Multivalent influenza nanoparticle vaccines

Abstract

The present invention discloses multivalent nanoparticle vaccine compositions suitable for use in influenza vaccines. The nanoparticles comprise an effective amount of an influenza glycoprotein that provides an enhanced immune response as compared to commercially available influenza vaccine compositions. The invention also provides vaccine formulation strategies that are cost-effective and convenient for clinical use. Also disclosed are methods of administering the nanoparticle vaccine compositions to a subject.

Description

[0001] This divisional application of the present invention is based on the patent application for invention titled "Multivalent Influenza Nanoparticle Vaccine" with an application date of March 19, 2019, application number 201980032462.2 (International Application Number PCT / US2019 / 022930).

[0002] Cross - reference to related applications

[0003] 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 content of each of the said patent applications is incorporated herein by reference in its entirety for all purposes.

[0004] This application also incorporates by reference in its entirety for all purposes the content 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. Technical field

[0005] The present disclosure generally relates to influenza vaccine compositions that can be used to stimulate an immune response against influenza. Background art

[0006] The substantial global burden imposed by seasonal influenza is well - documented. In the United States alone, approximately 140,000 to 10,000 hospitalizations and 12,000 to 56,000 deaths each year are attributable to influenza, with a relatively large proportion among the elderly, accounting for 62% of hospitalizations and 72% of deaths. Seasonal influenza vaccination has been a mainstay of prevention efforts and has been generally recommended in the United States since 2010.

[0007] However, recent developments, including the dominant and severe 2017 - 2018 A(H3N2) in the U.S. influenza season, illustrate that the vaccine efficacy is poor, at least in Australia, Canada, and the United States. Other issues, such as antigenic mismatch associated with egg - based influenza vaccines and the ongoing challenge of antigenic drift, also mean that there is a need to provide more effective vaccination strategies.

[0008] Accordingly, there is a continuing interest in producing vaccines against influenza viruses, and there remains a need to produce effective vaccines, particularly using methods that are free of recombinant egg proteins. Summary of the invention

[0009] The present disclosure provides multivalent influenza compositions. The compositions stimulate an immune response against multiple influenza strains. Advantageously, the immune response can include broadly neutralizing antibodies against variants of strains different from those used to prepare the compositions. Influenza mutations result in "drift strains", and the disclosed compositions provide protection against "drift strains". In addition, the compositions exhibit excellent stability, can be stored for long periods, and can be produced in pre-filled syringes ready for administration. In some aspects, the pre-filled syringe (PFS) may already contain an adjuvant and influenza antigen and can be stored for long periods. The compositions disclosed herein do not require refrigeration (2 - 8 °C) and exhibit good stability at higher temperatures (e.g., room temperature, about 25 °C). Thus, the compositions disclosed herein provide excellent convenience as well as an excellent immune response.

[0010] In certain aspects, prior to administration to a subject, nanoparticles containing influenza antigen are co-formulated into a vaccine composition by mixing with an ISCOM matrix adjuvant (also referred to herein as "matrix") for a period of time to form HaSMaN (hemagglutinin saponin matrix nanoparticles). Compositions containing HaSMaN exhibit good stability and immunogenicity and are thus well-suited for packaging, for example, in pre-filled syringes. Indeed, 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 the ISCOM matrix adjuvant involved mixing the vaccine composition with the adjuvant at the bedside immediately prior to administration. In certain aspects, the compositions disclosed herein eliminate this requirement and thus provide an improved method for inducing an immune response. It has surprisingly been found that the formation of HaSMaN varies according to the influenza type. When the composition contains hemagglutinin (HA) protein from an influenza A strain, HaSMaN is generally formed, but not when the HA protein is from an influenza B strain.

[0011] In some embodiments, the ISCOM matrix adjuvant in the multivalent influenza composition is Matrix M, which is a combination of two types of matrices, the first type being Matrix A containing saponin fraction A (also referred to herein as fraction A matrix), and the second type being Matrix C containing saponin fraction C (also referred to herein as fraction C matrix). In certain aspects, Matrix M may comprise at least about 85% (w / w) fraction A matrix, with the remainder being fraction C matrix. Unless otherwise stated, Matrix M (also referred to herein as Matrix M1) used herein comprises fraction A matrix and fraction C matrix in a ratio of 85:15 (w / w).

[0012] In some embodiments, each influenza HA protein in the tetravalent 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 Matrix M adjuvant, but the subtype B strain is not complexed with Matrix M adjuvant.

[0013] While the present disclosure contemplates multivalent compositions, particularly for seasonal vaccines, monovalent vaccine compositions can also be produced using HaSMaN for vaccination against, for example, pandemic influenza strains that emerge from time to time.

[0014] Stability, especially stability at room temperature, is a valuable property as 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 extended periods, 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 Matrix M adjuvant) means that the vaccine composition is suitable for delivery to the clinical setting in a ready-to-use form; for example, a pre-filled syringe formulation.

[0015] Specifically, the present invention includes but is not limited to the following:

[0016] 1. A multivalent immunogenic influenza composition comprising

[0017] (a) a detergent core nanoparticle, wherein the detergent core nanoparticle comprises a recombinant influenza hemagglutinin (HA) glycoprotein from an influenza B strain; and

[0018] (b) a hemagglutinin saponin matrix nanoparticle (HaSMaN), wherein the HaSMaN comprises a recombinant influenza HA glycoprotein from an influenza A strain and an ISCOM matrix adjuvant; and

[0019] (c) a pharmaceutically acceptable buffer.

[0020] 2. The composition according to item 1, further comprising one or more detergent core nanoparticles and / or one or more HaSMaNs.

[0021] 3. The composition according to item 1, wherein each of the nanoparticles is an antitrypsin nanoparticle.

[0022] 4. The composition according to item 1, wherein the ISCOM matrix adjuvant is Matrix M.

[0023] 5. The composition according to item 6, wherein the Matrix M comprises fraction A matrix and fraction C matrix (85:15; w / w).

[0024] 6. The composition according to item 1, wherein the detergent is PS-80.

[0025] 7. The composition according to item 6, wherein the PS-80 detergent is present in an amount of about 0.03% to about 0.5%.

[0026] 8. The composition according to item 6, wherein the PS-80 detergent is present in an amount of about 0.04%.

[0027] 9. The composition according to item 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.

[0028] 10. The composition according to item 1, wherein the pharmaceutically acceptable buffer comprises (i) about 25 mM sodium phosphate; (ii) about 150 mM sodium chloride; (iii) about 100 mM arginine hydrochloride; (iv) about 5% trehalose; wherein the pH of the composition is about 7.5.

[0029] 11. A prefilled syringe or blow-fill-seal container comprising the composition according to item 1.

[0030] 12. The prefilled syringe or blow-fill-seal container according to item 10, wherein the composition is stable for at least 12 months.

[0031] 13. The prefilled syringe or blow-fill-seal container according to item 11, wherein the composition is stable at 25°C.

[0032] 14. A method of stimulating an immune response against influenza, comprising administering a multivalent influenza composition according to any one of items 1-10.

[0033] 15. The method according to item 14, wherein the composition is administered intramuscularly.

[0034] 16. A HaSMaN (hemagglutinin saponin matrix nanoparticle) comprising ISCOM matrix particles and recombinant influenza HA glycoprotein trimers, wherein the HA glycoprotein is from an influenza A strain, wherein the HA glycoprotein tail region is associated with the particles, and wherein the HA glycoprotein head extends distally from the particles.

[0035] 17. The HaSMaN according to item 16, 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.

[0036] 18. A composition comprising the HaSMaN as described in item 16 and a pharmaceutically acceptable buffer or carrier.

[0037] 19. The composition as described in item 1, wherein the adjuvant is present in an amount of about 50 μg to about 75 μg per dose.

[0038] 20. The composition as described in item 1, wherein both the recombinant influenza HA glycoprotein from the influenza B strain and the recombinant influenza HA glycoprotein from the influenza A strain are present in an amount of about 60 μg.

[0039] 21. The composition as described in item 1, wherein both the recombinant influenza HA glycoprotein from the influenza B strain and the recombinant influenza HA glycoprotein from the influenza A strain are wild-type influenza HA glycoproteins.

[0040] 22. The composition as described in item 1, wherein both the recombinant influenza HA glycoprotein from the influenza B strain and the recombinant influenza HA glycoprotein from the influenza A strain are expressed in a host cell.

[0041] 23. The composition as described in item 22, wherein the host cell is an insect Sf9 cell.

[0042] 24. The composition as described in item 22, wherein both the recombinant influenza HA glycoprotein from the influenza B strain and the recombinant influenza HA glycoprotein from the influenza A strain are expressed using baculovirus.

[0043] 25. A method for preparing HaSMaN, which comprises mixing detergent core nanoparticles with a saponin-based matrix adjuvant.

[0044] 26. The method as described in item 25, wherein the saponin-based matrix adjuvant is an ISCOM matrix adjuvant.

[0045] 27. The method as described in item 26, wherein the ISCOM matrix adjuvant is Matrix M.

[0046] 28. The method as described in item 27, wherein Matrix M comprises Fraction A matrix and Fraction C matrix (85:15; w / w).

[0047] 29. The method as described in item 25, wherein the detergent is PS-80.

[0048] 30. The method as described in item 25, which comprises incubating the detergent core nanoparticles with the saponin-based matrix adjuvant for at least about 4 hours.

[0049] 31. The method according to item 30, which comprises incubating the detergent core nanoparticles with the saponin-based matrix adjuvant at a temperature of about 25 °C for at least about 24 hours.

[0050] 32. The method according to item 25, wherein the detergent core nanoparticles comprise recombinant influenza hemagglutinin (HA) glycoprotein from an influenza A strain.

[0051] 33. The method according to item 32, 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 Examples of transmission electron microscope (TEM) images showing the influenza HA detergent core nanoparticles alone (left), the matrix M alone (middle), and the combination of HA nanoparticles and matrix M (which forms hemagglutinin saponin matrix nanoparticles (HaSMaN)) (right).

[0053] Figure 2 Reductive SDS-PAGE gel images of influenza antigens of high-dose 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 4 °C at T = 0, 6 months, and 12 months are shown. The abbreviations in the figure are PFS: prefilled syringe; M1: matrix M.

[0054] Figure 3 Non-reductive and reductive SDS-PAGE gel images of influenza antigens of high-dose bedside vial formulations (240 μg / mL / strain) and PFS formulations (120 μg / mL / strain) with or without matrix M at 4 °C at time = 0 are shown.

