Live attenuated influenza vaccine composition and process for its preparation

Abstract

The present disclosure provides compositions and methods for producing and obtaining live attenuated influenza vaccine (LAIV) compositions that can be delivered intranasally and provide protection against influenza virus infection. The LAIV strains are based on the cold-adapted, temperature-sensitive, and attenuated phenotype of Master Donor Virus (MDV), which contains the surface glycoprotein genes of wild-type pandemic or seasonal influenza strains. The LAIV strains are further adapted for growth in MDCK (Madin-Darby Canine Kidney) cells. The use of eggs is avoided in large-scale vaccine production. The purification process lacks a chromatography step. The LAIV compositions contain one or more live attenuated influenza vaccine viruses and lack polymers and surfactants.

Description

[Technical field]

[0001] The present disclosure relates to the field of viral vaccine manufacturing, and more particularly to a live attenuated influenza vaccine composition and a method for preparing the same.The present disclosure relates to a method for producing a virus or viral antigen produced by cell culture, a virus or viral antigen obtainable by this method, and a vaccine containing such a virus or viral antigen. [Background technology]

[0002] The following background information herein is relevant to the present disclosure but is not necessarily prior art. Epidemics and pandemics caused by infectious agents have been occurring for centuries and have caused great disruption with various morbidity and mortality rates. Influenza viruses are one of the main players in the history of pandemics. In the last century, four influenza pandemics occurred, and at least 15 influenza pandemics have been recorded to date, with an estimated 50 million people killed in 1918-19 alone.

[0003] Vaccines play an important role in controlling the spread of the virus, and inactivated influenza vaccines (IIV) as well as live attenuated influenza vaccines (LAIV) have been used for many years. The widely used parenterally administered inactivated influenza vaccine induces serum antibody responses that effectively prevent influenza disease when the circulating strain is antigenically matched to the vaccine. In contrast, live attenuated influenza vaccines (LAIV) are administered intranasally, mimicking natural infection, and induce both local and systemic humoral and cellular immune responses, conferring protection against matched strains as well as drifted strains. Furthermore, needle-free application of LAIV may lower the threshold for vaccination acceptance, thereby increasing influenza vaccine coverage.

[0004] So far, LAIVs have been licensed in the United States (since 2003), Europe (since 2010), Russia (since the 1980s), and India (since 2010). LAIVs are based on attenuated influenza A and B Master Donor Viruses (MDVs), which were developed independently, but in essentially the same way, in the United States and Russia in the 1960s. MedImmune's seasonal and pandemic LAIV viruses are currently produced by reverse genetics (RG). In contrast, Russian LAIVs are produced by classical genetic reassortment in embryonated eggs. Russian LAIVs have recently been registered for use in China and Thailand.

[0005] Russian LAIVs consist of reassortant viruses that contain hemagglutinin (HA) and in most cases neuraminidase (NA) gene fragments from the circulating wild-type (WT) virus of interest in a 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 MDVs for LAIVs. MDVs contain mutations in multiple gene fragments that make them cold-adapted (ca), temperature-sensitive (ts), and attenuated (att). The surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) of contemporaneous strains are incorporated into these MDVs by reassortantment. Their ca, ts, and att phenotypes indicate that LAIVs replicate at low temperatures, stop replicating at high temperatures (>38°C), and restrict replication to the upper respiratory tract. Compared to the MDV used in the United States, the Russian MDV contains fewer mutations located at different sites in the gene fragment, indicating that they may be differentially attenuated. Direct comparisons in animals and humans have shown that the Russian MDV and single-strain reassortants derived from it are more immunogenic than their counterparts.

[0006] Over the years, Russian LAIVs have been safely administered to more than 75 million people and have been shown to be safe in terms of attenuation (genetic stability) and transmission. Reversion of virulence (loss of the attenuated phenotype) has never been observed and is highly unlikely as it requires reversion of multiple mutations in more than one gene segment. No neurovirulence has been reported for Russian LAIV viruses, and both MDV and its derived reassortant have been shown to be devoid of neurovirulent properties. Following administration of LAV, there have been no reports of serious adverse events associated with immunization, except for self-limited influenza-like symptoms (runny nose, stuffy nose, sore throat, cough, headache, and low-grade fever) reported in rare cases. In addition to being safe, LAIVs have been shown to be effective in protecting against diseases caused by influenza virus infection. The immunity induced by LAIVs is broad, with protection against floating strains shown. In particular, in children, LAIVs have been shown to be more effective than inactivated influenza vaccines in protecting against clinical and / or culture-confirmed influenza, and have also been shown to confer herd immunity.

[0007] Like inactivated influenza vaccines, Russian LAIVs are produced using embryonated chicken eggs, with the inherent disadvantage of limited suppliers of vaccine quality and specific pathogen-free eggs, requiring advance ordering with a minimum of 4 months lead time before eggs are available for large-scale vaccine production. Specific facilities for egg incubation, harvesting, etc. limit production capacity to scale up rapidly. The long time required to produce egg-dependent vaccines may result in too few doses being available to combat a pandemic situation, such as the pandemic that occurred in 2009, or to stop a pandemic resulting from a severely pathogenic influenza virus. Thus, current vaccine production systems are inadequate to respond to an influenza pandemic, and thus novel emergency vaccine production processes are required. In theory, production of influenza vaccines in cell culture would offer important advantages in the event of a pandemic. Vaccine production on a large scale could be easily achieved using existing tissue culture production units for other viral vaccines.

[0008] Control of substrate consistency and availability of adaptability in production, emergency scale-up, which is especially important in case of pandemic, and no dependency on egg supply and maintenance of flocks are the main advantages of tissue culture methods. Egg shells are porous structures and eggs are non-sterile outside. The manufacturing process of growing influenza virus in eggs requires outside manipulations for penetration of the egg shell for inoculation and harvesting with inherent risks of contamination. Furthermore, cell culture is a more controlled system with defined cell culture media and valid cell banks according to Good Manufacturing Practice (GMP). Therefore, attempts have been made to move from egg production to cell culture production.

[0009] Several cell lines are currently under investigation for cell culture-based influenza production, including MRC-5 cells (see de Ona et al. (1995) J Clin Microbiol 33:1948-49), HepG2 cells (see Ollier et al. (2004) J Clin Microbiol 42(12):5861-5), LLC-MK2 cells (see Schepetiuk & Kok (1993) J Virol Methods 42(2-3):241-50), Madin-Darby canine kidney (MDCK) cells (see Tobita et al. (1975) Med Microbiol Immunol (Berl). 162(1):9-14 and 23-27), and African green monkey Vero cells (see 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 (see Cox, RJ et al.; Vaccine 2009, 27, 1889-1897). Previously, a 5.0 log 10 TCID 50 Low to moderate titers of the virus were reported with titers of up to 6.7 log / ml on MDCK cells. 10 TCID 50Titers of 1000 / mL were reported. Cell culture derived influenza vaccines (egg isolated influenza viruses adapted / optimized for growth in cell culture) have been licensed in Europe (Optaflu Novartis; 2007) and the United States (Flucelvax Novartis; 2012), and only a few influenza vaccines on the market are cell culture derived. This may be due to the inconsistent yields of influenza vaccines using cell culture production systems combined with classical purification methods and laborious stabilization practices. Theoretical safety concerns regarding the continued use of cell lines such as MDCK cells have been raised regarding their use in vaccine manufacturing (VRBPAC, 2008). These concerns are mainly related to residual cellular components (DNA and proteins) in vaccine drug production, especially for live attenuated influenza vaccine (LAIV) products that are not inactivated or subjected to extensive biochemical purification as is the case for traditional inactivated influenza vaccines. To minimize these risks, two strategies have been followed: cell line characterization and vaccine purification processing steps. A particular aspect of the purification process is to reduce the amount and size of residual host cell DNA within the vaccine product. The manufacture of Optaflu Novartis (2007) involves several steps to remove residual host cell DNA. These include cellulose sulfate ion exchange chromatography, which binds the influenza virus and allows the DNA to pass through, and a subsequent CTAB precipitation step, which specifically precipitates the DNA.

