Attenuated live influenza vaccine composition and method for preparing the same

Abstract

The present disclosure provides compositions and methods for manufacturing and obtaining live attenuated influenza vaccine (LAIV) compositions that can be delivered intranasally to provide protection against influenza virus infection. The LAIV strains are based on the cold-adapted, temperature-sensitive, and attenuated phenotypes of a master donor virus (MDV) comprising the surface glycoprotein genes of wild-type pandemic or seasonal influenza strains. Moreover, the LAIV strains are further adapted to grow in MDCK cells (Madin-Darby canine kidney cells). Eggs are avoided in large-scale vaccine production. The purification process has no chromatographic steps. The LAIV composition comprises one or more live attenuated influenza vaccine viruses and is free of polymers and surfactants.

Description

Technical Field

[0001] The present disclosure relates to the field of viral vaccine manufacture, and more particularly, to live attenuated influenza vaccine compositions and methods for preparing the same. The present disclosure relates to methods for producing viruses or viral antigens generated by cell culture, the viruses or viral antigens obtained by such methods, and vaccines comprising such viruses or viral antigens. Background Art

[0002] The background information hereinbelow is relevant to the present disclosure but not necessarily prior art.

[0003] Epidemics and pandemics caused by infectious pathogens have occurred for centuries, causing significant damage with varying morbidity and mortality. Influenza viruses have been one of the major players in pandemic history. Four influenza pandemics occurred in the last century, and at least 15 influenza pandemics have been recorded to date, with an estimated 50 million deaths in 1918 - 1919 alone.

[0004] Vaccines play an important role in the control of viral spread, and inactivated influenza vaccines (IIV) and live attenuated influenza vaccines (LAIV) have been used for many years. When the circulating strains are antigenically matched to the vaccine, the widely used parenterally administered inactivated influenza vaccines induce serum antibody responses and effectively prevent influenza disease. In contrast, live attenuated influenza vaccines (LAIV) are administered intranasally, mimic natural infection, and induce local and systemic humoral and cellular immune responses, providing protection against both the matched strains and drifted strains. Additionally, the needle - free administration of LAIV can lower the acceptance threshold and thus increase the coverage of influenza vaccines.

[0005] To date, LAIV has been licensed in the United States (since 2003), Europe (since 2010), Russia (since the 1980s), and India (since 2010). LAIV is based on master donor viruses (MDV) of attenuated influenza A and B developed independently in the United States and Russia in the 1960s in substantially the same manner. MedImmune seasonal and pandemic LAIV viruses are currently generated by reverse genetics (RG). In contrast, the Russian LAIV is generated by traditional genetic recombination in chicken embryos. The Russian LAIV is recently being registered for use in China and Thailand.

[0006] The Russian LAIV consists of reassortant viruses that contain the hemagglutinin (HA) gene segment and in most cases the neuraminidase (NA) gene segment from a circulating wild-type (WT) virus of interest on the backbone of the remaining six internal protein genes (PB1, PB2, PA, NP, M, and NS) from (MDV). A / Leningrad / 134 / 17 / 57 (H2N2) (Len-MDV) and B / USSR / 60 / 69 MDV are currently used in Russia as the MDV for LAIV. The MDVs contain mutations in multiple gene segments that render them cold-adapted (ca), temperature-sensitive (ts), and attenuated (att). The surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) of contemporary strains are incorporated into these MDVs by reassortment. Their ca, ts, and att phenotypes indicate that LAIV replicates at low temperatures and stops replicating at higher temperatures (>38 °C), restricting replication to the upper respiratory tract. Compared to the MDVs used in the United States, the Russian MDVs contain fewer mutations present at different sites in the gene segments, suggesting that they may be attenuated differently. Direct comparisons in animals and humans suggest that the Russian MDV and its derived single-strain reassortants are more immunogenic than their equivalents.

[0007] For many years, the Russian LAIV has been safely administered to over 75 million people and has proven to be safe in terms of attenuation (genetic stability) and spread. Reversion of virulence (loss of the attenuated phenotype) has never been observed and is highly unlikely to occur because it would require the reversion of multiple mutations in multiple gene segments. Neurotoxicity of the Russian LAIV virus has never been reported, and both the MDV and its derived reassortants have been shown to not possess neurotoxic properties. After administration of LAIV, no reports of serious immune-related adverse events have been made, except for self-limiting influenza-like symptoms (runny nose, nasal congestion, sore throat, cough, headache, and low-grade fever) reported in a few cases. In addition to being safe, LAIV has been shown to be effective in preventing disease caused by influenza virus infection. The immunity induced by LAIV is broad and has been shown to provide protection against drifted strains. Particularly in children, LAIV has been shown to be more effective than inactivated influenza vaccine in terms of clinical protection and / or protection against culture-confirmed influenza and has also been shown to have herd immunity effects.

[0008] Like the inactivated influenza vaccine, the Russian LAIV is produced using chicken embryos. Its inherent drawbacks limit the vaccine quality suppliers, and eggs free of specific pathogens need to be ordered at least 4 months in advance before they can be used for large-scale vaccine production. The specialized facilities for egg incubation, harvesting, etc. limit the ability to rapidly scale up. The extended time required to produce egg-dependent vaccines may result in too few available doses to respond to pandemic situations (such as what happened in 2009) or to stop a pandemic originating from highly pathogenic influenza viruses. Therefore, the current vaccine production system is insufficient to cope with influenza pandemics, and a new type of rapid emergency vaccine production process is needed. Theoretically, producing influenza vaccines in cell culture offers important advantages in the case of a pandemic. The large-scale production of vaccines can be easily achieved using pre-existing tissue culture manufacturing units for other viral vaccines.

[0009] The availability of controlling substrate consistency and production flexibility, rapid scale-up, which is particularly important in the case of a pandemic, and not relying on egg supply and the maintenance of chicken flocks are the main advantages of the tissue culture method. The eggshell is a porous structure and the outside of the egg is not sterile. The manufacturing process of culturing influenza viruses in eggs requires piercing the eggshell for inoculation and open handling for harvesting, which has an inherent risk of contamination. In addition, cell culture is a more controlled system with a defined cell culture medium and a validated cell bank compliant with Good Manufacturing Practice (GMP). Therefore, attempts have been made to transfer production from eggs to cell culture.

[0010] Several cell lines are currently being investigated for use in the cell culture-based production of influenza virus, and the use of MRC-5 cells (reference: de Ona et al. (1995) J Clin Microbiol 33:1948-49), HepG2 cells (reference: Ollier et al. (2004) J Clin Microbiol 42(12):5861-5), LLC-MK2 cells (reference: Schepetiuk and Kok (1993) J Virol Methods 42(2-3):241-50), Madin-Darby canine kidney (MDCK) cells (reference: Tobita et al. (1975) Med Microbiol Immunol (Berl). 162(1):9-14 and 23-27), African green monkey Vero cells (reference: Monto et al. (1981) J Clin Microbiol 13(1):233-235 and Govorkova et al. (1995) J Infect Dis. 172(1):250-3) and PER.C6 cells (reference: Cox, R.J. et al.; Vaccine 2009, 27, 1889-1897) has been reported. Previously, for MRC-5, WI-38 and FRhL cells, viruses with low to moderate titers equal to or lower than 5.0 log 10 TCID 50 / ml have been reported, while for MDCK cells, titers up to 6.7 log 10 TCID 50Virus titers of / mL. Although cell culture-derived influenza vaccines (egg-derived influenza viruses adapted / optimized for cell culture growth) have been licensed in Europe (Optaflu Novartis; 2007) and the United States (Flucelvax Novartis; 2012), only a small fraction of influenza vaccines on the market are cell culture-derived. This may be due to inconsistent yields of influenza vaccines using cell culture production systems in combination with conventional purification methods and cumbersome stabilization practices. Theoretical safety concerns associated with the use of continuous cell lines such as MDCK cells have been raised in relation to their use in vaccine production (VRBPAC, 2008). These concerns are mainly related to residual cellular components (DNA and proteins) in the vaccine drug product, especially for live attenuated influenza vaccine (LAIV) products that, like traditional inactivated influenza vaccines, are neither inactivated nor extensively biochemically purified. Two strategies have been adopted to minimize these risks: cell line characterization and vaccine purification processing steps. A specific aspect of the purification process is to reduce the quantity and size of residual host cell DNA in the vaccine product. The manufacture of Optaflu Novartis (2007) includes multiple steps to eliminate residual host cell DNA. These include cellulose sulfate ion exchange chromatography that binds influenza virus and allows DNA to pass through and subsequent CTAB precipitation steps, especially for precipitating DNA.

[0011] Previously reported purification processes are costly and time-consuming because they employ one or more chromatographic methods from hydroxyapatite, affinity, anion exchange, and size exclusion. The overall recovery of virus has been reported to be unsatisfactory for conventional vaccine production using chromatographic methods. In addition, due to antigenic drift and antigenic shift observed in this virus, the antigenic characteristics of virus particles change for circulating influenza viruses. These changes affect the physicochemical properties of the virus and, in turn, affect the binding ability of the virus to the chromatographic matrix. This can lead to highly variable yields of drifted or shifted strains, rendering chromatographic methods unsuitable for conventional production. Another method is used to remove / reduce host cell DNA.

[0012] Using higher concentrations of Benzonase (e.g., 50 U / ml, 100 U / ml), which is expensive.

[0013] Typical nonionic surfactants used in pharmaceutical formulations include Triton TM X-100, F-68, F-88, and F-127 (poloxamer), Brij 35 (polyoxyethylene alkyl ether), polyoxyethylene stearate 40, EL and α-tocopherol TPGS. Each of these surfactants has a common fact that they all contain a polyoxyethylene moiety and thus more or less exhibit similar problems, that is, the polyoxyethylene moiety will auto-oxidize to produce reactive peroxides, which will lead to an increase in unwanted protein immunogenicity (see Edward T. Maggio et al.; Polysorbates, peroxides, protein aggregation, immunogenicity - a growing concern; Journal of Excipients and Food Chemicals 3(2):46 - 53; 2012).

[0014] Various stabilizers are used to stabilize vaccine formulations to achieve the desired shelf life. Stabilizers such as polyvinylpyrrolidone (PVP), trehalose, and sorbitol are also used in viral formulations. However, it has been reported that PVP disrupts the stability of live attenuated virus formulations. (Reference: JA White et al.; Development of a stable liquid formulation of live - attenuated influenza vaccine; Vaccine Volume 34, Issue 32, 12 July 2016, Pages 3676 - 3683; 2016).

[0015] Trehalose is costly; it must be combined with other sugars and protein additives (gelatin) to achieve stability. Moreover, other stabilizers are superior to trehalose in enhancing the shelf - life stability of freeze - dried vaccines.

[0016] Sorbitol has a low glass transition temperature (Tg) (-1.6 °C), so it cannot be used as a main formulation component. The low Tg of sorbitol limits its use. Sorbitol must be combined with other sugars and protein additives (gelatin) to achieve stability.