[0055] Figure 4 Non-reductive SDS-PAGE gel images of influenza antigens of high-dose and low-dose bedside vial formulations (240 μg / mL / strain and 60 μg / mL / strain) and PFS formulations (120 μg / mL / strain and 30 μg / mL / strain) with or without matrix M at 4 °C and 25 °C at the 3-month time point are shown.

[0056] Figure 5Shows the results of single radial immunodiffusion assay (SRID) of doses of 120 μg / mL / strain of PFS formulation with or without matrix M at 2 - 8°C for 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. Abbreviations in the figure are MXM: matrix M; HK: Hong Kong; MI: Michigan; Bris: Brisbane; Phu: Phuket.

[0057] Figure 6 Shows the SRID results of doses of 120 μg / mL / strain of PFS formulation with or without matrix M at 25°C for A / Hong Kong, A / Michigan, B / Brisbane, and B / Phuket strains at T = 0, 2 weeks, 1 month, 3 months, and 6 months.

[0058] Figure 7 Shows the hemagglutination inhibition antibody responses in mice following administration of Quad - NIV co - formulation or bedside mixed formulation with or without matrix M compared to administration of a 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.

[0059] Figure 8 Shows the hemagglutination inhibition antibody responses in mice following administration of Quad - NIV co - formulation or bedside mixed formulation with or without matrix M against A / Michigan / 45 / 2015 compared to administration of a commercial influenza vaccine.

[0060] Figure 9 Shows the hemagglutination inhibition antibody responses in mice following administration of Quad - NIV co - formulation or bedside mixed formulation with or without matrix M against B / Brisbane / 60 / 2008 compared to administration of a commercial influenza vaccine.

[0061] Figure 10 Shows the hemagglutination inhibition antibody responses in mice following administration of Quad - NIV co - formulation or bedside mixed formulation with or without matrix M against B / Phuket / 3073 / 2013 compared to administration of a commercial influenza vaccine.

[0062] Figure 11Shows the hemagglutination inhibition antibody responses in mice following administration of the Quad-NIV premix formulation or the point-of-care mix formulation with Matrix M against A / Hong Kong / 4801 / 2014, A / Michigan / 45 / 2015, B / Brisbane / 60 / 2008, and B / Phuket / 3073 / 2013. The PFS formulation was stored at 4 °C for 3 months. For the point-of-care mixed vaccine, the viral antigens were stored at -60 °C for 3 months and mixed with Matrix M adjuvant just prior to administration. Antibody responses are expressed as HAI GMT titers. GMT results use the 95% control lower limit and 95% control upper limit. Abbreviations in the figure are HAI: hemagglutination inhibition; HA: hemagglutinin; GMT: geometric mean titer; LCL: control lower limit; UCL: control upper limit.

[0063] Figure 12 Shows the hemagglutination inhibition antibody responses against the A / Michigan strain in mice at day 21 following administration of the Quad-NIV co-formulation or the point-of-care mix formulation with or without Matrix M (100 μg / mL). The premixed Quad-NIV co-formulation was stored at the specified temperature for 6 months.

[0064] Figure 13 Shows the hemagglutination inhibition antibody responses against the A / Michigan strain in mice at day 35 following administration of the Quad-NIV co-formulation or the point-of-care mix formulation with or without Matrix M (100 μg / mL). The premixed Quad-NIV co-formulation was stored at the specified temperature for 6 months.

[0065] Figure 14 Shows the hemagglutination inhibition antibody responses against A / Hong Kong / 4801 / 2014 in mice at day 21 following administration of the Quad-NIV co-formulation or the point-of-care mix formulation with or without Matrix M (100 μg / mL). The premixed Quad-NIV co-formulation was stored at the specified temperature for 6 months.

[0066] Figure 15 Shows the hemagglutination inhibition antibody responses against the A / Hong Kong strain in mice at day 35 following administration of the Quad-NIV co-formulation or the point-of-care mix formulation with or without Matrix M (100 μg / mL). The premixed Quad-NIV co-formulation was stored at the specified temperature for 6 months.

[0067] Figure 16 Shows the hemagglutination inhibition antibody responses against the B / Brisbane strain in mice at day 21 following administration of the Quad-NIV co-formulation or the point-of-care mix formulation with or without Matrix M (100 μg / mL). The premixed Quad-NIV co-formulation was stored at the specified temperature for 6 months.

[0068] Figure 17 Shows the hemagglutination inhibition antibody responses against the B / Brisbane strain in mice administered the Quad-NIV co-formulation or the bedside mixed formulation with or without Matrix M (100 μg / mL) at day 35. The antibody responses are expressed as HAI titers. The pre-mixed Quad-NIV co-formulation was stored at the specified temperature for 6 months.

[0069] Figure 18 Shows the hemagglutination inhibition antibody responses against the B / Phuket strain in mice administered the Quad-NIV co-formulation or the bedside mixed formulation with or without Matrix M (100 μg / mL) at day 21. The pre-mixed Quad-NIV co-formulation was stored at the specified temperature for 6 months.

[0070] Figure 19 Shows the hemagglutination inhibition antibody responses against the B / Phuket strain in mice administered the Quad-NIV co-formulation or the bedside mixed formulation with or without Matrix M (100 μg / mL) at day 35. The antibody responses are expressed as HAI titers. The pre-mixed Quad-NIV co-formulation was stored at the specified temperature for 6 months.

[0071] Figure 20 Shows the hemagglutination inhibition antibody responses against A / Hong Kong / 4801 / 2014 in mice administered the Quad-NIV co-formulation or the bedside mixed formulation with or without Matrix M (100 μg / mL) at day 21. The pre-mixed Quad-NIV co-formulation was stored at 4°C for 12 months.

[0072] Figure 21 Shows the hemagglutination inhibition antibody responses against the A / Hong Kong strain in mice administered the Quad-NIV co-formulation or the bedside mixed formulation with or without Matrix M (100 μg / mL) at day 35. The pre-mixed Quad-NIV co-formulation was stored at 4°C for 12 months.

[0073] Figure 22 Shows the hemagglutination inhibition antibody responses against the A / Michigan strain in mice administered the Quad-NIV co-formulation or the bedside mixed formulation with or without Matrix M (100 μg / mL) at day 21. The pre-mixed Quad-NIV co-formulation was stored at 4°C for 12 months.

[0074] Figure 23Shows the hemagglutination inhibition antibody response against the A / Michigan strain in mice administered the Quad-NIV co-formulation or the bedside mixed formulation with or without Matrix M (100 μg / mL) at 35 days. The pre-mixed Quad-NIV co-formulation was stored at 4 °C for 12 months.

[0075] Figure 24 Shows the hemagglutination inhibition antibody response against the B / Brisbane strain in mice administered the Quad-NIV co-formulation or the bedside mixed formulation with or without Matrix M (100 μg / mL) at 21 days. The pre-mixed Quad-NIV co-formulation was stored at 4 °C for 12 months.

[0076] Figure 25 Shows the hemagglutination inhibition antibody response against the B / Brisbane strain in mice administered the Quad-NIV co-formulation or the bedside mixed formulation with or without Matrix M (100 μg / mL) at 35 days. The antibody response is expressed as the HAI titer. The pre-mixed Quad-NIV co-formulation was stored at 4 °C for 12 months.

[0077] Figure 26 Shows the hemagglutination inhibition antibody response against the B / Phuket strain in mice administered the Quad-NIV co-formulation or the bedside mixed formulation with or without Matrix M (100 μg / mL) at 21 days. The pre-mixed Quad-NIV co-formulation was stored at 4 °C for 12 months.

[0078] Figure 27 Shows the hemagglutination inhibition antibody response against the B / Phuket strain in mice administered the Quad-NIV co-formulation or the bedside mixed formulation with or without Matrix M (100 μg / mL) at 35 days. The antibody response is expressed as the HAI titer. The pre-mixed Quad-NIV co-formulation was stored at 4 °C for 12 months.

[0079] Figure 28 Shows the AUC sedimentation curves of the Quad-NIV PFS formulation (120 μg / mL) at 4 °C at T = 0, 3 months, 6 months, 9 months or 12 months and the Quad-NIV PFS formulation with 100 μg / mL Matrix M1 at 4 °C at T = 3 months, 6 months, 9 months or 12 months.

[0080] Figure 29 Shows the analytical ultracentrifugation (AUC) sedimentation curves of the Quad-NIV PFS formulation with or without Matrix M1 (100 μg / mL) at the start of the study (T0), 1 month, 3 months and 6 months. The formulations were incubated at 25 °C.

[0081] Figure 30 TEM images of only high-dose Quad-NIV (120 μg / mL / strain), only matrix M (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.

[0082] Figure 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.

[0083] Figure 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 are circled.

[0084] Figure 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 are circled.

[0085] Figure 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 are circled.

[0086] Figure 35 TEM images of the low-dose PFS group (30 μg / mL / strain) with or without matrix M (100 μg / mL) incubated for 1 month at various temperatures (2 - 8 °C, 25 °C, and 37 °C) are shown.

[0087] Figure 36The DSC curves of the Quad-NIV formulation at a dose of 120 μg / mL / strain with or without matrix M1 (100 μg / mL) are shown. The molar heat capacity (Cp: kJ / mol.k) of the quadrivalent vaccine antigen incubated at 4 °C for 3 months, 6 months and 12 months and at 25 °C for 3 months and 6 months is shown.

[0088] Figure 37 The formation of HaSMaN measured by the reduction of nHA (i.e., detergent core nanoparticles) using the HA proteins from strain A, strain B and the C-terminally modified strain A fused with foldon is shown. Detailed Description

[0089] Definitions

[0090] Unless the context clearly dictates otherwise, the singular forms "a", "an" and "the" as used in this specification and the appended claims include plural referents. Thus, for example, reference to "a protein" can 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 the like.

[0091] As used herein, the term "adjuvant" refers to a compound that, when used in combination with an immunogen, enhances or otherwise alters or modifies the immune response induced against the immunogen. Modification of the immune response can include enhancing or expanding the specificity of either or both of the antibody and cellular immune responses.

[0092] As used herein, the term "about" or "approximately" when preceding a numerical value indicates a range of plus or minus 10% of the value. For example, "about 100" encompasses 90 and 110.