[0010] Previously reported purification processes are costly and time-consuming, using one or more of hydroxyapatite, affinity, anion exchange and size exclusion chromatography. It has been reported that the overall recovery of virus is not optimal for routine vaccine production using chromatographic methods. Furthermore, circulating influenza viruses change the antigenicity of the virus particles due to antigenic drift and shift observed in the virus. These changes affect the physicochemical properties of the virus and subsequently its binding ability to the chromatographic matrix. This can result in high levels of alteration in the yield of drift or shifter strains, making chromatography unsuitable for routine production. Alternative methods used for removal / reduction of host cell DNA utilize higher concentrations of benzonase (e.g., 50U / ml, 100U / ml) and are expensive.

[0011] Typical non-ionic surfactants used in pharmaceutical formulations include Triton™ X-100, Pluronic® F-68, F-88, and F-127 (poloxamers), Brij 35 (polyoxyethylene alkyl ether), polyoxyl 40 stearate, Cremophor® EL, and alpha-tocopherol TPGS. Each of these surfactants has in common the fact that they all contain polyoxyethylene moieties and therefore, to a greater or lesser extent, exhibit similar problems in that the polyoxyethylene moieties can autooxidize to produce reactive peroxides, causing increased immunogenicity of unwanted proteins. (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).

[0012] Various stabilizers are used to stabilize vaccine preparations and achieve the desired shelf life. Stabilizers such as polyvinylpyrrolidone (PVP), trehalose, and sorbitol are also used in virus formulations. However, PVP has also been reported to destabilize live attenuated virus formulations. (Ref: 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).

[0013] Trehalose is costly; it must be combined with other sugar and protein additives (gelatin) to achieve stability, and other stabilizers are superior to trehalose in increasing the shelf-life stability of lyophilized vaccines.

[0014] Sorbitol has a low glass transition temperature (Tg) (-1.6°C) and therefore cannot be used as a major formulation component. The low Tg of sorbitol limits its use. Sorbitol needs to be combined with other sugars and protein additives (gelatin) to achieve stability.

[0015] It was suggested that the theoretical impact of residual host DNA on the safety of the product should be considered with respect to the route of vaccine administration, since tissue distribution and clearance rates may vary based on the mode of administration. Studies demonstrate that uptake and clearance of MDCK DNA from tissues varies depending on the route of administration. When DNA was administered intranasally, detectable DNA levels were lower at all time points compared to intramuscularly. Thus, the intranasal route of vaccine administration appears to reduce possible risks associated with residual host cell DNA that may be present in the final vaccine product produced by cell culture. (Ref: DE Tabor et al.; Biologicals 41 (2013) 247-253).

[0016] The present disclosure aims to overcome the aforementioned limitations as well as provide compositions and methods for producing MDCK (Madin-Darby Canine Kidney) cell-based intranasally delivered live attenuated influenza vaccines (LAIVs) to protect against influenza viruses. The present disclosure further provides an improved manufacturing process that utilizes low concentrations of endonucleases, more specifically benzonase, lacks chromatography steps, and is suitable for large-scale cell culture production. Additionally, the present disclosure provides polymer- and surfactant-free LAIV formulations that include one or more live attenuated influenza vaccine viruses. Summary of the Invention [Means for solving the problem]

[0017] This disclosure: a) one or more live attenuated influenza vaccine viruses derived by "classical" or "reverse genetics" methods of reassortment, where the live attenuated influenza vaccine virus strain consists essentially of the hemagglutinin (HA) and / or neuraminidase (NA) genes from a wild-type pandemic or seasonal influenza virus, as well as genes expressing the PB1, PB2, PA, NP, M, and NS proteins, and, in some cases, the NA protein from the Master Donor Virus (MDV), the A / Leningrad / 134 / 17 / 57 (H2N2) and / or B / USSR / 60 / 69 strains; b) one or more amino acids; c) one or more carbohydrates, and d) Gelatin The present invention provides an MDCK cell-based intranasally delivered live attenuated influenza vaccine (LAIV) composition comprising:

[0018] The present disclosure further provides methods for producing such vaccine compositions / formulations. the purpose Some objectives of the present disclosure that are met by at least one embodiment herein are as follows: It is an object of the present disclosure to ameliorate one or more of the problems in the prior art, or at least to provide a useful alternative.

[0019] Another object of the present disclosure is to provide vaccine compositions and methods for producing a live attenuated influenza vaccine (LAIV) that can be delivered intranasally. Yet another object of the present disclosure is to provide a Madin-Darby canine kidney (MDCK) cell-based live attenuated influenza vaccine (LAIV) composition, in which the use of eggs is avoided entirely.

[0020] It is yet another object of the present disclosure to provide an LAIV composition comprising one or more live attenuated influenza vaccine viruses and lacking polymers and surfactants. Yet another object of the present disclosure is to provide an LAIV composition comprising one or more live attenuated influenza vaccine viruses, where the LAIV strain is based on the cold-adapted, temperature-sensitive and attenuated phenotype of Master Donor Virus (MDV), which contains one or two surface glycoproteins of a wild-type pandemic or seasonal influenza strain.

[0021] Yet another object of the present disclosure is to provide an MDCK cell-based intranasally delivered live attenuated influenza vaccine (LAIV) composition, which composition retains the desired characteristics of the virus, including immunogenicity and stability.

[0022] Yet another object of the present disclosure is to provide an MDCK cell-based intranasally delivered live attenuated influenza vaccine (LAIV) composition / formulation suitable for treating or preventing influenza virus infection, or preventing, ameliorating, or delaying the onset or progression of clinical symptoms thereof.

[0023] Yet another object of the present disclosure is to provide improved methodologies in the field of MDCK cell-based live attenuated influenza vaccine production that is suitable for large-scale cell culture production.

[0024] Other objects and advantages of the present disclosure will be more apparent from the following description, which is not intended to limit the scope of the present disclosure. The present disclosure will now be described with the aid of the accompanying drawings, which are listed below. [Brief description of the drawings]