[0017] It has been suggested that for vaccine administration routes, the theoretical impact of host residual DNA on product safety should be considered because tissue distribution and clearance rates can vary based on the mode of administration. Studies have shown that the absorption and clearance of MDCK DNA from tissues differ depending on the administration route. When DNA is administered intranasally compared to intramuscular injection, detectable DNA levels are lower at all time points. Therefore, the intranasal administration route of vaccines seems to reduce the potential risk associated with residual host cell DNA that may be present in the final vaccine products produced by cell culture. (Reference: D.E. Tabor et al.; Biologicals 41(2013)247 - 253).

[0018] The object of the present disclosure is to overcome the above limitations and also provide compositions and methods for manufacturing Madin - Darby canine kidney (MDCK) cells for an intranasally delivered live attenuated influenza vaccine (LAIV) to prevent influenza virus. The present disclosure further provides an improved manufacturing method that utilizes a low concentration of endonuclease, more specifically Benzonase, and has no chromatographic steps suitable for large - scale cell culture production. Further still, the present disclosure provides an LAIV formulation that does not contain polymers and surfactants and includes one or more live attenuated influenza vaccine viruses. Summary of the Invention

[0019] The present disclosure provides an intranasally delivered live attenuated influenza vaccine (LAIV) composition based on MDCK cells, comprising:

[0020] a) one or more live attenuated influenza vaccine viruses;

[0021] wherein the live attenuated influenza vaccine strain is derived by a reassortment "classical" or "reverse genetics" method and consists essentially of a hemagglutinin (HA) gene and / or a neuraminidase (NA) gene from a wild - type pandemic or seasonal influenza virus and genes encoding PB1, PB2, PA, NP, M, and NS proteins, and in some cases, the NA protein is derived from the master donor virus (MDV) A / Leningrad / 134 / 17 / 57 (H2N2) and / or B / USSR / 60 / 69 strain,

[0022] b) one or more amino acids,

[0023] c) one or more carbohydrates, and

[0024] d) gelatin.

[0025] The present disclosure further provides a method for manufacturing such a vaccine composition / formulation.

[0026] Objectives

[0027] Some objectives of the present disclosure met by at least one embodiment herein are as follows:

[0028] The object of the present disclosure is to alleviate one or more problems of the prior art or at least provide a useful alternative.

[0029] Another object of the present disclosure is to provide a vaccine composition and a method for manufacturing a live attenuated influenza vaccine (LAIV) that can be delivered intranasally.

[0030] Yet another object of the present disclosure is to provide a live attenuated influenza vaccine (LAIV) composition based on Madin - Darby canine kidney (MDCK) cells, in which the use of eggs is completely avoided.

[0031] Another object of the present disclosure is to provide a LAIV composition comprising one or more live attenuated influenza vaccine viruses and free of polymers and surfactants.

[0032] Another object of the present disclosure is to provide a LAIV composition comprising one or more live attenuated influenza vaccine viruses, wherein the LAIV strain is based on the cold adapted, temperature sensitive, and attenuated phenotypes of a master donor virus (MDV), and the master donor virus (MDV) comprises surface glycoproteins of one or two wild-type pandemic or seasonal influenza strains.

[0033] Another object of the present disclosure is to provide a live attenuated influenza vaccine (LAIV) composition for intranasal delivery based on MDCK cells, wherein the composition retains the desired characteristics of the virus, including immunogenicity and stability.

[0034] Another object of the present disclosure is to provide a live attenuated influenza vaccine (LAIV) composition / formulation for intranasal delivery based on MDCK cells, which is suitable for treating or preventing influenza virus infection, or preventing, alleviating, or delaying the onset or progression of its clinical manifestations.

[0035] Another object of the present disclosure is to provide an improved method suitable for large-scale cell culture production in the field of production of live attenuated influenza vaccines based on MDCK cells.

[0036] Other objects and advantages of the present disclosure will become more apparent in the following description which is not intended to limit the scope of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The present disclosure will now be described with the aid of the accompanying drawings listed below:

[0038] Figure 1 A schematic diagram illustrating the generation of a reassortant vaccine strain by the reassortment of wild-type pandemic or seasonal influenza viruses and attenuated MDV, where (A) represents a wild-type viral infectious pathogen, (B) represents a master donor virus comprising temperature sensitive (ts), cold adapted (ca), and attenuated (at) phenotypic gene segments, and (C) represents a reassortant vaccine strain.

[0039] Figure 2 : The chart illustrates the stability at 37 °C of the viral log10 yield titer (EID 50 / 0.5 ml) of the influenza A strain (A / 17 / Turkey / Turkey / 05 / 133-A / H5N2) and the stabilizer composition 1 disclosed in Table 3B.

[0040] Figure 3 : The chart illustrates the viral log10 yield titer (EID of influenza A strains (A / 17 / Turkey / Turkey / 05 / 133-A / H5N2 and A / 17 / Anhui / 2013 / 61-A / H7N9)50 The stability of (××× / 0.5 ml) and Stabilizer Composition 1 disclosed in Table 3A at 2°C to 8°C.

[0041] Figure 4 : The graph illustrates the log10 virus yield titers (EID of influenza A virus strain (A / 17 / California / 2009 / 38 - A / H1N1) and influenza B virus strain (B / Texas / 02 / 13 - CDC). 50 The stability of (××× / 0.5 ml) and Stabilizer Composition 3 disclosed in Table 3B at 37°C.

[0042] Figure 5 : The graph illustrates the log10 virus yield titers (EID of influenza A virus strain (A / 17 / California / 2009 / 38 - A / H1N1) and influenza B virus strain (B / Texas / 02 / 13 - CDC). 50 The stability of (××× / 0.5 ml) and Stabilizer Composition 3 disclosed in Table 3A at 2°C to 8°C.

[0043] Figure 6 : The graph illustrates the log10 virus yield titers (EID of influenza A virus strain (A / 17 / California / 2009 / 38 - A / H1N1) and influenza B virus strain (B / Texas / 02 / 13 - CDC). 50 The stability of (××× / 0.5 ml) and Stabilizer Composition 4 disclosed in Table 3B at 37°C.

[0044] Figure 7 : The graph illustrates the log10 virus yield titers (EID of influenza A virus strain (A / 17 / California / 2009 / 38 - A / H1N1) and influenza B virus strain (B / Texas / 02 / 13 - CDC). 50 The stability of (××× / 0.5 ml) and Stabilizer Composition 4 disclosed in Table 3A at 2°C to 8°C.

[0045] Figure 8 : The graph illustrates the log10 virus yield titers (EID of influenza A virus strain (A / South Africa / 3626 / 13 - H1N1) and influenza B virus strain (B / Texas / 02 / 13 - CDC). 50 The stability of (××× / 0.5 ml) and Stabilizer Composition 2 disclosed in Table 3B at 37°C.

[0046] Figure 9 : The graph illustrates the log10 virus yield titers (EID of influenza A virus strain (A / South Africa / 3626 / 13 - H1N1) and influenza B virus strain (B / Texas / 02 / 13 - CDC). 50 The stability of (××× / 0.5 ml) and Stabilizer Composition 2 disclosed in Table 3A at 2°C to 8°C.

[0047] Figure 10 : The figure illustrates the stability at 37°C of the viral log10 productivity titers (EID 50 / 0.5 ml) of influenza A virus strain (A / South Africa / 3626 / 13-H1N1) and influenza B virus strain (B / Texas / 02 / 13-CDC) with stabilizer composition 1 disclosed in Table 3B.

[0048] Figure 11 : The figure illustrates the stability at 2°C to 8°C of the viral log10 productivity titers (EID 50 / 0.5 ml) of influenza A virus strain (A / South Africa / 3626 / 13-H1N1) and influenza B virus strain (B / Texas / 02 / 13-CDC) with stabilizer composition 1 disclosed in Table 3A.

[0049] Figure 12 : Testing of virus infection in turbinate and lung samples. Animals were inoculated with one or two doses of H5 LAIV, H7 LAIV, or placebo. Groups 1 to 4 were challenged with H5 / tk / Tk, groups 5 and 6 were challenged with H5 / Vt, and groups 7 to 10 were challenged with H7 / An. Turbinate samples (a) and lung samples (b) collected 4 days post-infection were titrated to determine the presence of replicable virus particles. Individual titers are shown as group means indicated by the solid black line.

[0050] Figure 13 : HI and VN antibody responses after immunization. Geometric mean antibody responses against the homologous challenge viruses H5 / tk / Tk for animals immunized with H5 LAIV and H7 / An for animals immunized with H7 LAIV on day 28 after the final immunization (day 28 for the one-dose regimen study and day 56 for the two-dose regimen study). (a) HI antibody titers, (b) VN antibody titers. N = 6 per group, error bars represent standard error of the mean. Statistical significance was determined by the Mann-Whitney U test. *p < 0.05 and **p < 0.01. Detailed Description

[0051] Although the present disclosure may be embodied in specifically different embodiments, certain embodiments are shown in the accompanying drawings and in the following detailed description. It should be understood that the present disclosure is considered an example of the principles of the present disclosure and is not intended to limit the scope of the disclosure to that set forth and disclosed in this specification.

[0052] Embodiments of the present disclosure will now be described with reference to the accompanying drawings.

[0053] Embodiments are provided to repeatably and completely convey the scope of the present disclosure to those skilled in the art. A large number of details related to specific components and methods are set forth to provide a complete understanding of the embodiments of the present disclosure. It will be apparent to those skilled in the art that the details provided in the embodiments should not be construed as limiting the scope of the present disclosure. In some embodiments, well-known processes, well-known device structures, and well-known technologies are not described in detail.

[0054] The terms used in the present disclosure are for the purpose of explaining specific embodiments only, and such terms should not be considered as limiting the scope of the present disclosure. Unless the context clearly indicates otherwise, as used in the present disclosure, the forms "a", "an", and "the" may also be intended to include the plural forms.

[0055] The terms first, second, third, etc. should not be construed as limiting the scope of the present disclosure, as the foregoing terms may be used only to distinguish one element, component, region, layer, or part from another component, region, layer, or part. Unless the present disclosure clearly indicates otherwise, when used herein, terms such as first, second, third, etc. do not denote a specific sequence or order.

[0056] As used herein, the term "influenza virus" refers to RNA viruses including influenza A, B, C, and D viruses representing the family Orthomyxoviridae. The influenza virus can be a live wild-type pandemic or seasonal influenza virus, a live attenuated influenza vaccine virus, an inactivated influenza virus, a chimeric influenza virus, or a recombinant influenza virus.

[0057] The present disclosure provides a composition comprising a live attenuated influenza vaccine (LAIV) virus for protection against influenza virus infection. The present disclosure further provides a method for manufacturing a composition comprising one or more influenza vaccine viruses.