[0093] As used herein, the terms "immunogen", "antigen" and "epitope" refer to substances capable of eliciting an immune response, such as proteins (including glycoproteins) and peptides.

[0094] As used herein, an "immunogenic composition" is a composition comprising an antigen, wherein administration of the composition to a subject results in the occurrence of a humoral and / or cellular immune response in the subject against the antigen.

[0095] As used herein, "Quad-NIV", "QuadNIV" or "quadrivalent nanoparticle influenza vaccine" refers to an influenza vaccine formulation comprising antigens from four influenza strains.

[0096] As used herein, the terms “bedside mixture,” “bedside formulation,” “bedside vaccine composition,” “bedside vial,” “bedside vial formulation” refer to a vaccine formulation prepared immediately prior to administration. Such vaccine formulations contain viral antigens and adjuvants, which are stored in separate containers and administered to a subject (e.g., by administering two consecutive injections, or by mixing the antigen and adjuvant into a single injection prior to administration).

[0097] As used herein, the terms “co-formulation mixture,” “co-formulation,” “co-formulation vaccine composition,” “prefilled syringe,” “premix” refer to a vaccine formulation that is prepared prior to the time of administration to a subject and stored for a short to long term. Such vaccine formulations contain a combination of influenza antigen and an ISCOM matrix adjuvant in the same container, and are prepared under conditions sufficient to form HaSMaN (hemagglutinin saponin matrix nanoparticles).

[0098] As used herein, the terms “treat,” “treatment,” and “treating” refer to methods for obtaining a beneficial or desired result (e.g., a clinical result). For the purposes of this disclosure, a beneficial or desired result may include inhibiting or suppressing the initiation or progression of an infection or disease; ameliorating the symptoms of an infection or disease or reducing its incidence; or a combination thereof.

[0099] As used herein, “prevention” and “prophylaxis” are used interchangeably and may mean completely preventing an infection or disease, or preventing the occurrence of symptoms of this infection or disease; delaying the onset of an infection or disease or its symptoms; or reducing the severity of a subsequent infection or disease or its symptoms.

[0100] As used herein, “effective dose” or “effective amount” refers to the amount of an immunogen sufficient to induce an immune response that reduces the symptoms of infection by at least one pathogen. The effective dose or effective amount can be determined, for example, by measuring the amount of neutralizing secreted antibodies and / or serum antibodies (e.g., by plaque neutralization, complement fixation, enzyme-linked immunosorbent assay (ELISA), micro-neutralization assay, and HAI titer).

[0101] As used herein, the term “vaccine” refers to an immunogenic composition, such as an immunogen derived from a pathogen, that is used to induce an immune response against the pathogen that provides protective immunity (e.g., immunity that protects a subject from infection by the pathogen and / or reduces the severity of a disease or condition caused by infection by the pathogen). The protective immune response can include the formation of antibodies and / or cell-mediated responses. Depending on the context, the term “vaccine” can also refer to a suspension or solution of an immunogen administered to a subject to produce protective immunity.

[0102] As used herein, the term "subject" includes humans and other animals. Generally, the subject is a human. For example, the subject can be an adult, an adolescent, a child (2 to 14 years old), an infant (1 month to 24 months old) or a neonate (up to 1 month old). In some aspects, an adult is an elderly person about 65 years old or older, or about 60 years old or older. In typical aspects, an adult is an adult about 60 to less than about 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, a non-human primate; for example, a baboon, a chimpanzee, a gorilla or a rhesus monkey. In certain aspects, the subject can be a pet, such as a dog or a cat.

[0103] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the United States federal or state government or listed in the United States Pharmacopeia, the European Pharmacopeia, or other generally recognized pharmacopeias for use in animals and more particularly for use in humans. These compositions can be used as vaccines and / or antigen compositions for inducing a protective immune response in vertebrates.

[0104] Nanoparticles: Structure and Morphology

[0105] The compositions disclosed herein comprise two types of nanoparticles. The first type is called detergent core nanoparticles and is substantially as previously described. See U.S. Serial No. 15 / 257,436, which is incorporated herein by reference for all purposes. Briefly, these nanoparticles are produced in insect cells by expressing the HA protein using a baculovirus expression system and then extracting the HA protein with a detergent. During purification, the first detergent is replaced with a second detergent (usually a non-ionic detergent), thereby generating nanoparticles with a non-ionic detergent core into which the terminal portions of the HA protein are embedded in a trimeric form. Figure 1 (Left panel) shows these structures as observed under an electron microscope.

[0106] The second type of nanoparticle is referred to herein as HaSMaN (hemagglutinin saponin matrix nanoparticle). Figure 1 (Right panel) shows these structures as observed under an electron microscope. The HA glycoprotein modifies the matrix cage structure. The HaSMaN structure is formed by preparing HA nanoparticles and then incubating them with ISCOM matrix adjuvant particles for a period of time. The ISCOM matrix particles are as Figure 1As shown in the central figure. It is worth noting that HaSMaN is easily formed by the influenza A HA protein, but not easily formed by the influenza B HA protein. Thus, in certain aspects, a composition comprising nanoparticles having HA proteins from both influenza A and B will contain nanoparticles of both types (i.e., detergent core nanoparticles and HaSMaN). In some aspects, the nanoparticles will transition between two states, and thus the composition may contain transitional nanoparticles.

[0107] In certain embodiments, the detergent core nanoparticles consist of multiple protein trimers surrounding a non-ionic detergent core. For example, each nanoparticle may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 15 trimers. In some embodiments, each nanoparticle contains 2 to 6 trimers. In certain embodiments, each nanoparticle contains 2 to 9 trimers.

[0108] The HA protein associates with the non-ionic detergent core of the nanoparticle. Generally, the detergent is selected from polysorbate 20 (PS20), polysorbate 40 (PS40), polysorbate 60 (PS60), polysorbate 65 (PS65), and polysorbate 80 (PS80). The presence of the detergent promotes the formation of the nanoparticle by forming a core that organizes and presents the antigen. Thus, in certain embodiments, the nanoparticle contains an antigen assembled into a glycoprotein-PS80 protein-detergent nanoparticle, with its head region protruding outward and the hydrophobic region and PS80 detergent forming the central core surrounded by the antigen. In certain embodiments, the non-ionic detergent in the influenza vaccine composition is PS80. The Z-ave size of the detergent core nanoparticles ranges from about 25 nm to about 30 nm. However, once the detergent core nanoparticles are mixed with matrix M for a period of time, HaSMaN is formed, and the formed HaSMaN is slightly larger than the detergent core nanoparticles, about 50 nm to 60 nm. Unless otherwise specified, the particle size (Z-ave) is measured by dynamic light scattering (DLS) using a Malvern Zetasizer.

[0109] Nanoparticles: Production

[0110] The detergent core nanoparticles and HaSMaN of the present disclosure are non-naturally occurring products, and their components do not exist together in nature. Generally, the methods disclosed herein use a detergent exchange method, in which a first detergent is used to isolate the protein, and then this first detergent is exchanged for a second detergent to form the nanoparticles.

[0111] Glycoprotein antigens in nanoparticles (usually HA) are typically produced by recombinant expression in host cells. Generally, a baculovirus system is used to express glycoproteins in insect host cells. In a preferred embodiment, the baculovirus is a cathepsin-L knockout baculovirus. In other preferred embodiments, the baculovirus is a chitinase knockout baculovirus. In still other preferred embodiments, the baculovirus is a double knockout of both cathepsin-L and chitinase. High-level expression can be obtained in the 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 cells are Sf9 cells or derivatives thereof.

[0112] Typical transfection and cell growth methods can be used to culture the cells. A vector, e.g., a vector containing a polynucleotide encoding a fusion protein, can be transfected into the host cells according to methods well known in the art. For example, introduction of nucleic acids into eukaryotic cells can be achieved by calcium phosphate co-precipitation, electroporation, microinjection, lipofection, and transfection using polyamine transfection reagents.

[0113] 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. Generally, cell culture is carried out 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 value 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, N.J.). In other embodiments, the pre-sterilized plastic bag is a bag of about 50L to about 3500L.

[0114] Detergent extraction and purification of nanoparticles

[0115] After the host cells have grown, the protein can be harvested from the host cells using detergents and purification protocols. Once the host cells have grown for 48 to 96 hours, the cells are separated from the culture medium and a solution containing a detergent is added to lyse the cell membrane, thereby releasing the protein into the detergent extract. Both Triton X-100 and tergitol (also known as NP-9) are preferred detergents for extraction. The detergent 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%.

[0116] In other aspects, different first detergents can be used to isolate the protein from the host cells. For example, the first detergent can be Bis(polyethylene glycol bis[imidazoylcarbonyl]), nonoxynol-9, Bis(polyethylene glycol bis[imidazoylcarbonyl]), 35, 56, 72, 76, 92V, 97, 58P, EL, decaethylene glycol monododecyl ether, N-decanoyl-N-methylglucamine, n-decyl α-D-glucopyranoside, decyl β-D-maltopyranoside, n-dodecanoyl-N-methylglucamide, n-dodecyl α-D-maltoside, n-dodecyl β-D-maltoside, n-dodecyl β-D-maltoside, heptaethylene glycol monodecyl ether, heptaethylene glycol monododecyl ether, heptaethylene glycol monomyristyl ether, n-hexadecyl β-D-maltoside, hexaethylene glycol monododecyl ether, hexaethylene glycol monocetyl ether, hexaethylene glycol monooctadecyl ether, hexaethylene glycol monomyristyl ether, Igepal CA-630, Igepal CA-630, methyl-6-O-(N-heptylcarbamoyl)-α-D-glucopyranoside, nonaethylene glycol monododecyl ether, N-nonanoyl-N-methylglucamine, N-nonanoyl-N-methylglucamine, octaethylene glycol monodecyl ether, octaethylene glycol monododecyl ether, octaethylene glycol monocetyl ether, octaethylene glycol monooctadecyl ether, octaethylene glycol monomyristyl ether, octyl-β-D-glucopyranoside, pentaethylene glycol monodecyl ether, pentaethylene glycol monododecyl ether, pentaethylene glycol monocetyl 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 isocetyl ether, polyoxyethylene 20 oleyl ether, polyoxyethylene 40 stearate, polyoxyethylene 50 stearate, polyoxyethylene 8 stearate, polyoxyethylene bis(imidazolylcarbonyl), polyoxyethylene 25 propylene glycol stearate, saponins from the bark of Quillaja 20、 40、 60, 65、 80、 85, Tergitol type 15-S-12, Tergitol type 15-S-30, Tergitol type 15-S-5, Tergitol type 15-S-7, Tergitol type 15-S-9, Tergitol type NP-10, Tergitol type NP-4, Tergitol type NP-40, Tergitol type NP-7, Tergitol type NP-9, Tergitol type TMN-10, Tergitol type TMN-6, Triton X-100, or a combination thereof.