[0025] [Figure 1] FIG. 1 is a schematic diagram of the reassortment of wild-type pandemic or seasonal influenza virus and attenuated MDV to generate a reassortant vaccine strain, where (A) represents the wild-type virus infectious pathogen, (B) represents the master donor virus containing temperature-sensitive (ts), cold-adapted (ca), and attenuated (at) phenotypic gene segments, and (C) represents the reassortant vaccine strain. [Diagram 2] FIG. 2 is a graph showing the stability of influenza A strain (A / 17 / turkey / Turkey / 05 / 133-A / H5N2) virus log yield titer (EID50 / 0.5 ml) at 37° C. by stabilizer composition 1 as disclosed in Table 3B. [Diagram 3] FIG. 3 is a graph showing the stability of influenza A strains (A / 17 / turkey / Turkey / 05 / 133-A / H5N2 and A / 17 / Anhui / 2013 / 61-A / H7N9) virus log yield titers (EID50 / 0.5 ml) at 2-8° C. with stabilizer composition 1 as disclosed in Table 3A. [Figure 4] FIG. 4 is a graph showing the stability of influenza A strain (A / 17 / California / 2009 / 38-A / H1N1) and influenza B strain (B / Texas / 02 / 13-CDC) virus log yield titers (EID50 / 0.5 ml) at 37° C. with stabilizer composition 3 as disclosed in Table 3B. [Diagram 5]FIG. 5 is a graph showing the stability of influenza A strain (A / 17 / California / 2009 / 38-A / H1N1) and influenza B strain (B / Texas / 02 / 13-CDC) virus log yield titers (EID50 / 0.5 ml) at 2-8° C. with stabilizer composition 3 as disclosed in Table 3A. [Figure 6] FIG. 6 is a graph showing the stability of influenza A strain (A / 17 / California / 2009 / 38-A / H1N1) and influenza B strain (B / Texas / 02 / 13-CDC) virus log yield titers (EID50 / 0.5 ml) at 37° C. with stabilizer composition 4 as disclosed in Table 3B. [Figure 7] FIG. 7 is a graph showing the stability of influenza A strain (A / 17 / California / 2009 / 38-A / H1N1) and influenza B strain (B / Texas / 02 / 13-CDC) virus log yield titers (EID50 / 0.5 ml) at 2-8° C. with stabilizer composition 4 as disclosed in Table 3A. [Figure 8] FIG. 8 is a graph showing the stability of influenza A strain (A / SouthAfrica / 3626 / 13-H1N1) and influenza B strain (B / Texas / 02 / 13-CDC) virus log yield titers (EID50 / 0.5 ml) at 37° C. with stabilizer composition 2 as disclosed in Table 3B. [Figure 9] FIG. 9 is a graph showing the stability of influenza A strain (A / SouthAfrica / 3626 / 13-H1N1) and influenza B strain (B / Texas / 02 / 13-CDC) virus log yield titers (EID50 / 0.5 ml) at 2-8° C. with stabilizer composition 2 as disclosed in Table 3A. [Figure 10] FIG. 10 is a graph showing the stability of influenza A strain (A / SouthAfrica / 3626 / 13-H1N1) and influenza B strain (B / Texas / 02 / 13-CDC) virus log yield titers (EID50 / 0.5 ml) at 37° C. with stabilizer composition 1 as disclosed in Table 3B. [Figure 11]FIG. 11 is a graph showing the stability of influenza A strain (A / SouthAfrica / 3626 / 13-H1N1) and influenza B strain (B / Texas / 02 / 13-CDC) virus log yield titers (EID50 / 0.5 ml) at 2-8° C. with stabilizer composition 1 as disclosed in Table 3A. [Figure 12] Figure 12 shows infectious virus tested in nasal turbinate and lung samples. Animals were vaccinated with 1 or 2 doses of H5 LAIV, H7 LAIV, or placebo. Groups 1-4 were challenged with H5 / tk / Tk, groups 5 and 6 were challenged with H5 / Vt, and groups 7-10 were challenged with H7 / An. Nasal turbinate samples (a) and lung samples (b) collected 4 days after infection were titrated for the presence of replication-competent virus particles. Individual titers are shown with group means indicated by solid black lines. [Figure 13] Figure 13: HI and VN antibody responses after immunization. Geometric mean antibody responses 28 days after the last immunization (28 test days for the 1 dose regime and 56 test days for the 2 dose regime) against homologous challenge viruses, H5 / tk / Tk for H5 LAIV immunized animals and H7 / An for H7 LAIV immunized animals. (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 Mann-Whitney U test. *p<0.05 and **P<0.01. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] While the present disclosure may be subject to different embodiments, specific embodiments are shown in the drawings and the following detailed description, with the understanding that the disclosure is considered an exemplification of the principles of the disclosure and is not intended to limit the scope of the disclosure as illustrated and disclosed in this description.

[0027] Embodiments of the present disclosure will now be described with reference to the accompanying drawings. The embodiments are provided to thoroughly and completely convey the scope of the present disclosure to those skilled in the art. Numerous details regarding specific components and methods are described to provide a thorough 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 equipment structures, and well-known technologies are not described in detail.

[0028] The terminology used in this disclosure is for the purpose of describing particular embodiments only, and such terms are not to be construed as limiting the scope of the disclosure. As used in this disclosure, the forms "a," "an," and "the" may be intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0029] The terms first, second, third, etc. should not be construed as limiting the scope of the present disclosure, as such terms may be used only to distinguish one element, component, region, layer or section from another component, region, layer or section. The terms first, second, third, etc., as used herein, do not imply a particular sequence or order unless expressly indicated by the present disclosure.

[0030] As used herein, the term "influenza virus" refers to an RNA virus that includes the Orthomyxoviridae family influenza viruses A, B, C, and D. The influenza virus can be a wild-type pandemic or seasonal live influenza virus, a live attenuated influenza vaccine virus, an inactivated influenza virus, a chimeric influenza virus, or a recombinant influenza virus.

[0031] The present disclosure provides compositions comprising live attenuated influenza vaccine (LAIV) viruses for protection against infection with influenza viruses. The present disclosure further provides methods for producing compositions comprising one or more influenza vaccine viruses.

[0032] According to a first embodiment of the present disclosure, an LAIV composition may comprise one or more live attenuated influenza vaccine viruses, one or more amino acids, one or more carbohydrates and gelatin.

[0033] The term "live" is used in its conventional sense; a live virus is one that has not been inactivated, i.e., one that is capable of replicating in permissive cells. A live attenuated influenza vaccine virus is one that does not induce disease caused by the corresponding wild-type virus in animals or humans and is capable of inducing a specific immune response.

[0034] According to a second embodiment of the present disclosure, one or more live attenuated influenza vaccine viruses may be derived by "classical" or "reverse genetics" methods of reassortment involving genetic fragments from one or more influenza virus strains.

[0035] According to a first aspect of the second embodiment, the reassortant live attenuated influenza vaccine virus is a reassortant LAIV virus comprising 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 the hemagglutinin (HA) and / or neuraminidase (NA) of a wild type pandemic or seasonal influenza A or B or C virus strain in a ratio of 1:7, 2:6, 3:5, 4:4, 5:3, 6:2 or 7:1.

[0036] More preferably, the reassortant live attenuated influenza vaccine virus may contain gene segments from a Master Donor Virus (MDV) strain as well as a wild type pandemic or seasonal influenza virus strain in a 6:2 ratio (as shown in FIG. 1).

[0037] According to a second aspect of the second embodiment, the reassortant live attenuated influenza vaccine virus may comprise genetic segments from a Master Donor Virus (MDV) derived from an influenza A virus of any subtype, or may be derived from an influenza B virus of any subtype.

[0038] The reassortant live attenuated influenza vaccine virus may comprise gene segments from a Master Donor Virus (MDV) selected from the group including: A / Leningrad / 134 / 17 / 57 (H2N2) influenza A strain, B / USSR / 60 / 69 influenza B strain.

[0039] More preferably, the reassortant live attenuated influenza A vaccine virus may comprise genetic segments from Master Donor Viruses (MDV), including the A / Leningrad / 134 / 17 / 57 (H2N2) influenza A strain.

[0040] More preferably, the reassortant live attenuated influenza A vaccine virus strain-A / 17 / California / 2009 / 38 may comprise gene segments from Master Donor Viruses (MDVs) including the A / Leningrad / 134 / 17 / 57 (H2N2) influenza A strain.

[0041] More preferably, the reassortant live attenuated influenza A vaccine virus strain-A / 17 / turkey / Turkey / 05 / 133 may contain genetic segments from Master Donor Viruses (MDVs), including influenza A / Leningrad / 134 / 17 / 57 (H2N2).