[0058] According to a first embodiment of the present disclosure, the LAIV composition may comprise one or more live attenuated influenza vaccine viruses, one or more amino acids, one or more carbohydrates, and gelatin.

[0059] The term "live" is used in its conventional meaning, and a live virus is a virus that has not been inactivated, that is, a virus capable of replicating on permissive cells. A live attenuated influenza vaccine virus is a virus that does not induce the disease caused by the corresponding wild-type virus in animals or humans and is capable of inducing a specific immune response.

[0060] According to a second embodiment of the present disclosure, one or more live attenuated influenza vaccine viruses may be derived by a "classical" or "reverse genetics" rearrangement method comprising gene segments from one or more influenza strains.

[0061] According to the first aspect of the second embodiment, the reassortant live attenuated influenza vaccine virus is a reassortant LAIV virus that comprises cold-adapted, temperature-sensitive and / or attenuated phenotypic gene segments (PB1, PB2, PA, NP, M and / or NS proteins) of a master donor virus (MDV) strain and a hemagglutinin (HA) gene segment and / or a neuraminidase (NA) gene segment of a wild-type pandemic or seasonal influenza A virus or influenza B virus or influenza C virus strain in a ratio of 1:7, 2:6, 3:5, 4:4, 5:3, 6:2 or 7:1.

[0062] Still preferably, the reassortant live attenuated influenza vaccine virus may comprise gene segments from a master donor virus (MDV) strain and the wild-type pandemic or seasonal influenza virus strain (as Figure 1 illustrated) in a ratio of 6:2.

[0063] According to the second aspect of the second embodiment, the reassortant live attenuated influenza vaccine virus may comprise gene segments of a master donor virus (MDV) derived from any subtype of influenza A virus or any subtype of influenza B virus from which it can be derived.

[0064] The reassortant live attenuated influenza vaccine virus may comprise gene segments from a master donor virus (MDV) selected from the group comprising the influenza A strain A / Leningrad / 134 / 17 / 57 (H2N2) and the influenza B strain B / USSR / 60 / 69.

[0065] Still preferably, the reassortant live attenuated influenza A vaccine virus may comprise gene segments from a master donor virus (MDV) comprising the influenza A strain A / Leningrad / 134 / 17 / 57 (H2N2).

[0066] Still preferably, the reassortant live attenuated influenza A vaccine strain A / 17 / California / 2009 / 38 may comprise gene segments from a master donor virus (MDV) comprising the influenza A strain A / Leningrad / 134 / 17 / 57 (H2N2).

[0067] Still preferably, the reassortant live attenuated influenza A vaccine strain A / 17 / Turkey / Turkey / 05 / 133 may comprise gene segments from a master donor virus (MDV) comprising the influenza A strain A / Leningrad / 134 / 17 / 57 (H2N2).

[0068] Still preferably, the reassortant live attenuated influenza A vaccine strain A / 17 / Anhui / 2013 / 61 may comprise gene segments from a master donor virus (MDV) comprising the influenza A strain A / Leningrad / 134 / 17 / 57 (H2N2).

[0069] Still preferably, the reassortant attenuated live influenza vaccine A strain A / 17 / New York / 15 / 5364 may include gene segments from a master donor virus (MDV) that includes influenza A / Leningrad / 134 / 17 / 57 (H2N2).

[0070] Still preferably, the reassortant attenuated live influenza vaccine A strain A / 17 / Hong Kong / 2014 / 8296 may include gene segments from a master donor virus (MDV) that includes influenza A / Leningrad / 134 / 17 / 57 (H2N2).

[0071] Still preferably, the reassortant attenuated live influenza vaccine A strain A / South Africa / 3626 / 2013-CDC-LV14A may include gene segments from a master donor virus (MDV) that includes influenza A / Leningrad / 134 / 17 / 57 (H2N2).

[0072] Still preferably, the reassortant attenuated live influenza B vaccine virus may include gene segments from a master donor virus (MDV) that includes influenza B strain B / USSR / 60 / 69.

[0073] Still preferably, the reassortant attenuated live vaccine influenza B strain B / Texas / 02 / 2013-CDC-LV8B may include gene segments from a master donor virus (MDV) that includes influenza B strain B / USSR / 60 / 69.

[0074] Still preferably, the reassortant attenuated live vaccine influenza B strain B / Phuket / 3073 / 2013 may include gene segments from a master donor virus (MDV) that includes influenza B strain B / USSR / 60 / 69.

[0075] Still preferably, the reassortant attenuated live vaccine influenza B strain B / 56 / Brisbane / 60 / 08 may include gene segments from a master donor virus (MDV) that includes influenza B strain B / USSR / 60 / 69.

[0076] According to the third aspect of the second embodiment, the reassortant attenuated live influenza vaccine virus may include the hemagglutinin (HA) gene and / or the neuraminidase (NA) gene from an influenza A virus or an influenza B virus or an influenza C virus.

[0077] Still preferably, the reassortant live attenuated influenza A vaccine strain may comprise a hemagglutinin (HA) gene from any other reported HA subtype of influenza A virus HA subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17 or H18 and / or a neuraminidase (NA) gene from any other reported NA subtype of influenza A virus NA subtypes N1, N2, N3, N4, N5, N6, N7, N8, N9, N10 or N11.

[0078] Still preferably, the reassortant live attenuated influenza vaccine virus may comprise a hemagglutinin (HA) gene and / or a neuraminidase (NA) gene from a pandemic influenza strain or a potential pandemic influenza strain.

[0079] Still preferably, the reassortant live attenuated influenza vaccine virus may comprise a hemagglutinin (HA) gene and / or a neuraminidase (NA) gene from a seasonal influenza strain.

[0080] Still preferably, the reassortant live attenuated influenza vaccine strain may comprise a hemagglutinin (HA) gene and / or a neuraminidase (NA) gene from influenza A virus subtypes H1N1, H2N2, H3N2, H5N1, H5N2, H9N2, H7N1, H7N3, H7N7, H6N1, H7N9 and H10N8 or any other previously reported or newly detected strain.

[0081] Still preferably, the reassortant live attenuated influenza vaccine strain may comprise a hemagglutinin (HA) gene and / or a neuraminidase (NA) gene from the influenza A virus pdmH1N1 strain.

[0082] Still preferably, the reassortant live attenuated influenza vaccine virus strain may comprise a hemagglutinin (HA) gene and / or a neuraminidase (NA) gene from an influenza A virus A / California / 07 / 2009 (referred to as A / Cal) - like strain.

[0083] Still preferably, the reassortant live attenuated influenza vaccine virus strain may comprise a hemagglutinin (HA) gene and / or a neuraminidase (NA) gene from an influenza A virus A / Michigan / 45 / 2015 - like strain.

[0084] Still preferably, the reassortant live attenuated influenza vaccine virus strain may comprise a hemagglutinin (HA) gene and / or a neuraminidase (NA) gene from an influenza A virus strain A / South Africa / 3626 / 2013 - like strain.

[0085] Still preferably, the reassorted live attenuated influenza vaccine virus strain may comprise the hemagglutinin (HA) gene and / or the neuraminidase (NA) gene from the influenza A virus H3N2-A / Hong Kong / 4801 / 2014-like strain.

[0086] Still preferably, the reassorted live attenuated influenza vaccine virus strain may comprise the hemagglutinin (HA) gene and / or the neuraminidase (NA) gene from influenza B viruses belonging to two different lineages, Yamagata-like or Victoria-like.

[0087] Still preferably, the reassorted live attenuated influenza vaccine virus strain may comprise the hemagglutinin (HA) gene and / or the neuraminidase (NA) gene from the influenza B virus Victoria lineage B / Brisbane / 60 / 2008-like strain.

[0088] Still preferably, the reassorted live attenuated influenza vaccine virus strain may comprise the hemagglutinin (HA) gene and / or the neuraminidase (NA) gene from the influenza B virus Yamagata lineage B / Phuket / 3073 / 2013-like strain.

[0089] There are two methods for generating reassortants

[0090] 1. The typical reassortment method

[0091] The reassortment process of the wild-type pandemic or seasonal influenza vaccine virus of the reassortant strain and the attenuated MDV involves co-infecting a culture host (usually eggs) with the MDV strain and the wild-type virus strain. The reassortant virus is selected by adding antibodies specific to the HA and / or NA proteins of MDV so as to select the reassortant virus containing the HA and / or NA proteins of the wild-type virus strain. After several generations of such treatment, a fast-growing reassortant virus containing the HA and / or NA segments of the wild-type pandemic or seasonal influenza vaccine virus strain and the internal genes of MDV can be selected.

[0092] 2. The reverse genetics method of reassortment

[0093] Reverse genetics is a method of generating infectious virus particles from DNA replication. The six internal genes of MDV and the HA and NA genes from the wild-type strain recommended for inclusion in the vaccine are cloned in plasmids, which can produce full-length viral RNA when transfected in cultured cells. These plasmids are transfected into cultured cells together with four plasmids expressing viral polymerase subunits. The expression of the polymerase subunits and the production of full-length viral RNA result in virus assembly and the release of infectious virus particles in the supernatant. This rescued virus exhibits the antigenic characteristics of the recommended strain as well as the ca, ts, att phenotypes of MDV.

[0094] The reassorted LAIV strains were purchased from the Institute of Experimental Medicine (IEM) in St. Petersburg, Russia or WHO Collaborating Centers such as the Centers for Disease Control and Prevention (CDC) in Atlanta.

[0095] According to a third embodiment of the present disclosure, the LAIV composition may include one or more carbohydrates selected from, but not limited to, the group consisting of: natural carbohydrates, synthetic carbohydrates, polyols, glass transition accelerators, monosaccharides, disaccharides, trisaccharides, oligosaccharides, and their corresponding sugar alcohols, polyhydroxy compounds such as carbohydrate derivatives and chemically modified carbohydrates, hydroxyethyl starch, and sugar copolymers. Both natural carbohydrates and synthetic carbohydrates are suitable for use. Synthetic carbohydrates include, but are not limited to, those in which the glycosidic bond is replaced by a thiol bond or a carbon bond. Carbohydrates in both D-form and L-form may be used. The carbohydrates may be non-reducing or reducing. When using reducing carbohydrates, it is preferred to add Maillard reaction inhibitors. Reducing carbohydrates suitable for use in the composition are those known in the art and include, but are not limited to, glucose, sucrose, maltose, lactose, fructose, galactose, mannose, maltulose, and lactulose. Non-reducing carbohydrates include, but are not limited to, non-reducing glycosides of polyhydroxy compounds selected from sugar alcohols and other straight-chain polyols. Other useful carbohydrates include raffinose, stachyose, melezitose, dextran, cellobiose, mannotriose, and sugar alcohols. Sugar alcohol glycosides are preferably monoglycosides, especially compounds obtained by reducing disaccharides such as lactose, maltose, lactulose, and maltulose. Glass formers are selected from the group consisting of: sucrose, mannitol, trehalose, mannose, raffinose, lactitol, lactobionic acid, glucose, maltulose, isomaltulose, maltose, lactosorb, glucose, fructose, glycerol, or combinations thereof.