[0117] Then, the nanoparticles can be isolated from the cell debris using centrifugation. In some embodiments, gradient centrifugation can be used, such as using cesium chloride, sucrose, and iodixanol. Other techniques can be used as alternatives or supplements, such as standard purification techniques including, for example, ion exchange chromatography, affinity chromatography, and gel filtration chromatography.

[0118] For example, the first column can be an ion exchange chromatography resin, such as EMD TMAE (EMD Millipore), the second column can be a lentil (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 in purification processes to remove detergents, but may have a negative impact on the methods disclosed herein.

[0119] Detergent exchange

[0120] To form the detergent core nanoparticles, the first detergent used to extract the protein from the host cell is substantially replaced by a second detergent to form the nanoparticle structure. NP-9 is the preferred extraction detergent. Generally, when measured by HPLC, the nanoparticles do not contain detectable NP-9. The second detergent is typically selected from the group consisting of PS20, PS40, PS60, PS65, and PS80. Preferably, the second detergent is PS80.

[0121] In certain aspects, affinity chromatography is used to perform detergent exchange by binding glycoproteins through their sugar moieties. For example, an affinity chromatography can use a lectin column. Lectins are proteins initially identified in plants and found to interact specifically and reversibly with sugar residues. See, e.g., Sharon and Lis, “Legume lectins--a large family of homologous proteins,” FASEB J. November 1990; 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, onobrychis viciaefolia lectin, and lentil lectin. Lentil lectin is preferred for detergent exchange columns due to its binding properties. Lectin columns are commercially available; for example, Capto Lentil Lectin, available from GE Healthcare. In some aspects, the lentil lectin column can use a recombinant lectin. At the molecular level, it is thought that the sugar moiety binds to the lentil lectin, releasing the amino acids of the protein to associate around the detergent, thus forming a detergent core that provides nanoparticles with multiple antigen copies, such as glycoprotein oligomers, which can be dimers, trimers, or tetramers anchored in the detergent.

[0122] When the detergent is incubated with the protein during the detergent exchange process to form nanoparticles, the detergent can be present at up to about 0.1% (w / v) in the early purification steps.

[0123] Typically, influenza detergent core nanoparticles are prepared separately as a monovalent single strain product. For example, a non-ionic detergent (e.g., PS80) can be about 0.03% to about 0.5% non-ionic detergent. In certain aspects, the monovalent drug substance can contain 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 particular tetravalent composition, as measured by single radial immunodiffusion assay (SRID), the protein concentration can be about 0.48 mg / mL, and the amount of PS80 calculated is about 0.04%.

[0124] Detergent exchange can be performed with the purified protein as discussed above, and the protein is purified, stored frozen, and then thawed for detergent exchange.

[0125] Formation of HaSMaN

[0126] The HaSMaN disclosed herein is produced by incubating a detergent core nanoparticle with an ISCOM matrix adjuvant comprising a saponin fraction, cholesterol, and phospholipids.

[0127] Typically, formation requires incubation at 4 °C or 25 °C for about 24 to 48 hours. Higher temperatures promote the formation of HaSMaN. See, for example, Figure 33 and Figure 35 . Thus, in certain aspects, HaSMaN is formed by incubating at about 25 °C for at least 24 hours. Mixing the detergent core nanoparticle with the ISCOM matrix adjuvant shortly before administration to a subject - i.e., bedside mixing - does not produce HaSMaN. Longer incubation periods do not have a negative impact on the formation of HaSMaN.

[0128] Nanoparticle influenza HA protein

[0129] The HA glycoprotein used as an influenza antigen can be from any influenza virus strain. In almost every winter in the United States, human influenza A and B viruses cause seasonal epidemics. Influenza A viruses are further divided into subtypes based on two proteins on the virus surface: hemagglutinin (HA) and neuraminidase (NA).

[0130] 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.

[0131] In certain aspects, the influenza nanoparticle is an antitrypsin nanoparticle produced using neutral pH purification. During the purification and formulation of HA nanoparticles, trypsin resistance is achieved through a neutral pH range of above 6.9 to 8.5. Trypsin - resistant influenza glycoproteins and trypsin - resistant influenza nanoparticles; and methods for their manufacture are described in detail in U.S. Application No. 15 / 819,962, the content of which is incorporated herein by reference in its entirety for all purposes.

[0132] Modified antigen

[0133] Typically, the antigen is the full-length wild-type sequence. However, the antigen can be a variant or mutant of the wild-type antigen. In some aspects, the antigen can share identity with the disclosed antigens; for example, the percentage of identity can be at least 80%, at least 90%, at least 95%, at least 97% or at least 98%. The percentage of identity can be calculated using the alignment program ClustalW2, which is available at www.ebi.ac.uk / Tools / msa / clustalw2 / . The following default parameters can be used for pairwise alignment: protein weight matrix = Gonnet; gap open = 10; gap extend = 0.1.

[0134] Other variants can 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 external part of the molecule outside the virus; a single segment of 27 residues defines the transmembrane (TM) domain; and approximately 10 residues constitute the cytoplasmic tail region (CT). Although the antigen can have some variations, the formation of both detergent core nanoparticles and HaSMaN requires an intact transmembrane (TM) domain. Thus, in certain instances, the modified HA protein sequence has 100% identity in the TM and CT domains (i.e., is wild-type), 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 identified by homology to the amino acid sequences of the TM domain and CT of Japan / 305 / 57 HA as shown in Melikyan et al. (Mol Biol Cell. 1999 Jun;10(6):1821-1836), Figure 1 but it should be noted that the boundaries between the extracellular domain, TM and CT domains may vary by up to three amino acids between different HA proteins.

[0135] Vaccine stability

[0136] Advantageously, the disclosed nanoparticle influenza vaccine formulations are stable and suitable for long-term storage. The formulations provided herein can be particularly suitable for co-formulation strategies (i.e., where the antigen and matrix adjuvant are thoroughly mixed before administration; for example, in a pre-filled syringe). As disclosed herein, co-formulation influenza vaccine strategies can provide advantages for clinical practice and can be a cost-effective alternative to currently used bedside mixing formulations.

[0137] In some embodiments, the influenza antigen in the co-formulated vaccine composition is stable when the co-formulated vaccine composition is stored for up to 12 months. Although the vaccine compositions can be stored at 2 - 8°C, they exhibit excellent stability at 25°C.

[0138] The stability of pre-mixed (co-formulated) formulations can be evaluated by methods and protocols known in the art. Stability can be measured by the level of degradation of the vaccine antigen and the immunogenicity of the vaccine. Stability can be detected by evaluating the thermal stability of the viral antigen in the vaccine.

[0139] The long-term stability of vaccine formulations at room temperature (25 °C) is beneficial for cost-effective vaccine strategies, especially in regions where cold chain distribution and storage may be limited. As stated in the WHO guidelines for the evaluation of vaccine stability, controlling the temperature chain during manufacture, distribution, and storage is important for ensuring vaccine efficacy ("Guidelines for the evaluation of the stability of vaccines used in the controlled temperature chain; available at www.who.int / biologicals / WHO_CTC_first_draft_22_Dec_2014_amended.pdf).

[0140] Without being bound by theory, it is believed that the HaSMaN structure can contribute to the stability of co-formulated vaccine formulations. (See Figure 36 , which shows better thermal stability in the presence of the matrix adjuvant; that is, it shows that the HaSMaN particles have better stability than formulations using only the detergent core). Mixing the detergent core nanoparticles with matrix M to provide HaSMaN thus provides a vaccine composition that has an increase in temperature of about 0.5 °C to about 1.0 °C (measured by differential scanning calorimetry) compared to a vaccine composition containing only the detergent core nanoparticles.

[0141] This increased thermal stability helps to extend the shelf life of the vaccine. In certain aspects, the vaccine formulation can be stable for up to 6 months or up to 12 months. A test sample is considered "stable" in the context of the present disclosure if, after storage at 2 - 8 °C for 12 months, the HAI titer for the test sample is not statistically different from the HAI titer of a freshly prepared sample; and if, after storage at 2 - 8 °C for 12 months, the value obtained by single radial immunodiffusion (SRID) assay is at least about 70%. Statistical significance analysis of HAI titer measurements is performed as follows. Geometric mean titers (GMT) and associated 95% confidence intervals (CI) are calculated by group. Using Tukey HSD analysis with JMP13 software, the mean values of the log10-transformed HAI titer measurements between groups were compared. A p-value < 0.05 indicates a statistically significant difference between the two comparison groups.

[0142] Vaccine composition

[0143] The compositions disclosed herein can be used prophylactically or therapeutically. This disclosure includes methods for preventing influenza virus infection. The methods include administering to a subject a therapeutically or prophylactically effective amount of the immunogenic composition of this disclosure. Preferably, the pharmaceutical composition is a vaccine composition that provides a protective effect. In some aspects, the protective effect may include ameliorating symptoms related to infection in a certain percentage of the exposed population. For example, compared to untreated subjects, the composition can prevent or reduce one or more influenza symptoms selected from: fever, fatigue, muscle pain, headache, and sore throat.

[0144] The vaccine composition can contain various excipients, pharmaceutically acceptable buffers, etc. For example, the pharmaceutically acceptable buffer in the vaccine composition can contain one or more of sodium phosphate, sodium chloride, histidine, arginine hydrochloride, and trehalose. Sodium phosphate can be present at about 10 mM to about 50 mM, about 15 mM to about 30 mM. In certain cases, about 25 mM of sodium phosphate is present. Histidine can be present in the range of about 0.1% (w / v) to about 2.5% (w / v); for example, histidine can 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).

[0145] Sodium chloride can be in the range of about 50 mM to about 300 mM. Generally, when present in the composition, sodium chloride is about 150 mM.

[0146] Arginine hydrochloride can 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 certain cases, about 100 mM of arginine hydrochloride is present.

[0147] Trehalose can 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%.