[0042] More preferably, the reassortant live attenuated influenza A vaccine virus strain-A / 17 / Anhui / 2013 / 61 may contain gene segments from Master Donor Viruses (MDVs), including influenza A / Leningrad / 134 / 17 / 57 (H2N2).

[0043] More preferably, the reassortant live attenuated influenza A vaccine virus strain-A / 17 / New York / 15 / 5364 may contain genetic segments from Master Donor Viruses (MDVs), including influenza A / Leningrad / 134 / 17 / 57 (H2N2).

[0044] More preferably, the reassortant live attenuated influenza A vaccine virus strain-A / 17 / Hong-Kong / 2014 / 8296 may contain gene segments from Master Donor Viruses (MDVs), including A / Leningrad / 134 / 17 / 57 (H2N2) influenza A.

[0045] More preferably, the reassortant live attenuated influenza A vaccine virus strain-A / South-Africa / 3626 / 2013-CDC-LV14A may contain gene segments from Master Donor Viruses (MDVs), including A / Leningrad / 134 / 17 / 57 (H2N2) influenza A.

[0046] More preferably, the reassortant live attenuated influenza B vaccine virus may comprise genetic segments from Master Donor Viruses (MDV), including the B / USSR / 60 / 69 influenza B strain.

[0047] More preferably, the reassortant live attenuated influenza B vaccine virus strain-B / Texas / 02 / 2013-CDC-LV8B may contain gene segments from Master Donor Viruses (MDVs), including the B / USSR / 60 / 69 influenza B strain.

[0048] More preferably, the reassortant live attenuated influenza B vaccine virus strain-B / Phuket / 3073 / 2013 may contain gene segments from Master Donor Viruses (MDVs), including the B / USSR / 60 / 69 influenza B strain.

[0049] More preferably, the reassortant live attenuated influenza B vaccine virus strain-B / 56 / Brisbane / 60 / 08 may contain gene segments from Master Donor Viruses (MDVs), including the B / USSR / 60 / 69 influenza B strain.

[0050] According to a third aspect of the second embodiment, the reassortant live attenuated influenza vaccine virus may comprise 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.

[0051] More preferably, the reassortant live attenuated influenza A vaccine virus strain may comprise a hemagglutinin (HA) gene from an influenza A virus HA subtype H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17 or H18, or any other reported HA subtype, and / or a neuraminidase (NA) gene from an influenza A virus NA subtype N1, N2, N3, N4, N5, N6, N7, N8, N9, N10 or N11, or any other reported NA subtype.

[0052] More preferably, the reassortant live attenuated influenza vaccine virus may comprise the hemagglutinin (HA) gene and / or the neuraminidase (NA) gene from a pandemic or potentially pandemic influenza virus strain.

[0053] More preferably, the reassortant live attenuated influenza vaccine virus may comprise the hemagglutinin (HA) gene and / or the neuraminidase (NA) gene from a seasonal influenza virus strain.

[0054] More preferably, the reassortant live attenuated influenza vaccine virus strains may contain the hemagglutinin (HA) gene and / or the 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 viral strains.

[0055] More preferably, the reassortant live attenuated influenza vaccine virus strain may contain the hemagglutinin (HA) gene and / or the neuraminidase (NA) gene from influenza A virus strain pdmH1N1.

[0056] More preferably, the reassortant live attenuated influenza vaccine virus strain may contain the hemagglutinin (HA) gene and / or the neuraminidase (NA) gene from an influenza A virus A / California / 07 / 2009 (designated A / Cal)-like strain.

[0057] More preferably, the reassortant live attenuated influenza vaccine virus strain may contain the hemagglutinin (HA) gene and / or the neuraminidase (NA) gene from an influenza A virus A / Michigan / 45 / 2015-like strain.

[0058] More preferably, the reassortant live attenuated influenza vaccine virus strain may contain the hemagglutinin (HA) gene and / or the neuraminidase (NA) gene from an influenza A virus strain-A / South-Africa / 3626 / 2013-like strain.

[0059] More preferably, the reassortant live attenuated influenza vaccine virus strain may contain the hemagglutinin (HA) gene and / or the neuraminidase (NA) gene from an influenza A virus H3N2-A / HongKong / 4801 / 2014-like strain.

[0060] More preferably, the reassortant live attenuated influenza vaccine virus strain may contain the hemagglutinin (HA) gene and / or the neuraminidase (NA) gene from influenza B viruses belonging to two different lineages, either Yamagata-like or Victoria-like.

[0061] More preferably, the reassortant live attenuated influenza vaccine virus strain may contain the hemagglutinin (HA) gene and / or the neuraminidase (NA) gene from the Victoria lineage of influenza B virus-B / Brisbane / 60 / 2008-like strain.

[0062] More preferably, the reassortant live attenuated influenza vaccine virus strain may contain the hemagglutinin (HA) gene and / or the neuraminidase (NA) gene from the Yamagata lineage of influenza B virus-B / Phuket / 3073 / 2013-like strain. There are two methods for generating reassortants 1. Classical method of reassortment The process of reassortment of wild-type epidemic or seasonal influenza vaccine virus and attenuated MDV to generate reassortant strains involves co-infection of a culture host, usually eggs, with an MDV strain and a wild-type virus strain. Reassortant viruses are selected by adding antibodies with specificity for the HA and / or NA proteins of MDV to select for reassortant viruses containing the HA and / or NA proteins of the wild-type virus strain. Over several passages of this procedure, fast-growing reassortant viruses containing the HA and / or NA fragments of the wild-type epidemic or seasonal influenza vaccine virus strain and the internal genes of MDV can be selected. 2. Reverse Genetics Method of Reassortment Reverse genetics is a method of generating infectious viral particles from DNA copies. Six internal genes of MDV recommended for inclusion in the vaccine as well as the HA and NA genes from the wild-type strain are cloned into plasmids capable of generating full-length viral RNA when transfected into cultured cells. These plasmids are transfected into cultured cells along with four plasmids expressing the polymerase subunits of the virus. Expression of the polymerase subunits and generation of full-length viral RNA leads to virus assembly and release of infectious viral particles into the supernatant. The rescued viruses display antigenic characteristics of the recommended strains and the ca, ts, and att phenotypes of MDV.

[0063] Reassortant LAIV strains are produced from WHO collaborating centers such as the Institute of Experimental Medicine (IEM) in St. Petersburg, Russia, or the Centers for Disease Control and Prevention (CDC) in Atlanta.

[0064] According to the third embodiment of the present disclosure, the LAIV composition may include one or more carbohydrates selected from the group of, but not limited to, natural carbohydrates, synthetic carbohydrates, polyols, glass transition promoters, monosaccharides, disaccharides, trisaccharides, oligosaccharides and their corresponding sugar alcohols, polyhydroxyl compounds such as carbohydrate derivatives and chemically modified carbohydrates, hydroxyethyl starch, and sugar copolymers. Both natural and synthetic carbohydrates are suitable for use. Synthetic carbohydrates include, but are not limited to, those with glycosidic bonds replaced by thiol or carbon bonds. Both D- and L-forms of carbohydrates may be used. Carbohydrates may be non-reduced or reduced. When reduced carbohydrates are used, the addition of an inhibitor of the Maillard reaction is preferred. Reduced carbohydrates suitable for use in the composition are known to those skilled in the art and include, but are not limited to, glucose, sucrose, maltose, lactose, fructose, galactose, mannose, maltulose, and lactulose. Non-reducing sugars include non-reducing glycosides of polyhydroxyl compounds selected from, but not limited to, sugar alcohols and other linear polyalcohols. Other useful sugars include raffinose, stachyose, melezitose, dextran, cellibiose, mannobiose and sugar alcohols. The sugar alcohol glycosides are preferably monoglycosides, in particular compounds obtained by reduction of disaccharides such as lactose, maltose, lactulose and maltulose. The glass former is selected from the group consisting of sucrose, mannitol, trehalose, mannose, raffinose, lactitol, lactobionic acid, glucose, maltulose, iso-maltulose, maltose, lactose, sorbitol, dextrose, fructose, glycerol, or combinations thereof.