[0096] However, according to a preferred aspect of the third embodiment, the LAIV composition may include sucrose in the range of 1% and 20% weight / volume, preferably between 1% - 10%, more preferably between 3% - 6%, and most preferably equal to 4% (w / v) as a suitable carbohydrate stabilizer.

[0097] According to a fourth embodiment of the present disclosure, the LAIV composition may include one or more amino acids selected from, but not limited to, the group consisting of: tris(hydroxymethyl)methylglycine, arginine, leucine, isoleucine, histidine, glycine, glutamine, lysine, alanine, peptides, hydrolyzed proteins, or proteins such as serum albumin.

[0098] Still according to a preferred aspect of the fourth embodiment, the LAIV composition may include, alone or in combination, tris(hydroxymethyl)methylglycine, arginine, histidine, and alanine as suitable amino acids.

[0099] Still according to a preferred aspect of the fourth embodiment, one or more amino acids may include tris(hydroxymethyl)methylglycine in the range of between 0.1% and 2% weight / volume (w / v), preferably between 0.1% - 1%, more preferably between 0.1% - 0.5%, and most preferably equal to 0.3% (w / v).

[0100] Still according to a preferred aspect of the fourth embodiment, one or more amino acids may include histidine in the range of between 0.1% and 2% (w / v), preferably between 0.1% - 1%, more preferably between 0.1% - 0.5%, and most preferably equal to 0.21% (w / v).

[0101] Still according to a preferred aspect of the fourth embodiment, one or more amino acids may include alanine in the range of between 0.01% and 1% weight / volume, preferably between 0.05% - 0.5%, more preferably between 0.08% - 0.2%, and most preferably equal to 0.1% (w / v).

[0102] And, according to a preferred aspect of the fourth embodiment, the one or more amino acids may include arginine in the range of between 0.1% and 10% weight / volume, preferably between 0.1 - 5%, more preferably between 0.1 - 3%, and most preferably equal to 2.1% (w / v).

[0103] According to a fifth embodiment of the present disclosure, the LAIV composition may include gelatin in the range of between 0.1% and 10% weight / volume, preferably between 0.1% - 5%, more preferably between 0.1% - 3%, and most preferably equal to 0.85% (w / v).

[0104] As used herein, the term "gelatin" refers to a sterile and pyrogen - free protein preparation (e.g., fraction) produced by partial acid hydrolysis (type A gelatin) or partial alkali hydrolysis (type B gelatin) of animal collagen, with the most common sources being bovine, porcine, and fish. Gelatin with different molecular weight ranges can be obtained. Recombinant gelatin sources can also be used.

[0105] According to a sixth embodiment of the present disclosure, the LAIV composition may additionally include a buffer selected from the group consisting of: HEPES, citrate - phosphate, carbonate, phosphate, citrate, lactate, gluconate, and tartrate buffers, as well as more complex organic buffers including phosphate buffers, where the phosphate buffer contains sodium phosphate and / or potassium phosphate in selected ratios to achieve the desired pH value. In another example, the buffer contains tris(hydroxymethyl)aminomethane or "Tris", which is formulated to achieve the desired pH value. In yet another example, the buffer can be minimal essential medium with Hank's salts.

[0106] According to a seventh embodiment of the present disclosure, the single-dose composition is free of preservatives, and the multi-dose composition may additionally include a preservative selected from the group consisting of: 2-phenoxyethanol, benzethonium chloride (Phemerol), phenol, m-cresol, thimerosal, formaldehyde, parabens (e.g., methyl-, ethyl-, propyl-, or butyl-paraben), benzalkonium chloride, benzyl alcohol, chlorobutanol, parachloro-m-cresol or benzyl alcohol or a combination thereof. The vaccine composition may include materials for single immunization, or may include materials for multiple immunizations (i.e., a "multi-dose" kit). A preservative is preferably included in the multi-dose arrangement. As an alternative to (or in addition to) including a preservative in the multi-dose composition, the composition may be contained in a container having a sterile adapter for removing the material.

[0107] According to an eighth embodiment of the present disclosure, the LAIV composition may additionally include a pharmaceutically acceptable transporter, excipient, binder, carrier, isotonic agent, emulsifier or wetting agent, wherein the pharmaceutically acceptable excipient is selected from the group consisting of: surfactants, polymers and salts. Examples of surfactants may include nonionic surfactants such as polysorbate 20, polysorbate 80, etc. Examples of polymers may include dextran, carboxymethyl cellulose, hyaluronic acid, cyclodextrin, etc. Examples of salts may include NaCl, KCl, KH2PO4, Na2HPO4·2H2O, CaCl2, MgCl2, etc.

[0108] According to a ninth embodiment of the present disclosure, the LAIV composition may additionally include an adjuvant selected from the group consisting of: aluminum hydroxide, aluminum phosphate, hydroxyaluminum phosphate and aluminum potassium sulfate or a mixture thereof.

[0109] According to a tenth embodiment of the present disclosure, the LAIV composition additionally includes an immunostimulatory component selected from the group consisting of: oil-in-water emulsions, MF-59, liposomes, lipopolysaccharides, saponins, lipid A, lipid A derivatives, monophosphoryl lipid A, 3-deacylated monophosphoryl lipid A, AS01, AS03, oligonucleotides, oligonucleotides including at least one unmethylated CpG and / or liposomes, Freund's adjuvant, Freund's complete adjuvant, Freund's incomplete adjuvant, polymers, copolymers such as polyoxyethylene-polyoxypropylene copolymers, including block copolymers, polymer p1005, CRL-8300 adjuvant, muramyl dipeptide, TLR-4 agonist, flagellin, flagellin derived from Gram-negative bacteria, TLR-5 agonist, fragments of flagellin capable of binding to the TLR-5 receptor, α-C-galactosylceramide, chitosan, interleukin-2, QS-21, ISCOMs, combinations of saponins with sterols and lipids.

[0110] According to an eleventh embodiment of the present disclosure, a method of preparing a LAIV composition based on an MDCK cell culture may include any subset or all of the following steps:

[0111] a) Initially passage the LAIV candidate vaccine virus in SPF chicken embryos to produce an egg-based master seed virus (MSV).

[0112] b) The egg-based master seed virus is adapted to grow on a cell culture host to prepare a cell-based working seed virus (WSV). This cell-based WSV is cultured and propagated in different cell culture vessels / systems, such as tissue culture flasks (TCFs) with a surface area of 175 cm 2 , roller bottles (RBs) with a surface area of 850 cm 2 , cell factories (CFs) with a surface area of 6320 cm 2 and fixed-bed bioreactors (e.g., bioreactors from Life Sciences Corporation (Port Washington, New York), such as Nano bioreactors and 500 / 100 bioreactors).

[0113] c) Harvest the cultured virus.

[0114] d) Filter the virus harvest by dead-end filtration (DFF) through at least one clarification filter to obtain a clarified virus pool (CVP).

[0115] e) Treat the CVP with a non-specific endonuclease to degrade cellular DNA.

[0116] f) Subject the treated endonuclease-treated CVP to tangential flow filtration.

[0117] g) Stabilize the TFF concentrate with a stabilizer composition comprising one or more carbohydrates, one or more amino acids, and gelatin to form a stabilized virus harvest.

[0118] h) Sterilize the stabilized TFF concentrate by DFF through at least one sterilizing-grade filter to obtain a sterilized clarified monovalent virus pool (CMVP).

[0119] i) Store the sterilized CMVP in polycarbonate bottles at -60 °C or lower.

[0120] j) Fill the sterilized formulation into vials and store at 2 °C to 8 °C.

[0121] According to the first aspect of the eleventh embodiment, the egg-based LAIV virus candidate suitable for growth in a cell culture host can be any eukaryotic cell. Still preferably, the cell culture host can be mammalian or avian cells. Suitable mammalian cells include, but are not limited to, hamster, bovine, primate (including human and monkey), and dog cells. Various cell types include, but are not limited to, kidney cells, fibroblasts, retinal cells, and lung cells. Examples of suitable hamster cells are cell lines named BHK21 or HKCC. Suitable monkey cells are, for example, African green monkey cells, such as kidney cells in the Vero cell line. Suitable dog cells are, for example, kidney cells in the CLDK and MDCK cell lines.

[0122] Further suitable cells include, but are not limited to: CHO; 293T; BHK; MRC 5; PER.C6; FRhl.2; WI-38, etc. Suitable cells are widely obtained from, for example, the American Type Culture Collection (ATCC) depository center, the Coriell Cell Repositories, or the European Collection of Cell Cultures (ECACC). For example, ATCC provides various different Vero cells with catalog numbers CCL 81, CCL 81.2, CRL 1586, and CRL-1587, and it provides MDCK cells with catalog number CCL 34. PER.C6 can be obtained from ECACC, with the deposit number 96022940.

[0123] Still preferably, the cell culture host can be Madin-Darby Canine Kidney (MDCK) cells, selected from, but not limited to, ATCC CCL-34, MDCK 33016 cell line (DSMACC 2219), MDCK (ATCC CCL34MDCK(NBL2)), MDCK 33016 (DSMACC2219), DSMACC3309, ATCC CRL-12042, ATCC PTA-7909, ATCC PTA-7910, ATCC PTA-6500, ATCC PTA-6501, ATCC PTA-6502, ATCC PTA-6503, "MDCK-S", "MDCK-SF101", "MDCK-SF102", "MDCK-SF103", and FERM BP-7449.

[0124] Still preferably, the cell culture host can be Madin-Darby Canine Kidney (MDCK) cells ATCC CCL 34 (NBL2).

[0125] According to the second aspect of the eleventh embodiment, MDCK cells can be cultured in Minimum Essential Medium (MEM) containing 10% fetal bovine serum (FBS). The cells can be cultured at 37°C ± 1°C. The pH value of the medium during the proliferation of the cells before infection can be in the range of pH 6.8 and pH 7.6, and more preferably between the values of pH 7.0 and pH 7.4.

[0126] Still, MDCK cells can be cultured in serum-free or protein-free media.

[0127] According to the third aspect of the eleventh embodiment, before infection, MDCK cells can be rinsed with MEM and then rinsed with MEM containing a protease in the range of 5 U / ml to 25 U / ml.

[0128] However, the protease can be selected from but not limited to trypsin, chymotrypsin, fungal protease, pepsin, papain, bromelain, and subtilisin.

[0129] Still preferably, the protease can be trypsin obtained from porcine, bovine, fungal, or bacterial sources.