[0148] The pH range of the influenza vaccine composition is generally near neutral and is maintained above pH 6.9 during purification and in the composition. For example, the pH of the buffer and the composition can be about pH 7.2 to about pH 7.8, more preferably about 7.2 to about 7.5. In certain aspects, the pH is about pH 7.5. Avoid pH values below 6.9 because they can have a negative impact on the stability of the HA structure.

[0149] Adjuvant

[0150] 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 thus be administered as adjuvant-free compositions.

[0151] The immunogenicity of a particular composition can be enhanced by using a non-specific stimulator of the immune response, called an adjuvant. Effective amounts of adjuvants have long been used to promote a general increase in immunity against antigens (e.g., U.S. Patent No. 4,877,611). Adjuvants have been used in immunization protocols for many years to stimulate responses and, as such, are well known to those of ordinary skill in the art. Some adjuvants affect the way antigens are presented. For example, when a protein antigen is precipitated with alum, the immune response is enhanced. Emulsification of the antigen also prolongs the duration of antigen presentation. It is contemplated that any adjuvant described in Vogel et al., "A Compendium of Vaccine Adjuvants and Excipients (2nd Edition)" is within the scope of the present disclosure, and the reference is incorporated herein by reference in its entirety for all purposes.

[0152] While various adjuvants can be included, an ISCOM matrix adjuvant that remains adjuvantly effective after administration to a subject is used to produce HaSMaN. Thus, in some aspects, no additional adjuvant needs to be added to the formulation. As described above, this formulation method allows for the preparation of pre-filled syringes containing the vaccine without the need to mix the antigen with the adjuvant at the bedside.

[0153] Saponin adjuvant for the matrix

[0154] ISCOM matrices are prepared using saponin fractions. Saponins are glycosides derived from the bark of the Quillaja saponaria Molina tree. Generally, saponins are prepared using a multi-step purification process, resulting in multiple fractions. As used herein, the term "saponin fraction from Quillaja saponaria Molina" generally refers to a semi-purified or defined saponin fraction of Quillaja saponaria or a substantially pure fraction thereof.

[0155] Saponin fraction

[0156] There are several methods applicable to the production of 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 (crude aqueous Quillaja saponaria Molina extract) 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 elution is carried out using a gradient of 25% to 60% acetonitrile in acidic water. The fraction referred to herein as "Fraction A" or "QH-A" is, or corresponds to, the fraction eluted at approximately 39% acetonitrile. The fraction referred to herein as "Fraction B" or "QH-B" is, or corresponds to, the fraction eluted at approximately 47% acetonitrile. The fraction referred to herein as "Fraction C" or "QH-C" is, or corresponds to, the fraction eluted at approximately 49% acetonitrile. Additional information regarding fraction purification can be found in U.S. Patent No. 5,057,540. When prepared as described herein, Fractions A, B, and C of Quillaja saponaria Molina each represent a group or family of chemically closely related molecules with definable properties. Under the chromatographic conditions for obtaining the fractions, batch-to-batch reproducibility is highly consistent in terms of the elution curve and biological activity.

[0157] Other saponin fractions have been described. Fractions B3, B4, and B4b are described in EP 0436620. Fractions QA1 - QA22 are described in EP03632279B2, Q-VAC (Nor-Feed, AS Denmark), Quillaja saponaria Molina Spikoside (Isconova AB, Ultunaallén 2B, 756 51 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 of EP 0 3632 279B2 can be used, especially QA-7, QA-17, QA-18, and QA-21. The fractions are obtained as described in EP 0 3632 279B2, especially on page 6 and in Example 1 on pages 8 and 9.

[0158] The saponin fractions described herein and used to form adjuvants are generally substantially pure fractions; that is, the fractions are substantially free from contamination by other substances. In certain aspects, the substantially pure saponin fraction 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.

[0159] Other saponin fractions, such as QS-7 and QS-21 fractions, are produced and used as described in U.S. Patent Nos. 5,057,540; 6,231,859; 6,352,697; 6,524,584; 6,846,489; 7,776,343 and 8,173,141. These fractions can be used in the methods and compositions disclosed herein.

[0160] Matrix particles

[0161] The matrix particle adjuvants disclosed herein, due to their adjuvant properties, can be used to generate HaSMaN or used as discrete particles. The ISCOM matrix contains lipids and at least one saponin fraction. The lipid is at least a sterol, such as cholesterol. In certain aspects, the lipid is a phospholipid. The ISCOM matrix complex may also contain one or more other immunomodulatory (adjuvant-active) substances (not necessarily glycosides) and can be produced as described in EP0436620B1.

[0162] The saponin fraction can be fraction A, fraction B or fraction C of Quillaja saponaria, a semi-purified product of Quillaja saponaria, a purified product of Quillaja saponaria or any purified sub-fraction, such as QA 1-21.

[0163] The matrix particles can contain a mixture of saponin fractions, or the particles can be formed using only one saponin fraction. The compositions disclosed herein can contain multiple particles, where each particle contains only one saponin fraction. That is, certain compositions can contain one or more different types of ISCOM matrices, where each individual particle contains one saponin fraction from Quillaja saponaria Molina, and the saponin fraction in one particle is different from the saponin fraction in other complex particles.

[0164] 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 contain at least two types of complexes or particles, each type having one type of saponin incorporated into physically distinct particles.

[0165] In the composition, a mixture of ISCOM matrix particles can be used, wherein one saponin fraction, Quillaja saponaria Molina, and another saponin fraction, Quillaja saponaria Molina, are incorporated into different ISCOM matrix complex particles and / or ISCOM complex particles, respectively.

[0166] ISCOM matrices each having one saponin fraction can be present in the composition in any combination by weight %. In certain aspects, the composition can 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 the ISCOM matrix containing the first saponin fraction, with the remainder consisting of ISCOM matrices containing different saponin fractions. In some aspects, the remainder is one or more ISCOM matrix particles containing only one saponin fraction. In other aspects, the ISCOM matrix particles can contain more than one saponin fraction.

[0167] In a preferred composition, the saponin fraction in the first ISCOM matrix is fraction A ("fraction A matrix"), while the saponin fraction in the second ISCOM matrix or ISCOM complex particle is fraction C ("fraction C matrix"). Thus, the preferred composition comprises fraction A matrix adjuvant and fraction C matrix adjuvant as adjuvants. The amount of each matrix in the composition can vary. For example, the amount of the fraction A matrix can 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 the fraction C matrix. An example of a suitable 85:15 combination of the fraction A matrix and the fraction C matrix (referred to herein as matrix M or matrix M1) can be obtained as Matrix-M TM Obtained from Novavax AB, Uppsala, Sweden.

[0168] 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.

[0169] In some embodiments, the amount of matrix M in each administration 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 certain aspects, the adjuvant can be present at about 50 μg to about 75 μg.

[0170] Other adjuvants

[0171] Exemplary adjuvants include complete Freund's adjuvant (containing a non-specific stimulator of the immune response that kills 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 in a 2% squalene / 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 paucilamellar lipid vesicles; for example, are paucilamellar non-phospholipid vesicles in the range of about 100 nm to about 500 nm. It includes Brij 72, cholesterol, oleic acid, and squalene. Novasome has been shown to be an effective adjuvant (see U.S. Patent Nos. 5,629,021, 6,387,373, and 4,911,928.

[0172] Administration and dosage of nanoparticle vaccine compositions

[0173] The compositions disclosed herein can be administered by systemic, mucosal, or transdermal routes or directly into a specific tissue. As used herein, the term "systemic administration" includes parenteral administration routes. In particular, parenteral administration includes subcutaneous, intraperitoneal, intravenous, intramuscular, or intrasternal injection. Generally, the compositions are administered by intramuscular injection. In certain aspects, the compositions can be administered mucosally. As used herein, the term "mucosal administration" includes oral, intranasal, vaginal, rectal, and intratracheal.

[0174] The compositions can be administered to a subject (usually a human) in need thereof.

[0175] The compositions can be administered in a single-dose regimen or a multi-dose regimen. Multiple doses can be used in a primary immunization regimen or a booster immunization regimen. In a multi-dose regimen, the various doses can be administered by the same or different routes, for example, parenteral primary and mucosal booster, mucosal primary and parenteral booster, etc. In some aspects, a subsequent booster dose is administered about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or about 6 weeks after a previous dose.

[0176] In some embodiments, at least one of the four strains of the quadrivalent influenza vaccine composition is an A strain. For example, the quadrivalent influenza vaccine can comprise three A strains and one B strain.

[0177] The total amount of influenza HA in the vaccine composition can 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 can be from about 5 μg per strain to about 80 μg per strain, from about 10 μg per strain to about 75 μg per strain, from about 15 μg per strain to about 70 μg per strain, from about 20 μg per strain to about 65 μg per strain, from about 25 μg per strain to about 60 μg per strain, from about 30 μg per strain to about 55 μg per strain, from about 35 μg per strain to about 50 μg per strain, from 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 a larger percentage of the initial amount remains as measured by SRID; for example, at least about 70%, at least about 75%, or at least about 80% of the initial amount.

[0178] In some embodiments, the present disclosure provides a co-formulation (i.e., pre-filled syringe or premix) 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 prior to administration. Due to concerns about the stability of influenza antigens and their subsequent immunogenic capabilities, pre-mixed, co-formulated, or pre-filled syringe strategies for influenza vaccines are less common. The present disclosure provides nanoparticle influenza vaccine compositions that can be pre-mixed and pre-stored. 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.

[0179] Immunogenicity of Nanoparticle Influenza Vaccines

[0180] The present disclosure provides methods for preventing influenza infection. The immunogenicity of the nanoparticle influenza vaccines disclosed herein can be determined using suitable methods, including performing HAI assays or by measuring neutralizing antibodies. In some embodiments, the immunogenicity of the nanoparticle influenza vaccines can be compared to commercially available influenza vaccine compositions. As used herein, "commercially available influenza vaccine compositions" can be any influenza vaccine composition that can be used for medical purposes. For example, commercially available influenza vaccine compositions can be formulated as trivalent or quadrivalent injectables. In some aspects, the formulation for injection can contain inactivated forms of the virus. In another example, commercially available influenza vaccine compositions can be formulated as nasal sprays. In some aspects, the formulation for nasal sprays can contain attenuated or weakened forms of the virus.

[0181] 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), the compositions disclosed herein provide a non-inferior immune response.