[0065] Further according to a preferred aspect of the third embodiment, the LAIV composition may comprise sucrose as a suitable carbohydrate stabilizer in the range of between 1% and 20% weight / volume, preferably between 1-10%, more preferably between 3-6%, and most preferably 4% (w / v).

[0066] According to a fourth embodiment of the present disclosure, the LAIV composition may comprise one or more amino acids selected from the group including, but not limited to, tricine, arginine, leucine, isoleucine, histidine, glycine, glutamine, lysine, alanine, peptides, hydrolyzed proteins or proteins such as serum albumin.

[0067] Further according to a preferred aspect of the fourth embodiment, the LAIV composition may include tricine, arginine, histidine and alanine as preferred amino acids, either individually or in combination.

[0068] Further according to a preferred aspect of the fourth embodiment, the one or more amino acids may comprise tricine in the range of between 0.1% and 2% weight / volume, preferably between 0.1-1%, more preferably between 0.1-0.5%, most preferably 0.3% (w / v).

[0069] Further according to a preferred aspect of the fourth embodiment, the one or more amino acids may include histidine in the range of between 0.1%-2% (w / v), preferably between 0.1-1%, more preferably between 0.1-0.5%, and most preferably 0.21% (w / v).

[0070] Further according to a preferred aspect of the fourth embodiment, the one or more amino acids may comprise 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%, most preferably 0.1% (w / v).

[0071] Further according to a preferred aspect of the fourth embodiment, the one or more amino acids may comprise arginine in the range of between 0.1% and 10% weight / volume, preferably between 0.1-5%, more preferably between 0.1-3%, most preferably 2.1% (w / v).

[0072] According to the fifth embodiment of the present disclosure, the LAIV composition may contain 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 0.85% (w / v).

[0073] As used herein, the term "gelatin" refers to a sterile, non-pyrogenic protein preparation (e.g., fraction) produced by partial acid hydrolysis (type A gelatin) or partial alkaline hydrolysis (type B gelatin) of animal collagen, most commonly from bovine, porcine, and fish sources. Gelatin is available in a variety of molecular weight ranges. Recombinant sources of gelatin may also be used.

[0074] According to a sixth embodiment of the present disclosure, the LAIV composition may further comprise a buffer selected from the group consisting of HEPES, citrate-phosphate, carbonate, phosphate, citrate, lactate, gluconate, and tartaric acid buffers, as well as more complex organic buffers including phosphate buffers containing sodium and / or potassium phosphate in ratios selected to achieve the desired pH. In another example, the buffer contains Tris(hydroxymethyl)aminomethane, or "Tris", formulated to achieve the desired pH. In yet another example, the buffer may be a minimum essential medium containing Hanks salts.

[0075] According to a seventh embodiment of the present disclosure, the single dose composition does not contain a preservative, and the multi-dose composition may further contain a preservative selected from the group including 2-phenoxyethanol, benzethonium chloride (femerol), phenol, m-cresol, thiomersal, formaldehyde, paraben esters (e.g., methyl-, ethyl-, propyl-, or butyl-paraben), benzalkonium chloride, benzyl alcohol, chlorobutanol, p-chloro-m-cresol, or benzyl alcohol, or combinations thereof. The vaccine composition may contain materials for a single immunization or may contain materials for multiple immunizations (i.e., a "multi-dose" kit). Including a preservative is preferred for multi-dose formulations. As an alternative (or in addition) to including a preservative in the multi-dose composition, the composition may be contained in a container with a preservative adapter for removal of the material.

[0076] According to an eighth embodiment of the present disclosure, the LAIV composition further comprises a pharma- ceutically acceptable transporter, excipient, binder, carrier, tonicity agent, emulsifier, or wetting agent, where the pharma- ceutically acceptable excipient is selected from the group consisting of surfactants, polymers, and salts. Examples of surfactants may include non-ionic surfactants such as polysorbate 20, polysorbate 80, etc. Examples of polymers may include dextran, carboxymethylcellulose, hyaluronic acid, cyclodextrin, etc. Examples of salts may include NaCl, KCl, KH2PO4, Na2HPO4, 2H2O, CaCl2, MgCl2, etc.

[0077] According to a ninth embodiment of the present disclosure, the LAIV composition may further comprise aluminum hydroxide, aluminum phosphate, aluminum hydroxyphosphate, and aluminum potassium sulfate, or mixtures thereof.

[0078] According to a tenth embodiment of the present disclosure, the LAIV composition comprises an oil and water emulsion, MF-59, liposomes, lipopolysaccharides, saponin, lipid A, lipid A derivatives, monophosphoryl lipid A, 3-desacyl monophosphoryl lipid A, AS01, AS03, oligonucleotides, oligonucleotides containing at least one unmethylated CpG and / or liposomes, Freund's adjuvant, Freund's complete adjuvant, Freund's incomplete adjuvant, polymers, copolymers, e.g., block copolymers. In some embodiments, the composition may further comprise an immunostimulatory component selected from the group consisting of polyoxyethylene-polyoxypropylene copolymers including polymer p1005, CRL-8300 adjuvant, muramyl dipeptide, a TLR-4 agonist, flagellin, flagellin from gram-negative bacteria, a TLR-5 agonist, a fragment of flagellin capable of binding to the TLR-5 receptor, alpha-C-galactosylceramide, chitosan, interleukin-2, OS-21, ISCOMS, saponins in combination with sterols, and lipids.

[0079] According to an eleventh embodiment of the present disclosure, a method for preparing an MDCK cell culture-based LAIV composition may include any subset or all of the following steps: a) LAIV candidate vaccine viruses are first passaged in SPF embryonated chicken eggs producing egg-based master seed virus (MSV).

[0080] b) The egg-based master seed virus is adapted to grow in a cell culture host to prepare a cell-based working seed virus (WSV). The cell-based WSV is grown on a 175 cm2 surface area plate. 2 Tissue culture flask (TCF), surface area 850 cm 2 Roller bottle (RB), surface area 6320 cm 2Different cell culture vessels / systems are used to subculture and grow host cells, such as Cell Factories (CF) from Cell Sciences, Inc., and fixed-bed bioreactors (e.g., iCELLis® bioreactors, e.g., Nano and 500 / 100 bioreactors from Pall® Life Sciences, Port Washington, NY).

[0081] c) Harvest the cultured virus. d) Filtering the virus harvest by direct flow filtration (DFF) through at least one clarification filter to obtain a clarified virus pool (CVP).

[0082] e) The CVP is treated with a non-specific endonuclease to degrade the cellular DNA. f) The endonuclease treated CVP is subjected to tangential flow filtration. g) stabilizing 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.

[0083] h) Sterilizing the stabilized TFF concentrate by DFF through at least one sterilizing grade filter to obtain a sterile clarified monovalent virus pool (CMVP).