[0130] Still preferably, the protease can be recombinant trypsin expressed in host cells of yeast, plants, or bacteria, and the host cells are selected from but not limited to Aspergillus, Streptomyces griseus, corn, Escherichia coli, and Pichia pastoris. Preferably, the recombinant trypsin is selected from Biogenomics (Escherichia coli as the host), D.K. Bio Pharma Pvt. Ltd (Escherichia coli as the host), Richcore (Pichia pastoris as the host), and Gibco (fungal).

[0131] Still, the preferred trypsin concentration is 12.5 U / ml.

[0132] According to the fourth aspect of the eleventh embodiment, before infection, the working seed virus can be diluted with MEM containing a protease in the range of 5 U / ml to 25 U / ml and incubated at a temperature of 31°C to 33°C for 10 minutes to 60 minutes.

[0133] The protease can be selected from but not limited to trypsin, chymotrypsin, fungal protease, pepsin, papain, bromelain, and subtilisin.

[0134] Still preferably, the protease can be trypsin obtained from porcine, bovine, fungal, or bacterial sources.

[0135] Still preferably, the protease can be a recombinant trypsin expressed in a host cell of yeast or plant or bacteria, and the host cell is selected from but not limited to Aspergillus, Streptomyces griseus, corn, Escherichia coli, Pichia pastoris. Preferably, the recombinant trypsin is selected from Biogenomics (Escherichia coli as the host), D.K.Bio Pharma Pvt.Ltd (Escherichia coli as the host), Richcore (Pichia pastoris as the host), and Gibco (fungus).

[0136] Still, the preferred trypsin concentration is 12.5 U / ml.

[0137] Still, the preferred trypsin concentration is 2000 to 3000 units of trypsin per roller bottle.

[0138] According to the fifth aspect of the eleventh embodiment, infecting MDCK cells with the LAIV candidate virus can occur at a preferred TCF of about 40 - 60×10 2 / TCF, RB of about 150 - 180×10 6 / RB, and 7000 - 10000×10 6 / BR (4m 6 ) at the MDCK cell density. 2 )

[0139] According to the sixth aspect of the eleventh embodiment, the LAIV candidate virus can be grown on MDCK cells in adherent culture or suspension culture mode.

[0140] According to the seventh aspect of the eleventh embodiment, infecting MDCK cells with the egg-based LAIV candidate virus can occur at an MOI between 1:100 and 1:10000.

[0141] According to the eighth aspect of the eleventh embodiment, the infected MDCK cells can be cultured in minimum essential medium (MEM) containing trypsin in the range of 5 U / ml to 25 U / ml and at a temperature of 32°C ± 1°C. The pH value of the medium after infection can be in the range of pH 6.8 and pH 7.6, and most preferably in the range of 7.2 to 7.6.

[0142] According to the ninth aspect of the eleventh embodiment, the cell supernatant can be harvested after an incubation period of 40 to 70 hours; more preferably, it can be harvested after 54 ± 8 hours.

[0143] Still optionally, before discarding the input material and separately processing it to obtain a clarified monovalent virus pool (CMVP), multiple harvests can be carried out about 4 to 5 times at appropriate time intervals.

[0144] After harvest, a virus yield of at least 7.0 to 9.2 Log EID 50 / 0.5 ml can be achieved.

[0145] According to the tenth aspect of the eleventh embodiment, the virus-containing culture medium can be clarified, usually by a filter with a reduced pore size (e.g., 6 μm, 5 μm, 0.8 μm, 0.65 μm, 0.45 μm, 0.2 μm). Suitable commercially available filters and filtration devices are well known in the art and can be selected by a person skilled in the art. Exemplary filtration devices can be made of polypropylene or cellulose acetate or polyethersulfone, and commercially available filters can be Millipak (Millipore), Kleenpak (Pall), and Sartobran (Sartorius) filtration devices.

[0146] According to the eleventh aspect of the eleventh embodiment, the filtered harvest can be treated with a non-specific endonuclease with a concentration varying between 0.5 units / ml and 2 units / ml, most preferably Benzonase, at a temperature ranging between 30 °C and 34 °C for 2 hours to 6 hours, and then at a temperature of 2 °C to 8 °C for 5 hours to 15 hours.

[0147] Still optionally, the filtered harvest can be treated with a non-specific endonuclease, most preferably Benzonase, in the presence of divalent cations in an amount between 0.1 mM and 100 mM, said divalent cations selected from the group consisting of Ca 2+ 、Mg 2+ 、Mn 2 + and Cu 2+ which make up the group.

[0148] Still optionally, the filtered harvest can be treated with a non-specific endonuclease, most preferably Benzonase, in the presence of a divalent cation Mg 2+ salt at a concentration of 1 mM to 3 mM.

[0149] According to the twelfth aspect of the eleventh embodiment, the Benzonase-treated harvest can be further subjected to tangential flow filtration (TFF), usually through a filter with a molecular weight cut-off (MWCO) between 100 kDa and 500 kDa, so as to obtain a virus harvest that is 2 to 10 times concentrated and further remove residual impurities.

[0150] Still preferably, the residual impurities can include residual DNA, residual bovine serum albumin (BSA), and residual host cell proteins.

[0151] According to the thirteenth aspect of the eleventh embodiment, the above method can produce a purified and concentrated LAIV virus harvest comprising trace residual cellular DNA (<10 ng / dose), residual BSA (<50 ng / dose), and residual cellular proteins. Additionally, according to the above method, the total recovery rate of the purified virus can be at least 40%.

[0152] According to the fourteenth aspect of the eleventh embodiment, the concentrated monovalent virus bulk (TFF concentrate) can be stabilized with a stabilizer composition to obtain a final LAIV composition comprising one or more carbohydrates, one or more amino acids, and gelatin.

[0153] Still preferably, the concentrated virus bulk (TFF concentrate) can be stabilized with a stabilizer composition comprising any combination of sucrose, histidine, alanine, tris(hydroxymethyl)methylglycine, arginine, and gelatin.

[0154] Still preferably, the concentrated virus bulk (TFF concentrate) can be stabilized with a stabilizer composition comprising sucrose at a concentration of 1% to 10% (w / v), histidine at a concentration of 0.1% to 2% (w / v), alanine at a concentration of 0.01% to 1% (w / v), tris(hydroxymethyl)methylglycine at a concentration of 0.1% to 1% (w / v), arginine at a concentration of 0.1% to 5% (w / v), and gelatin at a concentration of 0.1% to 5% (w / v).

[0155] Still preferably, the concentrated virus bulk (TFF concentrate) can be stabilized with a stabilizer composition comprising sucrose at a concentration of 3 to 6% (w / v), histidine at a concentration of 0.1% to 1% (w / v), alanine at a concentration of 0.05% to 0.5% (w / v), tris(hydroxymethyl)methylglycine at a concentration of 0.1% to 0.5% (w / v), arginine at a concentration of 0.1% to 3% (w / v), and gelatin at a concentration of 0.1% to 3% (w / v).

[0156] Still preferably, the concentrated virus bulk (TFF concentrate) can be stabilized with a stabilizer composition comprising 4% (w / v) sucrose, 0.21% (w / v) histidine, 0.1% (w / v) alanine, 0.3% (w / v) tris(hydroxymethyl)methylglycine, 2.1% (w / v) arginine, and 0.85% (w / v) gelatin.

[0157] According to the fifteenth aspect of the eleventh embodiment, a stable virus harvest can be sterilized by direct current filtration (DFF) through at least one sterilizing grade filter, preferably 0.2 μm. Suitable commercially available filters and filtration devices are well known in the art and can be selected by a person skilled in the art. Exemplary filtration devices can be made of polypropylene or cellulose acetate or polyethersulfone or polyvinylidene fluoride, and commercially available filters can be Millipak (Millipore), Kleenpak (Pall), and Sartobran TM P (Sartorius) filtration devices.

[0158] According to the sixteenth aspect of the eleventh embodiment, the LAIV composition can be multivalent, including more than one LAIV virus strain or subtype as disclosed in the previous embodiments. The LAIV composition can be bivalent or trivalent or quadrivalent.

[0159] Still optionally, the LAIV composition can be monovalent, including any one LAIV virus strain or subtype as disclosed in the previous embodiments.

[0160] According to the seventeenth aspect of the eleventh embodiment, the LAIV composition can contain a dose of 6 to 7 Log EID 50 / 0.5 ml of influenza virus.

[0161] Still preferably, the LAIV composition can include influenza A virus or any subtype, with a dose of 6 to 7 Log EID 50 / 0.5 ml; more preferably not less than 7 Log EID 50 / 0.5 ml.

[0162] Still preferably, the LAIV composition can contain influenza B virus or any subtype, with a dose of 6 to 7 Log EID 50 / 0.5 ml; more preferably not less than 6.5 Log EID 50 / 0.5 ml.

[0163] According to the twelfth embodiment, a method for preparing an immunogenic composition can include the following steps:

[0164] a) Infecting an MDCK cell culture host with an influenza virus at an MOI between 1:100 and 1:10000;

[0165] b) Harvesting the supernatant containing the influenza virus after incubating for 40 to 70 hours in MEM containing trypsin in the range of 5 U / ml to 25 U / ml;

[0166] c) Filtering the virus harvest by direct current filtration (DFF) through at least one clarification filter with a pore size between about 6 microns and about 0.45 microns;

[0167] d) Treat the CVP with a non-specific endonuclease at a temperature in the range of 30 °C to 34 °C for 2 to 6 hours, and then treat it at a temperature of 2 °C to 8 °C for 5 to 15 hours;

[0168] e) Concentrate the endonuclease-treated CVP by tangential flow filtration (TFF) using a membrane with a molecular weight cut-off (MWCO) of 100 kDa to 500 kDa.

[0169] f) Stabilize the TFF concentrate with a stabilizer composition comprising one or more carbohydrates, one or more amino acids, and gelatin to form a stable virus harvest.

[0170] g) Sterilize the stable TFF concentrate by depth filtration (DFF) through at least one sterilizing-grade filter with a pore size between about 0.8 microns and about 0.2 microns to form a sterilized CMVP.

[0171] wherein the total recovery rate of the purified virus is greater than or equal to 40%.

[0172] According to the first aspect of the twelfth embodiment, a method for preparing an immunogenic composition, wherein step (d) may include treating the virus harvest with a non-specific endonuclease, more particularly Benzonase, at a concentration in the range of 0.5 units / ml to 5 units / ml, in the presence of a divalent cation selected from the group consisting of Ca 2+ , Mg 2+ , Mn 2+ and Cu 2+ and in an amount between 0.1 mM and 100 mM.

[0173] According to the second aspect of the twelfth embodiment, a method for preparing an immunogenic composition, wherein step (d) may include treating the virus harvest with a non-specific endonuclease, more particularly Benzonase, at a concentration in the range of 0.5 units / ml to 5 units / ml, in the presence of a divalent cation Mg 2+ salt at a concentration of 1 mM to 3 mM.

[0174] According to the third aspect of the twelfth embodiment, a method for preparing an immunogenic composition, wherein step (e) may include concentrating the virus harvest by tangential flow filtration (TFF) to obtain a virus harvest concentrated at least 4-fold.

