w / v to 0.5% w / v.

7. The multivalent immunogenic influenza composition of claim 5, wherein the PS80 detergent is present at 0.04% w / v.

8. The multivalent immunogenic influenza composition of claim 1, wherein the influenza strain is a subtype selected from the group consisting of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and H18.

9. The multivalent immunogenic influenza composition of claim 1, wherein the pharmaceutically acceptable buffer comprises (i) 25 mM sodium phosphate; (ii) 150 mM sodium chloride; (iii) 100 mM arginine hydrochloride; (iv) 5% trehalose; wherein the pH of the composition is 7.

5.

10. A pre-filled syringe or a blow-fill seal container comprising the multivalent immunogenic influenza composition of claim 1.

11. The prefilled syringe or blow-fill seal container of claim 10, wherein the multivalent immunogenic influenza composition is stable for at least 12 months.

12. The prefilled syringe or blow-fill seal container of claim 11, wherein the multivalent immunogenic influenza composition is stable at 25°C.

13. Use of a multivalent immunogenic influenza composition according to any one of claims 1 to 9 in the preparation of a medicament for stimulating an immune response against influenza.

14. The use of claim 13, wherein the multivalent immunogenic influenza composition is administered intramuscularly.

15. A composition comprising: (i) a first HaSMaN (hemagglutinin saponin matrix nanoparticle), wherein the first HaSMaN is formed by incubating a first detergent core nanoparticle containing a first recombinant influenza HA glycoprotein trimer from an influenza A strain with a first ISCOM matrix particle comprising a Fraction A matrix and not comprising a Fraction C matrix for at least 4 hours, and wherein the first recombinant influenza HA glycoprotein trimer has an HA glycoprotein tail region and an HA glycoprotein head region, the HA glycoprotein tail region is associated with the first ISCOM matrix particle, and the HA glycoprotein head region extends distally from the first ISCOM matrix particle; (ii) a second HaSMaN, wherein the second HaSMaN is formed by incubating a second detergent core nanoparticle containing a second recombinant influenza HA glycoprotein trimer from an influenza A strain with a second ISCOM matrix particle comprising a Fraction C matrix and not comprising a Fraction A matrix for at least 4 hours, and wherein the second recombinant influenza HA glycoprotein trimer has an HA glycoprotein tail region and an HA glycoprotein head region, the HA glycoprotein tail region is associated with the second ISCOM matrix particle, and the HA glycoprotein head region extends distally from the second ISCOM matrix particle; and (iii) a pharmaceutically acceptable buffer or carrier; wherein each of the first and second HA glycoproteins comprises a transmembrane domain, and The first detergent and the second detergent are selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65 and polysorbate 80.

16. The composition of claim 15, wherein the influenza A strain is a subtype selected from the group consisting of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17 and H18.

17. The multivalent immunogenic influenza composition of claim 1, wherein the adjuvant is present at 50 µg to 75 µg per dose.

18. The multivalent immunogenic influenza composition of claim 1, wherein the recombinant influenza HA glycoprotein from the influenza type B strain and the recombinant influenza HA glycoprotein from the influenza type A strain are both present in an amount of 60 μg.

19. The multivalent immunogenic influenza composition of claim 1, wherein the recombinant influenza HA glycoprotein from the influenza type B strain and the recombinant influenza HA glycoprotein from the influenza type A strain are both wild-type influenza HA glycoproteins.

20. The multivalent immunogenic influenza composition of claim 1, wherein the recombinant influenza HA glycoprotein from the influenza type B strain and the recombinant influenza HA glycoprotein from the influenza type A strain are both expressed in a host cell.

21. The multivalent immunogenic influenza composition of claim 20, wherein the host cell is an insect Sf9 cell.

22. The multivalent immunogenic influenza composition of claim 20, wherein the recombinant influenza HA glycoprotein from the influenza type B strain and the recombinant influenza HA glycoprotein from the influenza type A strain are both expressed using baculovirus.

23. A method for preparing HaSMaN, comprising mixing detergent core nanoparticles with a saponin-based matrix adjuvant for at least 4 hours, wherein the detergent core nanoparticles comprise HA glycoprotein from an influenza A strain and a non-ionic detergent, wherein the saponin-based matrix adjuvant is an ISCOM matrix adjuvant, wherein the ISCOM matrix adjuvant comprises a fraction A matrix and a fraction C matrix, and wherein the detergent is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, and polysorbate 80.

24. The method of claim 23, wherein the Fraction A matrix and the Fraction C matrix are present in a ratio of 85:15; w / w.

25. The method of claim 23, wherein the detergent is PS80.

26. The method of claim 23, comprising combining the detergent core nanoparticles with the saponin-based matrix adjuvant at a temperature of 25°C for at least 24 hours.

27. The method of claim 23, wherein the influenza A strain is selected from the group consisting of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and H18.

28. The multivalent immunogenic influenza composition of claim 1, further comprising one or more detergent core nanoparticles and one or more HaSMaNs, wherein each detergent core nanoparticle comprises a type B strain HA glycoprotein, and each HaSMaN comprises a type A strain HA glycoprotein, and wherein each HA glycoprotein is from at least four different influenza strains.

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