[0084] i) Sterilized CMVP is stored in a polycarbonate bottle at -60°C or below. j) Fill the sterilized formulation into vials and store at 2-8°C. According to the first aspect of the eleventh embodiment, the egg-based LAIV virus candidate is adapted for growth in a cell culture host, which may be any eukaryotic cell. More preferably, the cell culture host may be either a mammalian or avian cell. Suitable mammalian cells include, but are not limited to, hamster, bovine, primate (including human and monkey), and canine cells. Various cell types include, but are not limited to, kidney cells, fibroblast cells, retinal cells, and lung cells. Examples of suitable hamster cells are cell lines named BHK21 or HKCC. Suitable monkey cells are African green monkey cells, such as kidney cells, e.g., Vero cell lines. Suitable canine cells are kidney cells, e.g., CLDK and MDCK cell lines.

[0085] 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 available, for example, from the American Type Culture Collection (ATCC), from the Coriell Cell Depository, or from the European Collection of Cell Cultures (ECACC). For example, ATCC supplies a variety of different Vero cells under catalog numbers CCL 81, CCL 81.2, CRL 1586, and CRL-1587, and supplies MDCK cells under catalog number CCL 34. PER.C6 is available from ECACC under deposit number 96022940.

[0086] More preferably, the cell culture host may be a Madin-Darby Canine Kidney (MDCK) cell selected from, but not limited to, ATCC CCL-34, MDCK 33016 cell line (DSM ACC 2219), MDCK (ATCC CCL34 MDCK(NBL2)), MDCK 33016 (DSM ACC 2219), DSM ACC3309, 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.

[0087] More preferably, the cell culture host can be Madin-Darby canine kidney (MDCK) cells of ATCC CCL 34 (NBL2). According to a second aspect of the eleventh embodiment, the MDCK cells may be cultured in Minimum Essential Medium (MEM) containing 10% fetal bovine serum (FBS). The culture of the cells may occur at 37° C.±1° C. The pH value of the medium during the growth of the cells before infection may range between values ​​of pH 6.8 and pH 7.6, and more preferably between pH 7.0 and pH 7.4.

[0088] Additionally, MDCK cells can be cultured in serum-free or protein-free medium. According to the third aspect of the eleventh embodiment, prior to infection, MDCK cells may be washed with MDM followed by MEM containing a protease in the range of 5-25 U / ml.

[0089] However, the protease may be selected from, but is not limited to, trypsin, chymotrypsin, fungal proteases, pepsin, papain, bromelain, and subtilisin.

[0090] More preferably, the protease may be trypsin obtained from porcine or bovine or fungal or bacterial origin. More preferably, the protease may be a recombinant trypsin expressed in a yeast or plant or bacterial host cell selected from, but not limited to, Aspergillus spp., Streptomyces griseus, maize, E. coli, Pichia pastoris.

[0091] Additionally, the preferred trypsin concentration is 12.5 U / ml. According to the fourth aspect of the eleventh embodiment, prior to infection, the working seed virus may be activated by diluting the virus in MEM containing protease in the range of 5-25 U / ml and incubating at a temperature of 31°C to 33°C for 10-60 minutes.

[0092] However, the protease may be selected from, but is not limited to, trypsin, chymotrypsin, fungal proteases, pepsin, papain, bromelain, and subtilisin.

[0093] More preferably, the protease may be trypsin obtained from porcine or bovine or fungal or bacterial origin. More preferably, the protease may be a recombinant trypsin expressed in a yeast or plant or bacterial host cell selected from, but not limited to, Aspergillus, Streptomyces griseus, Maize, Escherichia coli, Pichia pastoris. Preferably, said recombinant trypsin is selected from Biogenomics (E. coli as host), DKBio Pharma Pvt.Ltd (E. coli as host), Richcore (Pichia pastoris as host) and Gibco (fungal).

[0094] Additionally, the preferred trypsin concentration is 12.5 U / ml. Additionally, the preferred trypsin concentration is 2000-3000 units of trypsin per roller bottle.

[0095] According to the fifth aspect of the eleventh embodiment, the infection of MDCK cells with the LAIV virus candidate is preferably performed at a concentration of 40-60×10 6 Pieces / TCF, RB: 150-180 x 10 6 pcs / RB and bioreactor (4m 2 ) is 7000~10000×10 6 pieces / BR(4m 2 ) can occur at MDCK cell densities of

[0096] According to a sixth aspect of the eleventh embodiment, the LAIV virus candidate may be propagated in MDCK cells in an adherent or suspension culture mode. According to the seventh aspect of the eleventh embodiment, infection of MDCK cells with egg-based LAIV virus candidates may occur at an MOI of between 1:100 and 1:10000.

[0097] According to the eighth aspect of the eleventh embodiment, after infection, the MDCK cells may be cultured in Minimum Essential Medium (MEM) containing trypsin in the range of 5-25 U / ml at a temperature of 32° C.±1° C. The pH value of the medium after infection may be in the range of pH 6.8 and pH 7.6, most preferably in the range of 7.2-7.6.

[0098] According to the ninth aspect of the eleventh embodiment, after infection, the cell supernatant may be harvested after an incubation period of 40 to 70 hours; more preferably, 54±8 hours. Alternatively, multiple harvests can be performed at appropriate time intervals, approximately 4-5 times, before discarding the input material and processing it separately to obtain a clarified monovalent virus pool (CMVP).

[0099] At the time of collection, LogEID must be at least 7.0-9.2 50 A virus yield of 0.5 ml / ml can be achieved. According to the tenth aspect of the eleventh embodiment, the medium containing the virus can be clarified, typically through filters of decreasing pore size (e.g., 6μ, 5μ, 0.8μ, 0.65μ, 0.45μ, 0.2μ). Suitable commercially available filters and filtration devices are well known in the art and can be selected by the skilled artisan. Exemplary filtration devices are made of polypropylene or cellulose acetate or polyethersulfone, and commercially available filters can be Millipak (Millipore), Kleenpak (Pall) and Sartobran™ (Sartorius) filtration devices.

[0100] According to an eleventh aspect of the eleventh embodiment, the filtered harvest may be treated with a non-specific endonuclease, most preferably benzonase, at a concentration varying between 0.5 Units / ml and 2 Units / ml, at a temperature ranging between 30-34°C for 2-6 hours, followed by a temperature of 2-8°C for 5-15 hours.

[0101] Alternatively, the filtered harvest may be treated with a non-specific endonuclease, most preferably benzonase, in an amount between 0.1 mM and 100 mM Ca. 2+ , Mg 2+ , Mn 2+ , and Cu 2+ The treatment may be performed in the presence of a divalent cation selected from the group consisting of:

[0102] Alternatively, the filtered harvest may be purified by a non-specific endonuclease, most preferably benzonase, at a concentration of 1-3 mM, using a divalent cation, Mg 2+ The treatment may be carried out in the presence of a salt.

[0103] According to a twelfth aspect of the eleventh embodiment, the benzonase treated harvest may be further subjected to tangential flow filtration (TFF), typically through filters with molecular weight cut-off (MWCO) ranging between 100 KDa and 500 KDa, resulting in a 2x to 10x concentration of the viral harvest and further resulting in the removal of remaining impurities.

[0104] Further preferred remaining impurities may include residual DNA, residual bovine serum albumin (BSA), and residual host cell proteins. According to a thirteenth aspect of the eleventh embodiment, the above process can produce a purified and concentrated LAIV virus harvest containing residual cellular DNA (<10 ng / dose), residual BSA (<50 ng / dose), and traces of residual cellular protein. Furthermore, the above process can result in an overall recovery of purified virus of at least 40%.

[0105] According to a fourteenth aspect of the eleventh embodiment, the concentrated monovalent virus stock (TFF concentrated) is stabilized by a stabilizer composition to obtain a final LAIV composition comprising one or more carbohydrates, one or more amino acids, and gelatin.

[0106] More preferably, the concentrated virus stock (TFF concentrated) may be stabilized by a stabilizer composition comprising sucrose, histidine, alanine, tricine, arginine, and gelatin in any combination thereof.