[0182] Advantageously, the compositions disclosed herein induce neutralizing antibodies that bind to strains that have drifted (i.e., undergone minor mutations) with respect to the sequences used for the virus within the same influenza subtype. In certain aspects, one, two, three, four, or all of the strains used in the composition induce neutralizing antibodies against one drifted strain, against two drifted strains, against three drifted strains, against four drifted strains, or against five drifted strains. Without being bound by theory, it is believed that the presence of the matrix adjuvant in the composition promotes the exposure of additional antigens, thereby providing extended protection against drifted strains. Similarly, the formation of HaSMaN after incubation of the matrix adjuvant with the HA protein of subtype A is thought to contribute to this process.

[0183] Importantly, HaSMaN is at least as immunogenic as the detergent-core nanoparticles. For example, as shown in Table 6 below, on day 35, at 25°C, the HAI titer of 1.5 μg of the premixed formulation against A / Michigan was approximately 538, while at 25°C, the HAI titer of 1.5 μg of the bedside mixed formulation against A / Michigan was approximately 453. In another example shown in Table 8, on day 35, at 25°C, the HAI titer of 1.5 μg of the premixed formulation against A / Hong Kong was approximately 538, while at -60°C, the HAI titer of 1.5 μg of the bedside mixed formulation against A / Michigan was approximately 494. The similar immunogenicity between the premixed formulation and the bedside mixed formulation further indicates that the premixed formulation is stable at room temperature. Additionally, 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.

[0184] Container

[0185] A variety of containers can be used to store and transport the premixed formulation, including single-dose syringes and plastic ampoules. In some cases, blow-fill-seal manufacturing techniques or methods can be used to manufacture plastic ampoules. Generally, the blow-fill-seal (BFS) manufacturing method involves 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. Thus, BFS can be an automated process that can be carried out in a sterile environment without direct human intervention.

[0186] In some cases, the ability to aseptically manufacture sterile ampoules containing the desired liquid can make BFS-manufactured ampoules particularly suitable for the pharmaceutical industry. However, BFS technology is not compatible with all drug 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 can have an adverse effect on temperature-sensitive liquids and / or products such as vaccines, biologics, etc. However, advancements in cooled BFS technology have increased the variety of applicable products, liquids, etc., thus allowing some vaccines, biologics, and / or other temperature-sensitive drugs to be included in BFS ampoules.

[0187] In some cases, the size, shape, and / or configuration of the BFS ampoule can be at least partially based on the desired use and / or the desired pharmaceutical liquid or dose that the ampoule is configured to contain. For example, some known BFS ampoules can include a pierceable top, a break-off top, a top with a male Luer or a female Luer, etc. The size and / or shape of some known BFS ampoules can be based on the volume of the liquid or dose configured to be placed therein. Additionally, some known BFS ampoules can be manufactured as a strip of multiple temporarily connected ampoules, which can improve manufacturing, packaging, and / or storage efficiency, etc.

[0188] All patents, patent applications, references, and journal papers cited herein are hereby expressly incorporated by reference in their entirety for all purposes.

[0189] Examples

[0190] Example 1: Purification of HA Nanoparticles

[0191] The HA protein from a single strain was expressed in Sf9 cells by baculovirus infection and allowed to grow for 48 - 96 hours before harvest. The HA protein was then harvested by detergent extraction and converted into detergent-core nanoparticles during the purification process. Briefly, the TMAE column was pre-equilibrated with a buffer consisting of 25 mM Tris, pH 8.0, 1.5 M sodium chloride, 0.02% NP9. The sample was loaded at ≤90 cm / h (residence time 24 minutes), and then washed with EQ buffer (25 mM Tris, pH 8.0, 50 mM sodium chloride or 81 mM sodium chloride (for strain A and strain B respectively), 0.02% NP-9). The purified sample was then eluted using 1.5 CV of EQ buffer.

[0192] For strain A, the product from the TMAE column was nanofiltered and then applied to a lentil lectin affinity chromatography column, which was pre-equilibrated with 3 CV of a 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 4-minute residence time. After loading, it was washed with 3 CV of lentil lectin equilibration buffer. The product was collected at 75 cm / h and at an 8-minute residence time for 2 CV, 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.

[0193] For strain B, 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 with a 4-minute hold time and collected with 2 column volumes (CV) of the equilibration buffer. The product from the Capto Blue column was nanofiltrated 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 B respectively), 0.02% (w / v) NP-9, pH 8.0 (flow rate: 150 cm / h). The sample was loaded at a 4-minute hold time. After loading, it was washed with 3 CV of lentil lectin equilibration buffer. The product was collected at 75 cm / h with a 8-minute hold time for 2 CV 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.

[0194] The lentil lectin products for both strain A and B were concentrated to the target HA concentration and then buffer exchanged to the final API formulation buffer. Concentration and buffer exchange were performed by ultrafiltration and diafiltration.

[0195] Example 2 - SDS PAGE Analysis

[0196] To evaluate the stability of co-formulated Quad-NIV and ISCOMATRIX adjuvant (Matrix M) in pre-filled syringes and to understand the physical, chemical, and biological characteristics of this vaccine, formulations were prepared as shown in Table 1 below. The vaccine compositions tested were assigned to three groups: (1) glass bedside vials containing only the HA antigen; (2) pre-filled syringes (PFS) containing the HA vaccine antigen co-formulated with Matrix M1; and (3) PFS containing the HA vaccine antigen. The glass vials for the bedside mixtures and PFS were commercially obtained (Schott).

[0197] Table 1. Comparative study of bedside vial and PFS formulations

[0198]

[0199] Figure 2Shows a reducing SDS-PAGE gel image, which shows protein bands from bedside vials stored at 4°C; or protein bands from prefilled syringes (PFS) with or without matrix M1 stored at 4°C. At all tested time points, the band patterns of proteins from PFS were similar to those of proteins from bedside vials, indicating that the proteins in PFS were at least as stable as those in bedside vials. The presence or absence of matrix M did not change the band patterns among PFS groups, nor did it during storage for 6 months or even 12 months. The LD bedside vial group and the LD PFS group also had consistently similar band patterns at 4°C, although the LD group had weaker band patterns compared to its HD cohort at the same time points.

[0200] Figure 3 Shows the band patterns of proteins from PFS or bedside vials stored at 4°C, detected on non-reducing SDS-PAGE gels and reducing SDS-PAGE gels. The results showed that even on non-reducing gels, the protein band patterns of all groups were similar, further indicating that the proteins in PFS were similar to those in bedside vials at zero time.

[0201] In addition, the protein stability of the bedside vial group and the PFS group with or without matrix M stored at 4°C or 25°C for 3 months was detected using non-reducing SDS-PAGE assay ( Figure 4 ). Compared with those groups stored at a lower temperature (such as 4°C), some of the groups stored at a higher temperature (25°C) tended to show bands with higher molecular weights. Stronger expression of proteins was also observed in the groups stored at a lower temperature. The results showed that even after long-term storage (such as storage for 3 months), the proteins in PFS were at least as stable as those in bedside vials.

[0202] These data show that in terms of stability, the PFS co-formulation vaccine strategy is feasible. The PFS co-formulation vaccine is feasible for storage at 4°C and 25°C. The presence of matrix M1 and the duration of storage time (e.g., 3 months) do not have a negative impact on the stability of the vaccine formulation, thus confirming that the formation of HaSMaN does not interfere with the protein stability over a long time.

[0203] Example 3 - SRID Analysis

[0204] The stability of proteins was tested by performing SRID assays on various influenza A strains and influenza B strains formulated in PFS groups. The SRID assays were 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 6Shows SRID data of 120 μg / mL PFS formulations containing A / Hong Kong, A / Michigan, B / Brisbane, or B / Phuket with or without 100 μg / mL matrix M at 2 - 8 °C ( Figure 5 ) and 25 °C ( Figure 6 ).

[0205] The SRID immunoreactivity results of PFS with matrix M are similar to or better than those of PFS without matrix M. The data show that the overall trend of immunoreactivity over time when stored at 25 °C is not significantly different from that observed at 4 °C. The absence of a significant change in vaccine immunoreactivity indicates that the Quad - NIV PFS formulation is stable for at least 6 months at various temperatures. The stability of the Quad - NIV PFS formulation at room temperature (25 °C) advantageously provides a cost - effective vaccine strategy as the PFS formulation does not require long - term refrigeration or is limited to low - temperature handling. These data further show that it is a feasible approach to mass - produce a stable pre - mixed vaccine (containing influenza antigens and matrix M) long before vaccination in the clinic.

[0206] Example 4 - Comparative Immunogenicity Study of Quad - NIV and Commercial Influenza Vaccines in Ferrets

[0207] Ferrets studies were conducted to compare the immunogenicity of various test vaccine strategies (bedside and co - formulation) with commercially available influenza vaccines.

[0208] Table 2. Comparative Immunogenicity Study of Quad - NIV and Commercial Vaccines in Ferrets

[0209]

[0210]

[0211] * - The dose of each B strain is 90 μg; and the dose of each A strain is 60 μg.

[0212] # - Figures 7 - 10 The results depicted in

[0213] For the Quad-NIV formulation, the A / Michigan / 45 / 2015-H1N1 strain, A / Hong Kong / 4801 / 2014-H3N2 strain, B / Brisbane / 60 / 2008 strain, and B / Phuket / 3073 / 2013 strain are used. For Fluzone HD (TIV; Sanofi Pasteur), A / Michigan / 45 / 2015-H1N1, A / Hong Kong / 4801 / 2014-H3N2, and B / Brisbane / 60 / 2008 are used. For Fluzone HD (QIV; Sanofi Pasteur), A / Michigan / 45 / 2015-H1N1, A / Hong Kong / 4801 / 2014-H3N2, B / Brisbane / 60 / 2008, and B / Phuket / 3073 / 2013 are used. For Fluad (TIV; Seqirus), A / Singapore / GP1908 / 2015 (similar to A / Michigan / 45 / 2015-H1N1), A / Hong Kong / 4801 / 2014-H3N2, and B / Brisbane / 60 / 2008 are used. For Flublok (QIV; ProteinSciences), A / Michigan / 45 / 2015-H1N1, A / Hong Kong / 4801 / 2014-H3N2, B / Brisbane / 60 / 2008, and B / Phuket / 3073 / 2013 are 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) are used.