[0175] According to the fourth aspect of the twelfth embodiment, a method for preparing an immunogenic composition, wherein step (f) may include stabilizing the virus harvest with a stabilizer composition comprising sucrose at a concentration of 1% (w / v), histidine at a concentration of 0.1% to 2% (w / v), alanine at a concentration of 0.01% to 1% (w / v), tris(hydroxymethyl)methylglycine at a concentration of 0.1% to 2% (w / v), arginine at a concentration of 0.1% to 5% (w / v), and gelatin at a concentration of 0.11% to 5% (w / v).

[0176] According to the fifth aspect of the twelfth embodiment, a method for preparing an immunogenic composition, wherein step (f) may include stabilizing the virus harvest with a stabilizer composition comprising sucrose at a concentration of 3% to 6% (w / v), histidine at a concentration of 0.1% to 1% (w / v), alanine at a concentration of 0.05% to 0.5% (w / v), tris(hydroxymethyl)methylglycine at a concentration of 0.1% to 0.5% (w / v), arginine at a concentration of 0.1% to 3% (w / v), and gelatin at a concentration of 0.1% to 3% (w / v).

[0177] According to the sixth aspect of the twelfth embodiment, a method for preparing an immunogenic composition, wherein step (f) may include stabilizing the virus harvest with a stabilizer composition comprising 4% (w / v) sucrose, 0.21% (w / v) histidine, 0.1% (w / v) alanine, 0.3% (w / v) tris(hydroxymethyl)methylglycine, 2.1% (w / v) arginine, and 0.85% (w / v) gelatin.

[0178] According to the sixth aspect of the twelfth embodiment, a method for preparing an immunogenic composition, wherein step (f) may include stabilizing the virus harvest with a stabilizer composition comprising 4% (w / v) sucrose, 0.21% (w / v) histidine, 0.1% (w / v) alanine, 0.3% (w / v) tris(hydroxymethyl)methylglycine, 2.1% (w / v) arginine, and 1.0% (w / v) gelatin.

[0179] According to the seventh aspect of the twelfth embodiment, a method for preparing an immunogenic composition, wherein step (f) may include stabilizing the virus harvest with a stabilizer composition comprising 4% (w / v) sucrose, 0.21% (w / v) histidine, 0.1% (w / v) alanine, 0.3% (w / v) tris(hydroxymethyl)methylglycine, 1.6% (w / v) arginine, and 1.0% (w / v) gelatin.

[0180] According to a thirteenth embodiment of the present disclosure, the immunogenic composition may comprise a) one or more live attenuated influenza vaccine (LAIV) viruses, in a dose of 6 to 7 Log EID 50 / 0.5 ml; b) 1% to 10% (w / v) sucrose; c) 0.1% to 2% (w / v) histidine; d) 0.01% to 1% (w / v) alanine; e) 0.1% to 2% (w / v) tris(hydroxymethyl)methylglycine; f) 0.1% to 5% (w / v) arginine; g) 0.1% to 5% (w / v) gelatin.

[0181] Still preferably, the immunogenic composition may comprise a) one or more live attenuated influenza vaccine (LAIV) viruses, in a dose of 6 to 7 Log EID 50 / 0.5 ml; b) 3% to 6% (w / v) sucrose; c) 0.1% to 1% (w / v) histidine; d) 0.05% to 0.5% (w / v) alanine; e) 0.1% to 0.5% (w / v) tris(hydroxymethyl)methylglycine; f) 0.1% to 3% (w / v) arginine; g) 0.1% to 3% (w / v) gelatin.

[0182] Still preferably, the immunogenic composition may comprise a) one or more live attenuated influenza vaccine (LAIV) viruses, in a dose of 6 to 7 Log EID 50 / 0.5 ml; b) 4% (w / v) sucrose; c) 0.21% (w / v) histidine; d) 0.1% (w / v) alanine; e) 0.3% (w / v) tris(hydroxymethyl)methylglycine; f) 2.1% (w / v) arginine; g) 0.85% (w / v) gelatin.

[0183] Still preferably, the immunogenic composition may comprise a) one or more live attenuated influenza vaccine (LAIV) viruses, in a dose of not less than 6 to 7 Log EID 50 / 0.5 ml; b) 4% (w / v) sucrose; c) 0.21% (w / v) histidine; d) 0.1% (w / v) alanine; e) 0.3% (w / v) tris(hydroxymethyl)methylglycine; f) 2.1% (w / v) arginine; g) 1% (w / v) gelatin.

[0184] Still preferably, the immunogenic composition may comprise a) one or more live attenuated influenza vaccine (LAIV) viruses, in a dose of 6 to 7 Log EID 50 / 0.5 ml; b) 4% (w / v) sucrose; c) 0.21% (w / v) histidine; d) 0.1% (w / v) alanine; e) 0.3% (w / v) tris (hydroxymethyl) methylglycine; f) 1.6% (w / v) arginine; g) 1% (w / v) gelatin.

[0185] According to a fourteenth embodiment of the present disclosure, the LAIV composition can be fully liquid.

[0186] Still optionally, the LAIV composition can be a lyophilized or freeze-dried composition.

[0187] As used herein, the terms "lyophilize" or "lyophilize" or "lyophilization" relate to freeze-drying and refer to the process in which a suspension is frozen and then water is removed by sublimation at low pressure. As used herein, the term "sublimation" refers to a change in the physical properties of a composition in which the composition directly changes from a solid state to a gaseous state without becoming a liquid.

[0188] According to a fifteenth embodiment of the present disclosure, the LAIV composition can be formulated for a method of reducing the onset or preventing a health condition, including immunizing a human subject by intranasal or other routes with an effective amount of the LAIV composition.

[0189] According to a preferred aspect of the embodiment, the LAIV composition can be administered to a human subject by the intranasal route. In one embodiment, it is in the form of an intranasal dispersion device, such as an aerosol (intranasal spray) or a droplet delivery system. The liquid nasal preparation can be delivered by nasal spray, instillation and nasal catheter, compressed air nebulizer, squeeze bottle, metering pump spray (such as a multi-dose metering spray pump or a single / double-dose spray pump, a spray device connected to a syringe). Other dosage forms can be selected from nasal powders (blowers, dry powder inhalers), nasal gels, nasal drops, solutions, suspensions, co-solvent systems, microspheres, nanoparticles, microemulsions, nasal inserts.

[0190] The intranasal delivery device can be selected from, but not limited to, Becton Dickinson (BD) Accuspray TM delivery device, Bi-Directional Optinose nasal device, Teleflex's MAD intranasal mucosal atomization device, AeroLife TM and AeroVax (AerovectRx, Inc., Atlanta, GA), jet syringe- needleless syringe; MUNJIs multi-purpose nozzle jet syringe: Aquapuncture device, Single-dose syringe injector: Medi- J- Vitajet TM , LectraJet HS, M3, ZetaJet TM , Aktiv-DryPuffHaler TM and intranasal influenza vaccine devices.

[0191] According to the sixteenth embodiment of the present invention, the LAIV composition can be formulated for a method of reducing the onset or preventing the following health conditions, which include influenza A virus infection or its subtypes as disclosed in the previous embodiments of the present disclosure, influenza B virus infection or its subtypes as disclosed in the previous embodiments of the present disclosure, or influenza C virus infection or its subtypes as disclosed in the previous embodiments of the present disclosure.

[0192] According to the seventeenth embodiment of the present disclosure, the above LAIV composition can be administered intranasally in an effectively protective dose. The vaccine is administered in a manner compatible with the dosage form and in a prophylactically effective amount. The immunogenic composition of the present disclosure can be administered as a primary prophylactic agent in adults or children at risk of infection. For example, the live attenuated influenza vaccine composition disclosed herein can be used in adults or children at risk of influenza virus infection.

[0193] More preferably, the LAIV composition can be administered intranasally in a dose volume of about 0.1 ml to 0.5 ml.

[0194] According to the eighteenth embodiment of the present disclosure, the LAIV composition can be formulated into single-dose vials or multi-dose vials or multi-dose kits or prefilled syringes or nasal sprays, wherein the LAIV composition can be administered according to a single-dose regimen, or preferably a multi-dose regimen, in which 1 to 2 separate doses are administered at subsequent time intervals required to maintain and / or boost the immune response after the primary course of vaccination. For example, a second dose can be administered within 1 - 4 months if needed, and subsequent vaccinations are carried out several months or years or annually after vaccination. This dosing regimen will also depend at least in part on the need for booster doses required to confer protective immunity.

[0195] According to the nineteenth embodiment of the present disclosure, the final pH value of the immunogenic composition can include 6.5 to 8.

[0196] Other embodiments disclosed herein also include a vaccine kit, which includes a first container containing a lyophilized (freeze-dried) immunogenic composition and a second container containing an aqueous solution, which is optionally saline or WFI (water for injection), for reconstituting the lyophilized (freeze-dried) LAIV composition.

[0197] Throughout the specification, the word "comprising" or variations such as "comprises" or "comprising" will be understood to mean including the recited element, integer or step, or group of elements, integers or steps, but not excluding any other element, integer or step, or any other element of a group of elements, integers or steps, and may mean "including", "comprising", and the term is open-ended, allowing for more than what is recited, provided that the basic characterizing features or novel features being recited are not changed by the presence of more being recited, but not excluding prior art embodiments.

[0198] Throughout this specification, the term "immunogenic composition" encompasses any composition that elicits an immune response against an antigen or immunogen of interest expressed from a vector; for example, after administration to a subject, it elicits an immune response against the targeted immunogen or antigen of interest. The terms "vaccine composition" and "vaccine" encompass any composition that induces a protective immune response against an antigen of interest or effectively provides protection against an antigen; for example, after administration or injection to a subject, it elicits a protective immune response against the targeted antigen or immunogen or provides effective protection against the antigen or immunogen expressed from a vector.

[0199] The use of the expression "one or more" or "at least one" implies the use of one or more elements or ingredients or amounts, as this use may, in embodiments of the present invention, serve to achieve one or more desired purposes or results. Although certain embodiments of the present invention have been described, these embodiments are presented by way of example only and are not intended to limit the scope of the present invention. Those skilled in the art may conceive of changes or modifications to the compositions of the present invention within the scope of the present invention after reading the present disclosure. Such changes or modifications are also within the spirit of this disclosure.

[0200] Unless the specification states otherwise, the numerical values given for various physical parameters, dimensions and amounts are only approximate values, and it is contemplated that values higher than those specified for physical parameters, dimensions and amounts fall within the scope of the present invention.

[0201] Similarly, the components used in purification, such as filters, chromatography columns, are in no way intended to be restrictive or exclusive and may be replaced by other components at the discretion of the practitioner to achieve the same purpose.