[0107] More preferably, the concentrated virus stock (TFF concentrated) may be stabilized by a stabilizer composition comprising sucrose at a concentration of 1-10% (w / v), histidine at a concentration of 0.1%-2% (w / v), alanine at a concentration of 0.01%-1% (w / v), tricine at a concentration of 0.1%-1% (w / v), arginine at a concentration of 0.1-5% (w / v), and gelatin at a concentration of 0.1-5% (w / v).

[0108] More preferably, the concentrated virus stock (TFF concentrated) may be stabilized by a stabilizer composition comprising sucrose at a concentration of 3-6% (w / v), histidine at a concentration of 0.1%-1% (w / v), alanine at a concentration of 0.05%-0.5% (w / v), tricine at a concentration of 0.1%-0.5% (w / v), arginine at a concentration of 0.1-3% (w / v), and gelatin at a concentration of 0.1-3% (w / v).

[0109] More preferably, the concentrated virus stock (TFF concentrated) may be stabilized by a stabilizer composition comprising sucrose 4% (w / v), histidine 0.21% (w / v), alanine 0.1% (w / v), tricine 0.3% (w / v), arginine 2.1% (w / v), and gelatin 0.85% (w / v).

[0110] According to the fifteenth aspect of the eleventh embodiment, the stabilized virus harvest can be sterilized by direct flow filtration (DFF) through at least one sterilizing grade filter, preferably 0.2μ. Suitable commercially available filters and filtration devices are well known in the art and can be selected by the skilled artisan. Exemplary filtration devices are made of polypropylene or cellulose acetate or polyethersulfone or polyvinylidene difluoride, and commercially available filters can be Millipak (Millipore), Kleenpak (Pall) and Sartobran™ P (Sartorius) filtration devices.

[0111] According to a sixteenth aspect of the eleventh embodiment, the LAIV composition may be multivalent, comprising more than one LAIV virus strain or subtype, as disclosed in the previous embodiments. The LAIV composition may be bivalent or trivalent or quadrivalent.

[0112] Further alternatively, the LAIV composition can be monovalent, comprising any one LAIV virus strain or subtype, as disclosed in the preceding embodiments. According to a seventeenth aspect of the eleventh embodiment, the LAIV composition has a concentration of 6 to 7 LogEID 50 The vaccine may comprise a dose of influenza virus.

[0113] More preferably, the LAIV composition has a concentration of 6 to 7 LogEID per 0.5 ml. 50 more preferably NLT7 LogEID per 0.5 ml 50 The vaccine may comprise a dose of influenza A virus or any subtype.

[0114] More preferably, the LAIV composition has a concentration of 6 to 7 LogEID per 0.5 ml. 50 more preferably NLT6.5 LogEID per 0.5 ml 50 The vaccine may comprise a dose of influenza B virus or any subtype.

[0115] According to a twelfth embodiment, the method for preparing an immunogenic composition comprises the following steps: a) infecting an MDCK cell culture host with influenza virus at an MOI of between 1:100 and 1:10,000; b) harvesting the influenza virus-containing supernatant after an incubation period of 40-70 hours in MEM containing trypsin in the range of 5-25 U / ml; c) filtering the viral harvest by direct flow filtration (DFF) through at least one clarification filter having a pore size between about 6 micrometers and about 0.45 micrometers; d) treating the CVP with a non-specific endonuclease at a temperature ranging between 30-34° C. for 2-6 hours, followed by a temperature ranging between 2-8° C. for 5-15 hours; e) concentrating 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; f) stabilizing 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; g) sterilizing the stabilized TFF concentrate by DFF through at least one sterilizing grade filter having a pore size between about 0.8 micrometers and about 0.2 micrometers to form a sterile CMVP; Including, The overall recovery of the virus purified here is greater than 40%.

[0116] According to a first aspect of the twelfth embodiment, step (d) comprises treating the virus harvest with a non-specific endonuclease, more specifically benzonase having a concentration ranging from 0.5 units / ml to 5 units / ml, in an amount between 0.1 mM and 100 mM, Ca 2+ , Mg 2+ , Mn 2+ , and Cu 2+ A method of producing an immunogenic composition, which may include a step of treating in the presence of a divalent cation selected from the group consisting of:

[0117] According to a second aspect of the twelfth embodiment, step (d) comprises treating the virus harvest with a non-specific endonuclease, more specifically benzonase, having a concentration in the range of 0.5 units / ml to 5 units / ml, at a concentration of 1 to 3 mM, and divalent cation Mg 2+ A method of producing an immunogenic composition, which may include a step of treating in the presence of a salt.

[0118] According to a third aspect of the twelfth embodiment, the method of producing an immunogenic composition, wherein step (e) may comprise concentrating the virus harvest by tangential flow filtration (TFF) to result in a virus harvest of at least 4x concentration.

[0119] According to a fourth aspect of the twelfth embodiment, the method of producing an immunogenic composition, wherein step (f) may comprise stabilising the virus harvest with a stabiliser composition comprising sucrose at a concentration of 1-10% (w / v), histidine at a concentration of 0.1%-2% (w / v), alanine at a concentration of 0.01%-1% (w / v), tricine at a concentration of 0.1%-2% (w / v), arginine at a concentration of 0.1-5% (w / v) and gelatin at a concentration of 0.1-5% (w / v).

[0120] According to a fifth aspect of the twelfth embodiment, the method of producing an immunogenic composition, wherein step (f) may comprise stabilising the virus harvest with a stabiliser composition comprising sucrose at a concentration of 3-6% (w / v), histidine at a concentration of 0.1%-1% (w / v), alanine at a concentration of 0.05%-0.5% (w / v), tricine at a concentration of 0.1%-0.5% (w / v), arginine at a concentration of 0.1-3% (w / v), and gelatin at a concentration of 0.1-3% (w / v).

[0121] According to a sixth aspect of the twelfth embodiment, the method of producing an immunogenic composition, wherein step (f) may comprise stabilizing the virus harvest with a stabilizer composition comprising sucrose 4% (w / v), histidine 0.21% (w / v), alanine 0.1% (w / v), tricine 0.3% (w / v), arginine 2.1% (w / v), and gelatin 0.85% (w / v).

[0122] According to a sixth aspect of the twelfth embodiment, the method of producing an immunogenic composition, wherein step (f) may comprise stabilizing the virus harvest with a stabilizer composition comprising sucrose 4% (w / v), histidine 0.21% (w / v), alanine 0.1% (w / v), tricine 0.3% (w / v), arginine 2.1% (w / v), and gelatin 1.0% (w / v).

[0123] According to a seventh aspect of the twelfth embodiment, the method of producing an immunogenic composition, wherein step (f) may comprise stabilizing the virus harvest with a stabilizer composition comprising sucrose 4% (w / v), histidine 0.21% (w / v), alanine 0.1% (w / v), tricine 0.3% (w / v), arginine 1.6% (w / v), and gelatin 1.0% (w / v).

[0124] According to a thirteenth embodiment of the present disclosure, the immunogenic composition comprises: a) an immunogenicity of 6 to 7 LogEID 50a) one or more live attenuated influenza vaccine (LAIV) viruses at a dose of 0.1% to 10% (w / v); b) sucrose 1-10% (w / v); c) histidine 0.1% to 2% (w / v); d) alanine 0.01% to 1% (w / v); e) tricine 0.1% to 2% (w / v); f) arginine 0.1-5% (w / v); g) gelatin 0.1-5% (w / v).