[0214] Ferrets were immunized on Day 0 and Day 21 of the study. Blood was drawn on the day prior to the start of the study, on Day 21 and Day 42 of the study. HAI assays were performed using human red blood cells to evaluate the immunogenicity of the test formulation in ferrets. The experimental protocol is known in the art and has been discussed above. The following HAI reagents were used in the assays 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. The method for preparing influenza antigen VLP is disclosed, for example, in U.S. Patent Application No. 15 / 901,000, which is incorporated herein by reference in its entirety for all purposes.

[0215] All commercially available influenza vaccines had lower HAI titers compared to all Quad-NIV formulations in the presence of matrix M, regardless of the dosing regimen and formulation type (i.e., bedside and co-formulation). See Figure 7 (A / Hong Kong / 4801-2014), Figure 8 (A / Michigan / 45 / 2015), Figure 9 (B / Brisbane / 60 / 2008), and Figure 10 (B / Phuket / 3073 / 2013).

[0216] In summary, compared to commercial vaccines, for all tested influenza strains disclosed herein, Quad-NIV formulations elicited a stronger immunogenic effect in ferrets, and the difference was more significant with the presence of matrix M in the formulation. The Quad-NIV co-formulation (pre-mixed) containing HaSMaN had similar immunogenicity compared to the Quad-NIV bedside formulation. Among the co-formulation group, the formulations stored at 4°C and at 25°C generally had very similar immunogenicity.

[0217] Example 5 - Immunogenicity Assessment of Quadrivalent Nanoparticle Influenza Vaccine Stored at 4°C

[0218] Table 3 shows the mouse study design for detecting the immunogenicity and stability of the pre-mixed 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 Matrix M adjuvant just prior to administration.

[0219] Table 3. Quad-NIV formulation for immunogenicity assessment

[0220]

[0221] Immunizations were administered intramuscularly on days 0 and 21. Blood samples were collected on the day prior to the start of the study and again on day 42 of the study (or 21 days after the second immunization) for immunogenicity analysis. Immunogenicity was determined based on the hemagglutination inhibition (HAI) responses to 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 the Manual for the Laboratory Diagnosis and Virological Surveillance of Influenza (World Health Organization 2011, accessed 02 / 15 / 2018, at: www.who.int / influenza / gisrs_laboratory / manual_diagnosis_surveillance_influen za / en / , the disclosures of which are incorporated by reference).

[0222] Figure 11 Shows the HAI titers of the formulations listed in Table 3 against the four influenza strains for all HD groups at 3 months. The results showed that the pre-mixed formulation stored at 4 °C for 3 months had similar immunogenicity compared to the bedside-mixed formulation stored at -60 °C. The results also showed that Matrix M did not alter the immunogenicity of the tested Quad-NIV formulation even when the pre-mixed formulation was stored long-term (such as for 3 months).

[0223] Example 6 - Immunogenicity and long-term stability of the pre-mixture against Matrix M-containing bedside-mixed HA nanoparticles at 25 °C

[0224] Groups 1 to 14 were prepared as shown in Table 4 below. For the premixed groups, HA nanoparticles and matrix M (fraction A matrix and fraction C matrix at 85:15 w / w) were mixed and stored at 4 °C for 6 months or 12 months before administration, or stored at 25 °C for 6 months. For the bedside mixing groups, 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 immediately before administration to mice. The HA protein nanoparticles contain HA from the following strains: A / Michigan H1N1, A / Hong Kong-H3N2 B / Brisbane, and B / Phuket.

[0225] Table 4. 6 months and 12 months 3 Formulation stability analysis

[0226]

[0227]

[0228] 1 High dose: 120 μg / mL / strain (total 480 μg HA) + 100 μg / mL matrix.

[0229] 2 Low dose: 30 μg / mL / strain HA (total 120 μg HA) + 100 μg / mL matrix.

[0230] 3 The 12-month stability study did not include samples stored at 25 °C.

[0231] We measured the HAI titers for each of the four strains. Figures 12 - 19 And Tables 5 - 12 show the HAI titers of the HD and LD groups against the A / Hong Kong, A / Michigan, B / Brisbane, and B / Phuket strains at 6 months.

[0232] Table 5. A / Michigan H1N1 - HAI titer on day 21

[0233]

[0234] Table 6. A / Michigan H1N1 - HAI titer on day 35

[0235]

[0236] Table 7. A / Hong Kong - H3N2 HAI titer on day 21

[0237]

[0238]

[0239] Table 8. A / Hong Kong-H3N2 HAI titer on day 35

[0240]

[0241] Table 9. B / Brisbane HAI titer on day 21

[0242]

[0243] Table 10. B / Brisbane HAI titer on day 35

[0244]

[0245] Table 11. B / Phuket HAI titer on day 21

[0246]

[0247] Table 12. B / Phuket HAI titer on day 35

[0248]

[0249] Figures 20 - 27 Shows the HAI titers against A / Hong Kong strain, A / Michigan strain, B / Brisbane strain and B / Phuket strain in the high-dose (HD) group and low-dose (LD) group at 12 months.

[0250] Data show that, as measured by HAI titers, both the bedside-mixed vaccine and the pre-mixed vaccine produced similar immunogenicity in mice even after 6 months or 12 months of storage. The study also confirmed that the presence of matrix M favored the generation of a greater immune response. Even upon long-term storage, matrix M did not alter the immunogenicity of the tested Quad-NIV formulation.

[0251] The pre-mixed 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 pre-mixture 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). Combining the stability of the pre-mixed formulation upon long-term storage at 25 °C, the vaccine is thus particularly suitable for environments where cold-chain storage may be limited or lacking.

[0252] Figures 12 - 27The data shown in [Figure] indicate that, compared to the low-dose groups for all strains on day 21, the high-dose groups, particularly the premixed formulations, had higher HAI titer responses. 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., from day 21 to day 35, the A / Hong Kong HAI titer of the 1.5 μg premixed formulation at 25°C: 22 vs. 538), while minimal differences were observed between the high-dose and low-dose groups on day 35.

[0253] The data confirm that HaSMaN particles formed from the type A HA protein are at least as effective as HA nanoparticles with a detergent core in a non-HaSMaN form in inducing an immune response. The data show that the interaction of HA nanoparticles with the matrix to form HaSMaN does not negatively affect the adjuvant effect of the matrix or the immunogenicity of the HA protein itself. Although HA nanoparticles seem to retain the stability of the HA protein by inserting into the detergent core, the data thus suggest that HASMaN particles can provide a certain degree of protection for the HA protein structure and can also have a positive effect on the presentation to the immune system without the HA protein being embedded in the detergent core.

[0254] Example 7 - Sedimentation Coefficient Analysis

[0255] The sedimentation velocity (SV) was measured by analytical ultracentrifugation (AUC) to determine the particle size, and a tetravalent composition previously tested for stability in mice in Example 6 was analyzed. Figure 28 The results of Quad-NIV alone at 120 μg / mL / strain (Quad-NIV alone) are shown at 0, 3 months, 6 months, 9 months, and 12 months at 4°C; or the results of using Quad-NIV + 100 μg / mL M1 at 3 months, 6 months, 9 months, and 12 months at 4°C. The data show that over time, the amount of the rapidly sedimenting species (i.e., the structure appearing at higher values) increases, indicating the formation of HaSMaN from HA particles over time. Figure 29 The results of using Quad-NIV alone or in combination with 100 μg / mL M1 are shown at 0, 3 months, or 6 months at 25°C, and it is confirmed that the formation of HaSMaN is faster at higher temperatures.

[0256] Example 8 - Transmission Electron Microscopy (TEM) Analysis of HaSMaN Formation

[0257] As previously described, Figure 1Shows different structures of HA nanoparticles (Flu), matrix M, and HaSMaN (Flu-matrix interaction) observed using TEM. As expected, there is no formation of HaSMaN in HA nanoparticles. Specifically, HA nanoparticles exhibit a detergent core structure with HA proteins surrounding the core. Matrix M shows a cage-like structure that contains saponin fractions (fraction A or fraction C, but not both simultaneously), phospholipids, and cholesterol. In the HaSMaN structure, HA glycoproteins are attached to the cage-like matrix particles with the HA head portion extending outward.

[0258] Next, we observed the time course of various samples described below under TEM to visually evaluate the formation of HaSMaN under different conditions. Table 13 below shows the sample conditions and time points for testing the nanoparticle structures of pre-mixed Quad-NIV formulations (high dose HD and low dose LD) using TEM. The mark "X" indicates the sample conditions available at each time point. In addition, the LD formulation was tested at various temperatures at 1 month ( Figure 35 ).

[0259] Figures 30 - 34 Shows a representative image of the HaSMaN structure. 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 ( Figure 30 and Figure 31 ). By 24 hours, HaSMaN forms at both low temperature (4 °C) and high temperature (25 °C); however, the formation of HaSMaN was not observed in the high dose Quad-NIV at 4 °C. The high dose Quad-NIV shows the formation of HaSMaN at 48 hours at 4 °C ( Figure 33 ) and at day 7 ( Figure 34 ). The data also show that by 1 month, the HaSMaN structure remains in the LD group and is more prominent at higher temperatures, especially at 37 °C ( Figure 35 ).

[0260] Table 13. TEM images: Study design

[0261]

[0262]

[0263] Matrix M concentration: 100 μg / mL in all samples

[0264] These data show that the formation of HaSMaN requires co-incubation of HA nanoparticles with matrix M1 for more than about 4 hours. All conditions showed the formation of HASMaN within 48 hours. The formed HaSMaN particles are stable over the long term. Under conditions of higher temperature and lower dosage of Quad-NIV, the formation and development of HaSMaN are earlier.

[0265] This indicates that pre-filled syringe formulations containing HaSMaN can be stored at room temperature or higher temperatures (e.g., 37 °C), which will avoid the costs of low-temperature storage and transportation and prevent potential inconsistencies in mixing and preparing the vaccine just before vaccination at the clinic.

[0266] Example 9

[0267] Differential scanning calorimetry (DSC) curve of Quad-NIV

[0268] The stability of the Quad-NIV formulations described in Examples 6 and 7 was detected 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.

[0269] Figure 36 The formulations incubated at 25 °C showed a smaller shift in the melting point temperature (T m ) compared to the same group incubated at 4 °C, and had a wider peak width. Table 14 shows that the T m of the 120 μg / mL / strain Quad-NIV + matrix M1 formulation incubated at 25 °C was lower than that 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 for the formulation at 25 °C to reach the peak temperature was less than that at 4 °C, which further indicates that this formulation has relatively fewer HaSMaN complexes.