[0202] Although considerable emphasis has been placed herein on the specific features of the preferred embodiments, it should be understood that many additional features may be added and many changes may be made to the preferred embodiments without departing from the principles of this disclosure. These and other changes to the preferred embodiments of this disclosure will be apparent to those skilled in the art from the present disclosure, and it should thus be clearly understood that the foregoing description is to be construed as illustrative of this disclosure rather than as limiting.

[0203] Technical advantages

[0204] 1. Currently, almost all influenza vaccines produced use eggs as the host to prepare the virus bank. There are certain disadvantages in using eggs to manufacture LAIV, which can be overcome by using cell culture as the substrate. The limitations of using eggs as the substrate are:

[0205] ● The suppliers of vaccine-quality eggs and specific pathogen-free eggs are limited.

[0206] ● Orders need to be placed at least 4 months in advance before the eggs are available.

[0207] ● Some candidate pandemic strains can cause fatal infections in poultry, resulting in the unavailability of the substrate (eggs) for vaccine production.

[0208] ● Egg-based manufacturing requires specialized facilities for egg incubation, harvesting, etc., thus limiting the ability to rapidly scale up.

[0209] 2. Tissue culture-based manufacturing has the advantage of a fully controlled system and is easy to scale up.

[0210] 3. In the event of a pandemic, large-scale production of the vaccine can be easily achieved using the pre-existing tissue culture production units of other viral vaccines.

[0211] 4. The virus obtained from cell culture is more similar to the circulating strains, while the virus produced in eggs may have antigenic modifications.

[0212] 5. The components participating in the vaccine composition are minimal.

[0213] 6. It does not contain preservatives, polymers, and surfactants.

[0214] 7. The purification process uses a low concentration of endonuclease (Benzonase).

[0215] 8. The purification process does not have expensive and cumbersome chromatographic steps.

[0216] 9. The method for producing this stable composition / formulation has been improved, thus increasing the yield.

[0217] 10. Intranasal administration is the simplest immunization route because it does not require a high level of expertise and is suitable for multi-dose administration.

[0218] 11. It has not been reported to be associated with Guillain Barre syndrome and provides better protection due to delivery at the site of infection.

[0219] 12. It is not easy to present liquid vaccines, which helps to overcome the problems of limited freeze-drying capacity, the need to supply diluents for reconstitution, and the additional reconstitution steps required before vaccine delivery.

[0220] 13. The MDV backbone used to generate LAIV recombinants has well-established safety characteristics and is reported to provide a high level of protection.

[0221] Examples

[0222] The following examples are included to illustrate the preferred embodiments of the present invention. Those skilled in the art should understand that the compositions and techniques disclosed in the following examples represent techniques that the inventors have found to work well in the practice of the present invention and can thus be considered to constitute preferred modes of its practice. However, those skilled in the art should understand from this disclosure that many changes can be made to the specific embodiments disclosed without departing from the spirit and scope of the present invention and still obtain similar or analogous results.

[0223] The reassorted LAIV strains were procured from the Institute of Experimental Medicine (IEM) in St. Petersburg, Russia or a WHO Collaborating Center, such as the Centers for Disease Control and Prevention (CDC) in Atlanta.

[0224] Example 1: Stability Data of Reassorted LAIV Viral Immunogenic Compositions

[0225] Component 1 - Live Attenuated Influenza Vaccine Virus (LAIV)

[0226] The influenza vaccine virus is a reassorted LAIV virus derived by a typical reassortment method, which includes the cold-adapted, temperature-sensitive, and / or attenuated phenotypic gene segments of the master donor virus (MDV) and the hemagglutinin (HA) gene segment and / or neuraminidase (NA) gene segment of a wild-type pandemic or seasonal influenza A, B, or C virus strain in a ratio of 6:2 or 7:1.

[0227]

[0228]

[0229] For the LAIV immunogenic composition:

[0230] In the dose range of 6 to 7 log EID50 / 0.5 ml; more preferably, the dose of influenza A virus is 7 log EID50 / 0.5 ml

[0231] In the dose range of 6 to 7 log EID50 / 0.5 ml; more preferably, the dose of influenza B virus is 6.5 log EID50 / 0.5 ml

[0232] In the case of reassorted LAIV virus strains used in immunogenic compositions disclosed in any combination in Table 1, the LAIV composition is monovalent or multivalent (bivalent; trivalent; tetravalent).

[0233]

[0234]

[0235]

[0236]

[0237] Explanation:

[0238] Stabilizer composition 3 (1% w / v gelatin + 5% w / v sorbitol + 0.1% w / v L-alanine + 0.21% w / v L-histidine + 0.3% w / v tris(hydroxymethyl)methylglycine + 1.6% w / v L-arginine hydrochloride):

[0239] For influenza A / H1N1 and B vaccine strains, unacceptable degradation rates were observed for both stress stability at 37°C and real-time stability at temperatures between 2°C and 8°C. (Refer to Figure 4 and Figure 5 )

[0240] Stabilizer composition 4 (1% w / v gelatin + 5% w / v sorbitol + 0.1% w / v L-alanine + 0.21% w / v L-histidine + 0.9% w / v tris(hydroxymethyl)methylglycine + 1.6% w / v L-arginine hydrochloride):

[0241] For influenza A / H1N1 and B vaccine strains, unacceptable degradation rates were observed for both stress stability at 37°C and real-time stability at temperatures between 2°C and 8°C. (Refer to Figure 6 and Figure 7 )

[0242] Stabilizer composition 2 (0.85% w / v gelatin + 3% w / v sucrose + 0.1% w / v L-alanine + 0.21% w / v L-histidine + 0.3% w / v tris(hydroxymethyl)methylglycine + 2.1% w / v L-arginine hydrochloride):

[0243] Unacceptable degradation rates were observed for both stress stability at 37°C and real-time stability at temperatures between 2°C and 8°C. (Refer to Figure 8 and Figure 9 )

[0244] Stabilizer composition 1 (0.85% w / v gelatin + 4% w / v sucrose + 0.1% w / v L-alanine + 0.21% w / v L-histidine + 0.3% w / v tris(hydroxymethyl)methylglycine + 2.1% w / v L-arginine hydrochloride):

[0245] For both stress stability at 37 °C and real-time stability at 2 °C to 8 °C, acceptable degradation rates (values within an acceptable range) were observed. (See Figure 2 、 3 、10 and 11)

[0246] Example 2: MDCK cell-based LAIV virus manufacturing process

[0247] The method for preparing an LAIV composition based on MDCK cell cultures may include any subset or all of the following steps:

[0248] k) Initially passage the LAIV candidate vaccine virus in specific pathogen-free (SPF) chicken embryos that generate egg-based master seed virus (MSV).

[0249] l) Grow the egg-based master seed virus on an MDCK cell culture (ATCC CCL-34) host to prepare a cell-based working seed virus (WSV). This cell-based WSV is used to infect MDCK cell cultures at an MOI of 1:100 to 1:10,000 in different cell culture vessels / systems (such as tissue culture flasks (TCFs) with a surface area of 175 cm 2 , roller bottles (RBs) with a surface area of 850 cm 2 , cell factories (CFs) with a surface area of 6320 cm 2 and fixed-bed bioreactors (e.g., bioreactors from Life Sciences, Inc. (Port Washington, New York), such as the Nano bioreactor and the 500 / 100 bioreactor)). (MDCK cells are cultured in MEM with FBS; rinsed with MEM containing 5 U / ml to 25 U / ml of trypsin before inoculation; the WSV is inoculated into the cells at an MOI of 1:10 to 1:10,000 and incubated at 31 °C to 33 °C for 48 hours to 72 hours)

[0250] m) Harvest the cultured virus.

[0251] n) Filter the virus harvest through at least one clarification filter by depth filtration (DFF) to obtain a clarified virus pool (CVP).

[0252] o) Treat the CVP with a non-specific endonuclease (e.g., Benzonase) at a temperature in the range of 30 °C to 34 °C for 2 hours to 6 hours, and then at a temperature of 2 °C to 8 °C for 5 hours to 15 hours;

[0253] p) Concentrate the endonuclease-treated CVP by tangential flow filtration (TFF) using a membrane with a molecular weight cut-off (MWCO) of 100 kDa to 500 kDa;

[0254] q) Stabilize the TFF concentrate with a stabilizer composition comprising one or more carbohydrates, one or more amino acids, and gelatin to form a stable virus harvest.

[0255] r) Sterilize the stabilized TFF concentrate by depth filtration (DFF) through at least one sterilizing grade filter with a pore size of approximately 0.2 microns to obtain a sterilized CMVP (clarified monovalent virus pool).

[0256] s) Store the sterilized CMVP in polycarbonate bottles at a temperature of -60 °C or lower.

[0257] t) Fill the sterilized formulation into vials and store at 2 °C to 8 °C.

[0258]

[0259]

[0260] Cell culture medium: MEM with 10% FBS (pH adjusted to 7.0 to 7.4 with 1N HCl)

[0261] (Additional glutamine 350 mg / L)

[0262] Virus culture medium: MEM without FBS (pH adjusted to 7.2 to 7.6 with 1N HCl)

[0263] (Additional glucose 500 mg / L, glutamine 350 mg / L)

[0264] Add an additional 0.4% glucose to the CM and VM used in the bioreactor system

[0265]

[0266]

[0267]

[0268]

[0269]

[0270] Example 3: Effects of Virus Input (MOI) and Incubation Period after Inoculation on Yield

[0271]

[0272]

[0273] Infection:

[0274] A. Infection Time:

[0275] Based on the optimization study, the upper limit of the number of cells infected with the MDCK cell-derived influenza working seed virus was selected to be 120 million to 180 million cells per roller bottle and 7 billion to 10 billion cells in the bioreactor system. Microscopic observation of monolayer confluence was performed on the roller bottles with MDCK cells before the infection procedure.

[0276] B. MOI and Incubation Period after Inoculation:

[0277] Based on all the observations of the MOI optimization study, the MOI of influenza A (H1N1), influenza A (H3N2), and influenza B viruses was selected in the range of 1:100 to 1:10,000, and the incubation period after inoculation was between 48 and 72 hours.

[0278] Example 4: Effects of Different Concentrations of Trypsin on Yield

[0279]

[0280] Inference: Trypsin is required to activate the influenza virus for inoculating MDCK cells. From the above results, it can be seen that using 2000 to 3000 units of trypsin per roller bottle produces the maximum virus potency.

[0281] Example 5: Effects of the Concentration and Temperature of Benzonase on the Cellular DNA Content and Virus Titer

[0282] The degradation of host cell DNA by different concentrations of Benzonase at different temperatures was tested. The clarified virus pool (CVP) was treated with Benzonase at concentrations of 500 (containing 2 mM MgCl2), 500, 1000, 2500, and 5000 U / L, and the treated CVP was maintained at 32 °C for 3 hours and further treated overnight at 2 °C to 8 °C. Sampling was performed at each stage, and the following are the DNA content results at each stage.

[0283]

[0284]

[0285] Inference: As can be seen from the results, compared with the CVP treated with Benzonase at a concentration of 500 U / L without 2 mM MgCl2, the CVP treated with Benzonase at a concentration of 500 U / L in the presence of 2 mM MgCl2 showed higher DNA degradation. It can also be seen that higher Benzonase concentrations of 1000 U / L, 2500 U / L, and 5000 U / L showed a DNA degradation level comparable to that of the Benzonase concentration of 500 U / L (with 2 mM MgCl2).

[0286] Example 6: Virus Yield at Different Manufacturing Stages

[0287]

[0288] 1. CVP: Clarified virus bank (harvested after filtration),

[0289] 2. BCVP: CVP treated with Benzonase,

[0290] 3. CMVP: Clarified monovalent virus bank (after TFF, with stabilizer added and filtered through 0.2 μm)

[0291] Inference: The stage-wise virus concentrations of each seasonal influenza virus of influenza A (H1N1), influenza A (H3N2), and influenza B were examined, and it was observed that the initial virus concentration at the harvest level was maintained throughout the process until the final stage, i.e., the preparation of the vaccine batch (CMVP).

[0292]

[0293] Inference: The virus recovery rate can be calculated as the percentage of virus retention during the manufacturing process, where the harvest is the starting point and CMVP is the end point of manufacturing. From the results, it can be concluded that the average virus recovery rate is 44.67%, which is equivalent to a titer loss of 0.34 Log EID 50 / 0.5 ml. It was also observed that the final virus recovery rate (virus titer) at the CMVP level was within an acceptable range, and CMVP can be used for the production of the final product batch of MDCK-based LAIV virus.

[0294] Example 7: Comparative Data on Stage-wise Host Cell DNA Concentrations during CMVP Manufacturing

[0295] The clarified virus banks (CVP) of different strains were treated with Benzonase, and DNA content was sampled stage-wise. The following are the results of DNA content at each stage.

[0296]

[0297] Inference:

[0298] As can be seen from the results, CVP showed a significant reduction in DNA when treated with Benzonase. Furthermore, it was further observed that during the TFF process (diafiltration / concentration) and the CMVP preparation process, the residual DNA was effectively removed again. The DNA content at the final CMVP level was within the desired and acceptable limits.

[0299] Example 8: Various attempts of TFF experiments

[0300] Considering parameters such as the dilution medium (viral medium and PBS) used for the TFF process, the diafiltration and concentration procedures, various TFF experiments were carried out. The virus concentration of the TFF concentrate samples was tested.

[0301]

[0302] A / Cal: A / 17 / California / 2009 / 38;

[0303] B / Tex = B / Texas / 02 / 2013-CDC-LV8B;

[0304] A / HK = A / 17 / Hong Kong / 2014 / 8296;

[0305] VM: Viral medium

[0306] Explanation:

[0307] From the previous set of experiments, the TFF process was developed, and then the 2X DF and 4X concentration stages were selected to obtain the desired TFF concentrate with the best virus yield. PBS showed better stability for the virus compared to VM.

[0308] Example 9: Immunogenicity results

[0309] A study was conducted to evaluate the immune response and vaccine efficacy of egg-based and MDCK cell-cultured trivalent and quadrivalent seasonal influenza vaccines in a ferret model. All animals were immunized intranasally on day 0 with trivalent or quadrivalent preparations based on egg and MDCK cell cultures, which contained strains similar to A / Michigan / 45 / 2015 (H1N1), A / Hong Kong / 4801 / 2015 (H3N2), B / Brisbane / 60 / 2008, and B / Phuket / 3073 / 2013, and were challenged four weeks later (day 28).

[0310] Table 19: Geometric mean hemagglutination inhibition (HAI) and neutralization titers (NT) of sera collected on day 28

[0311]

[0312]

[0313] Inference: (Refer to Figure 12 and Figure 13 )

[0314] It was concluded that inactivated egg- and MDCK-based trivalent and quadrivalent vaccines containing pandemic H1N1 protected animals from H1N1 infection when challenged with homologous pandemic H1N1 virus.

[0315] Another study aimed to evaluate the immune response and efficacy of egg- and MDCK cell-based monovalent LAIVs (H5N2 and H7N9) in a ferret model. Animals in different groups were immunized with egg- and MDCK cell cultures of monovalent LAIVs based on H5N1 and H7N9 and challenged with homologous H5N1 and H7N9 viruses, respectively. The results showed that animals immunized with monovalent H5N2 LAIV were protected from homologous challenge with H5N1 virus. Animals immunized with monovalent H7N9 LAIV were protected from homologous challenge with H7N9 virus.

Claims

1. An immunogenic composition, comprising: a) One or more live attenuated influenza viruses at a dose of 6 to 7 Log EID 50 / 0.5 ml; and b) a stabilizer composition, comprising: i. 4% w / v sucrose; ii. 0.21% w / v histidine; iii. 0.1% w / v alanine; iv. 0.3% w / v tris (hydroxymethyl) methylglycine; v. 2.1% w / v arginine; and vi. 0.85% w / v gelatin, wherein the influenza virus is a reassortant LAIV virus, which comprises cold-adapted, temperature-sensitive and / or attenuated phenotypic gene segments of the master donor virus and hemagglutinin gene segments and / or neuraminidase gene segments of wild-type pandemic or seasonal influenza A, influenza B or influenza C strains in a ratio of 1:7, 2:6, 3:5, 4:4, 5:3, 6:2 or 7:1, and wherein the master donor virus is selected from the group consisting of A / Leningrad / 134 / 17 / 57 H2N2 influenza A strain, B / USSR / 60 / 69 influenza B strain.

2. The immunogenic composition according to claim 1, wherein the immunogenic composition is monovalent with respect to influenza viruses derived from influenza A, influenza B or influenza C.

3. The immunogenic composition according to claim 1, wherein the immunogenic composition is multivalent with respect to influenza viruses derived from influenza A, influenza B or influenza C.

4. The immunogenic composition according to claim 1, wherein the reassortant LAIV virus comprises a hemagglutinin gene and / or a neuraminidase gene from an influenza A virus.

5. The immunogenic composition according to claim 1, wherein the reassortant LAIV virus comprises hemagglutinin genes and / or neuraminidase genes from H1 to H18 and N1 to N11.

6. The immunogenic composition according to claim 1, wherein the reassortant LAIV virus comprises hemagglutinin genes and / or neuraminidase genes from H1N1, H2N2, H3N2, H5N1, H5N3, H9N2, H7N1, H7N3, H7N7, H6N1, H7N9 or H10N8.

7. The immunogenic composition according to claim 4, wherein the reassortant LAIV virus comprises hemagglutinin genes and / or neuraminidase genes from influenza A strain pdmH1N1 strains such as A / California / 07 / 2009-like strain, A / Michigan / 45 / 2015-like strain, A / South Africa / 3626 / 2013-like strain, A / 17 / Turkey / Turkey / 05 / 133, A / 17 / Anhui / 2013 / 61 or H3N2 - A / Hong Kong / 4801 / 2014-like strain.

8. The immunogenic composition according to claim 1, wherein the reassortant LAIV virus comprises hemagglutinin genes and / or neuraminidase genes from influenza B strain Victoria lineage B / Brisbane / 60 / 2008-like strain, B / Texas / 02 / 13-CDC-LV8B or Yamagata lineage B / Phuket / 3073 / 2013-like strain.

9. The immunogenic composition according to claim 1, wherein the immunogenic composition comprises a buffer selected from the group consisting of sodium chloride, carbonate, citrate, lactate, gluconate, tartrate, phosphate buffered saline, HEPES, citrate - phosphate, or TRIS.

10. The immunogenic composition according to claim 1, wherein the immunogenic composition comprises a pharmaceutically acceptable transporter, binder, carrier, isotonic agent, emulsifier, or wetting agent.

11. The immunogenic composition according to claim 1, wherein the immunogenic composition comprises an excipient.

12. The immunogenic composition according to claim 11, wherein the immunogenic composition comprises a pharmaceutically acceptable excipient selected from the group consisting of sugars; polyols; salts including NaCl, KCl, KH2PO4, Na2HPO4·2H2O, CaC12, or MgCl2; amino acids, or pH modifiers.

13. The immunogenic composition according to claim 1, wherein the final pH of the immunogenic composition comprises a pH of 6.5 to 8.

14. The immunogenic composition according to claim 1, wherein the immunogenic composition is formulated for use in a human subject by one of the intranasal, intramuscular, intravenous, subcutaneous, transdermal, or intradermal routes, and wherein the immunogenic composition is formulated as a single - dose vial or a multi - dose vial or a multi - dose kit or a pre - filled syringe or a nasal spray for reducing or preventing a health condition including influenza A virus infection, influenza B virus infection, or influenza C virus infection.

15. A method for preparing the immunogenic composition according to claim 1, the method comprising the following steps: a) Infecting a Madin - Darby canine kidney cell culture host with influenza virus at an MOI between 1:100 and 1:10000; b) Harvesting the supernatant comprising influenza virus after incubating in MEM containing trypsin in the range of 5 U / ml to 25 U / ml for 40 hours to 70 hours; c) Filtering the virus harvest by dead - end filtration through at least one clarification filter having a pore size between 6 microns and 0.45 microns to obtain a clarified virus bank; d) Treating the clarified virus bank with Benzonase at a temperature in the range of 30°C to 34°C for 2 hours to 6 hours and then at a temperature in the range of 2°C to 8°C for 5 hours to 15 hours; e) Concentrating the Benzonase - treated clarified virus bank by tangential flow filtration using a membrane with a molecular weight cut - off of 100KDa to 500KDa to produce a virus harvest that is at least 4 - fold concentrated; f) Stabilizing the tangential flow - filtered concentrate with the stabilizer composition of claim 1 to form a stable virus harvest; and g) Sterilizing the stable tangential flow - filtered concentrate by dead - end filtration through at least one sterile - grade filter having a pore size between 0.8 microns and 0.2 microns to form a sterilized clarified monovalent virus bank; wherein the total recovery rate of the purified virus is greater than or equal to 40%.

16. The method according to claim 15, wherein the influenza virus is propagated in Madin-Darby canine kidney cells ATCC CCL-34.

17. The method of manufacturing an immunogenic composition according to claim 15, wherein step (d) comprises treating the virus harvest with Benzonase at a concentration in the range of 0.5 units / ml to 5 units / ml in the presence of a divalent cation selected from the group consisting of Ca 2+ , Mg 2+ , Mn 2+ and Cu 2+ in an amount between 0.1 mM and 100 mM.

18. The method for manufacturing an immunogenic composition according to claim 17, wherein step (d) comprises treating the virus harvest with Benzonase at a concentration ranging from 0.5 units / ml to 5 units / ml in the presence of a divalent cation Mg 2+ salt at a concentration of 1 mM to 3 mM.

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