[0270] DSC curves and T m are a measure of the thermal stability of proteins. The results show that the formation of HaSMaN increases the T m of the HA protein by about 0.5 °C to about 1 °C, thus improving the thermal stability of the HA protein in HaSMaN compared to the detergent core nanoparticles.

[0271] Table 14. DSC curves of high-dose and low-dose Quad-NIV formulations

[0272]

[0273] Example 10 - A strain instead of B strain for HaSMaN

[0274] We studied the ability of HA glycoproteins from different influenza strains to form HaSMaN. As described in Example 1, detergent core nanoparticles were prepared and purified for the following strains: Type A: A / Hunan, A / Guangdong (both are H7N9 subtype) and A / Panama and A / Hong Kong / 4801 / 2014 (both are H3N2 subtype) and Type B: B / Brisbane / 60 / 2008.

[0275] The nanoparticles were mixed with Matrix M (85:15 w / w fraction A matrix and fraction C matrix) for up to 4 weeks. The final concentration of HA was 120 μg / mL (60 μg per A strain + 60 μg per B strain in 0.5 mL). Free nHA was measured. The results are as Figure 37 shown. The decrease in free nHA species corresponded to the formation of HaSMaN.

[0276] These data indicate that the HA glycoprotein of the Type A strain formed HaSMaN, while the HA glycoprotein of the Type B strain did 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.

[0277] The present invention further includes the following:

[0278] 1. A multivalent immunogenic influenza composition comprising:

[0279] (a) one or more detergent core nanoparticles, wherein the one or more detergent core nanoparticles comprise recombinant influenza hemagglutinin (HA) glycoproteins from one or more different Type B influenza strains;

[0280] (b) one or more hemagglutinin saponin matrix nanoparticles (HaSMaN), wherein the one or more HaSMaN are formed by combining recombinant influenza HA glycoproteins from one or more different Type A influenza strains with a saponin-based matrix adjuvant for at least 4 hours;

[0281] (c) a pharmaceutically acceptable buffer;

[0282] Wherein the composition comprises HA glycoproteins from at least 4 different influenza strains.

[0283] 2. The multivalent immunogenic influenza composition according to item 1, wherein one or more detergent core nanoparticles comprise detergent core particles containing recombinant HA glycoproteins from at least 2 different influenza B strains.

[0284] 3. The multivalent immunogenic influenza composition according to item 1, wherein one or more HaSMaN comprise HaSMaN containing recombinant HA glycoproteins from at least 2 different influenza A strains.

[0285] 4. The multivalent immunogenic influenza composition according to item 1, wherein each of the nanoparticles is an antitrypsin nanoparticle.

[0286] 5. The multivalent immunogenic influenza composition according to item 1, wherein the saponin-based matrix adjuvant comprises fraction A matrix and fraction C matrix.

[0287] 6. The multivalent immunogenic influenza composition according to item 5, wherein the fraction A matrix and fraction C matrix are present in a ratio of 85:15; w / w.

[0288] 7. The multivalent immunogenic influenza composition according to item 1, wherein the detergent core nanoparticles comprise PS80 as a detergent.

[0289] 8. The multivalent immunogenic influenza composition according to item 7, wherein the PS80 detergent is present at about 0.04%.

[0290] 9. The multivalent immunogenic influenza composition according to item 1, wherein one or more of the influenza A strains are 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.

[0291] 10. The multivalent immunogenic influenza composition according to item 1, wherein the pharmaceutically acceptable buffer comprises (i) about 25 mM sodium phosphate; (ii) about 150 mM sodium chloride; (iii) about 100 mM arginine hydrochloride; (iv) about 5% trehalose; wherein the pH of the composition is about 7.5.

[0292] 11. A pre-filled syringe or blow-fill sealed container comprising the multivalent immunogenic influenza composition according to item 1.

[0293] 12. The pre-filled syringe or blow-fill sealed container according to item 11, wherein the multivalent immunogenic influenza composition is stable for at least 12 months.

[0294] 13. The prefilled syringe or blow-fill sealed container according to item 11, wherein the multivalent immunogenic influenza composition is stable at 25 °C.

[0295] 14. Use of a multivalent immunogenic influenza composition according to any one of items 1-10 in the preparation of a medicament for stimulating an immune response against influenza.

[0296] 15. The use according to item 14, wherein the composition is administered intramuscularly.

[0297] 16. A composition comprising:

[0298] (i) A first HaSMaN (hemagglutinin saponin matrix nanoparticle) comprising:

[0299] (a) A first ISCOM matrix particle comprising fraction A matrix rather than fraction C matrix, and

[0300] (b) A first recombinant influenza HA glycoprotein trimer, wherein the HA glycoprotein tail region is associated with the first ISCOM matrix particle, and wherein the HA glycoprotein head extends distally from the first ISCOM matrix particle;

[0301] (iii) A pharmaceutically acceptable buffer or carrier;

[0302] (ii) A second HaSMaN, comprising:

[0303] (a) A second ISCOM matrix particle comprising fraction C matrix rather than fraction A matrix, and

[0304] (b) A second recombinant influenza HA glycoprotein trimer, wherein the HA glycoprotein tail region is associated with the second ISCOM matrix particle, and wherein the HA glycoprotein head extends distally from the second ISCOM matrix particle; and

[0305] (iii) A pharmaceutically acceptable buffer or carrier;

[0306] (iii) A pharmaceutically acceptable buffer or carrier;

[0307] wherein each of the first and second HA glycoproteins is from an influenza A strain; wherein each of the first and second glycoproteins comprises a transmembrane domain; and the first HaSMaN is formed by combining the first recombinant influenza HA glycoprotein trimer with the first ISCOM matrix particle for at least 4 hours, and / or the second HaSMaN is formed by combining the second recombinant influenza HA glycoprotein trimer with the second ISCOM matrix particle for at least 4 hours.

[0308] 17. The composition according to item 16, 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.

[0309] 18. The multivalent immunogenic influenza composition according to item 1, wherein the adjuvant is present in an amount of about 50 μg to about 75 μg per dose.

[0310] 19. The multivalent immunogenic influenza composition according to item 1, wherein the recombinant influenza HA glycoprotein from the influenza B strain and the recombinant influenza HA glycoprotein from the influenza A strain are both present in an amount of about 60 μg.

[0311] 20. The multivalent immunogenic influenza composition according to item 1, wherein the recombinant influenza HA glycoprotein from the influenza B strain and the recombinant influenza HA glycoprotein from the influenza A strain are both wild-type influenza HA glycoproteins.

[0312] 21. The multivalent immunogenic influenza composition according to item 1, wherein the recombinant influenza HA glycoprotein from the influenza B strain and the recombinant influenza HA glycoprotein from the influenza A strain are both expressed in a host cell.

[0313] 22. The multivalent immunogenic influenza composition according to item 21, wherein the host cell is an insect Sf9 cell.

[0314] 23. The multivalent immunogenic influenza composition according to item 21, wherein the recombinant influenza HA glycoprotein from the influenza B strain and the recombinant influenza HA glycoprotein from the influenza A strain are both expressed using a baculovirus.

[0315] 24. A method for preparing HaSMaN, which comprises combining a detergent core nanoparticle with a saponin-based matrix adjuvant for at least 4 hours, wherein the detergent core nanoparticle comprises an HA glycoprotein from an influenza A strain and a non-ionic detergent.

[0316] 25. The method according to item 24, wherein the saponin-based matrix adjuvant is an ISCOM matrix adjuvant.

[0317] 26. The method according to item 25, wherein the ISCOM matrix adjuvant comprises a fraction A matrix and a fraction C matrix.

[0318] 27. The method according to item 26, wherein the fraction A matrix and the fraction C matrix are present in a ratio of 85:15; w / w.

[0319] 28. The method according to item 24, wherein the detergent is PS80.

[0320] 29. The method according to item 25, which comprises incubating the detergent core nanoparticles with the saponin-based matrix adjuvant at a temperature of about 25 °C for at least about 24 hours.

[0321] 30. The method according to item 25, 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.

[0322] 31. The multivalent immunogenic influenza composition according to item 1, which further comprises one or more detergent core nanoparticles and one or more HaSMaNs, wherein each detergent core nanoparticle comprises a B strain HA glycoprotein, and each HaSMaN comprises an A strain HA glycoprotein, and wherein each HA glycoprotein is from at least 4 different influenza strains.

Claims

1. A multivalent immunogenic influenza composition comprising: (a) one or more detergent core nanoparticles, wherein said one or more detergent core nanoparticles comprise recombinant influenza hemagglutinin (HA) glycoproteins from one or more different influenza B strains; (b) one or more hemagglutinin saponin matrix nanoparticles (HaSMaN), wherein said one or more HaSMaN are formed by combining recombinant influenza HA glycoproteins from one or more different influenza A strains with a saponin-based matrix adjuvant for at least 4 hours; (c) a pharmaceutically acceptable buffer; wherein said composition comprises HA glycoproteins from at least 4 different influenza strains.

2. The multivalent immunogenic influenza composition according to claim 1, wherein said one or more detergent core nanoparticles comprise detergent core particles containing recombinant HA glycoproteins from at least 2 different influenza B strains.

3. The multivalent immunogenic influenza composition according to claim 1, wherein said one or more HaSMaN comprise HaSMaN containing recombinant HA glycoproteins from at least 2 different influenza A strains.

4. The multivalent immunogenic influenza composition according to claim 1, wherein each of said nanoparticles is an antirypsin nanoparticle.

5. The multivalent immunogenic influenza composition according to claim 1, wherein the saponin-based matrix adjuvant comprises fraction A matrix and fraction C matrix.

6. The multivalent immunogenic influenza composition according to claim 5, wherein said fraction A matrix and fraction C matrix are present in a ratio of 85:15; w / w.

7. The multivalent immunogenic influenza composition according to claim 1, wherein said detergent core nanoparticles comprise PS80 as a detergent.

8. The multivalent immunogenic influenza composition according to claim 7, wherein said PS80 detergent is present at about 0.04%.

9. The multivalent immunogenic influenza composition according to claim 1, wherein said one or more influenza A strains are 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.

10. The multivalent immunogenic influenza composition according to claim 1, wherein said pharmaceutically acceptable buffer comprises (i) about 25 mM sodium phosphate; (ii) about 150 mM sodium chloride; (iii) about 100 mM arginine hydrochloride; (iv) about 5% trehalose; wherein the pH of said composition is about 7.5.

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