[0125] More preferably, the immunogenic composition has a LogEID concentration of 6 to 7 LogEID per 0.5 ml. 50 dose of one or more live attenuated influenza vaccine (LAIV) viruses; b) sucrose 3-6% (w / v); c) histidine 0.1%-1% (w / v); d) alanine 0.05%-0.5% (w / v); e) tricine 0.1%-0.5% (w / v); f) arginine 0.1-3% (w / v); g) gelatin 0.1-3% (w / v).

[0126] More preferably, the immunogenic composition has a LogEID concentration of 6 to 7 LogEID per 0.5 ml. 50 a) one or more live attenuated influenza vaccine (LAIV) viruses at a dose of 0.21% (w / v); b) sucrose 4% (w / v); c) histidine 0.21% (w / v); d) alanine 0.1% (w / v); e) tricine 0.3% (w / v); f) arginine 2.1% (w / v); g) gelatin 0.85% (w / v).

[0127] More preferably, the immunogenic composition has a LogEID concentration of 6 to 7 LogEID per 0.5 ml. 50 a) sucrose 4% (w / v); c) histidine 0.21% (w / v); d) alanine 0.1% (w / v); e) tricine 0.3% (w / v); f) arginine 2.1% (w / v); g) gelatin 1% (w / v).

[0128] More preferably, the immunogenic composition has a LogEID concentration of 6 to 7 LogEID per 0.5 ml. 50a) one or more live attenuated influenza vaccine (LAIV) viruses at a dose of 0.21% (w / v); b) sucrose 4% (w / v); c) histidine 0.21% (w / v); d) alanine 0.1% (w / v); e) tricine 0.3% (w / v); f) arginine 1.6% (w / v); g) gelatin 1% (w / v).

[0129] According to a fourteenth embodiment of the present disclosure, the LAIV composition may be entirely liquid. Further alternatively, the LAIV composition can be a lyophilized or freeze-dried composition. As used herein, the term "freeze-drying" or "lyophilize" or "lyophilization" includes lyophilization and refers to a process whereby a suspension is frozen and then the 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, whereby the composition changes directly from a solid state to a gaseous state without becoming a liquid.

[0130] According to a fifteenth embodiment of the present disclosure, the LAIV composition may be formulated for use in a method of reducing the incidence of a health condition or preventing a health condition, comprising administration of an effective amount of the LAIV composition to a human subject intranasally or via other routes of immunization.

[0131] According to a preferred aspect of the embodiment, the LAIV composition can be administered to a human subject via the intranasal route. In one embodiment, the device is an intranasal dispensing device, such as a device in the form of an aerosol (intranasal spray) or a droplet delivery system. Liquid nasal formulations can be delivered via nasal drops, drops and nasal catheters, compressed air inhalers, squeeze bottles, metered pump sprays like multiple dose metered spray pumps or single / double dose spray pumps, spray devices attached to syringes. Other dosage forms can be selected from nasal powders (syringes, dry powder inhalers), nasal gels, nasal drops, solutions, suspensions, cosolvent systems, microparticles, nanoparticles, microemulsions, nasal inserts.

[0132] Intranasal delivery devices include, but are not limited to, Becton Dickinson (BD) Accuspray™ delivery device, Bi-Directional™ Optinose nasal device, MAD Intranasal Mucosal Atomization device by Teleflex, AeroLife™ and AeroVax™ (AerovectRx, Inc., Atlanta, GA), Jet injector-PharmaJet®, Stratis® Needle-Free Injector, MUNJIs Multi-use-nozzle jet injectors: Aquapuncture device, Hypospray®, MadaJet®, GentleJet®, Disposable-syringe Jet Injectors: Medi-Jector®, J-Tip®, Injex®, Vitajet™, LectraJet The spray nozzle may be selected from the following devices: Axial HS, LectraJet® M3, ZetaJet™, PharmaJet®, Aktiv-Dry PuffHaler™ and Nasal spray flu shot devices.

[0133] According to a sixteenth embodiment of the present disclosure, the LAIV composition may be formulated for use in a method of reducing the onset of or preventing a condition involving an influenza A virus infection or subtype thereof as disclosed in the previous embodiments of the present disclosure, an influenza B virus infection or subtype thereof as disclosed in the previous embodiments of the present disclosure, or an influenza C virus infection or subtype thereof as disclosed in the previous embodiments of the present disclosure.

[0134] According to the seventeenth embodiment of the present disclosure, the LAIV composition may be administered intranasally in a protectively effective dose. The vaccine is administered in a manner consistent with the dosage formulation and in an amount that will be prophylactically effective. The immunogenic composition of the present disclosure may 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 may be used in adults or children at risk of influenza virus infection.

[0135] More preferably, the LAIV composition may be administered intranasally in a dosage volume of about 0.1 to 0.5 ml. According to an eighteenth embodiment of the present disclosure, the LAIV composition is formulated as a single or multi-dose vial or kit, or as a pre-filled syringe or nasal spray, and said LAIV composition may be given in a single dose schedule, or preferably in a multi-dose schedule, where the main course of vaccination is followed by a subsequent dose at a time interval required to maintain and / or bolster the immune response, e.g., 1-4 months for a second dose, followed by a subsequent dose several months or years later, if necessary, or one or two separate doses given in an annual vaccination. The dosing regimen will also be determined, at least in part, by the need for booster doses required to confer protective immunity.

[0136] According to a nineteenth embodiment of the present disclosure, the final pH of the immunogenic composition may be comprised between 6.5 and 8. Other embodiments disclosed herein also include vaccine kits comprising a first container containing a lyophilized (freeze-dried) immunogenic composition and a second container containing an aqueous solution, optionally saline or WFI (water for injection), for reconstitution of the lyophilized (freeze-dried) LAIV composition.

[0137] Throughout this specification, the word "comprise" or variations such as "comprises" or "comprising" are understood to mean the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps, and can mean "includes", "including", and the term is open-ended and allows for the presence of more than what is recited, unless the basic or novel characteristics of what is recited are altered by the presence of more than what is recited, but excluding prior art embodiments.

[0138] Throughout this specification, the word "immunogenic composition" covers any composition that elicits an immune response against an antigen or immunogen of interest expressed from a vector; e.g., after administration to a subject, elicits an immune response against a targeted immunogen or antigen of interest. The terms "vaccine composition" and "vaccine" cover any composition that induces a protective immune response against an antigen of interest or that effectively protects against an antigen; e.g., after administration or injection to a subject, elicits a protective immune response against a targeted antigen or immunogen or provides effective protection against an antigen or immunogen expressed from a vector.

[0139] The use of the phrase "one or more" or "at least one" indicates the use of one or more elements or ingredients or amounts that may be used in an embodiment of the present invention to achieve one or more desired objectives or results. Although specific embodiments of the present invention are described, these embodiments are presented by way of example only and are not intended to limit the scope of the present invention. Modifications or alterations to the compositions of this invention may occur to those skilled in the art upon reference to the disclosure herein, within the scope of the present invention. Such modifications or alterations are well within the spirit of this disclosure.

[0140] The numerical values ​​given for various physical parameters, dimensions, and quantities are approximate only, and values ​​higher than the numerical values ​​assigned to physical parameters, dimensions, and quantities are considered to be within the scope of the invention, unless otherwise stated in the specification.

[0141] Similarly, components used in purification, e.g., filters, columns, are not intended to be limiting or exclusive in any way, and other components may be substituted to achieve the same purpose at the physician's discretion.

[0142] Although emphasis is placed herein on certain features of the preferred embodiments, it will be understood that many additional features may be added and many modifications may be made to the preferred embodiments without departing from the principles of the present disclosure. These and other modifications of the preferred embodiments of the present disclosure will be apparent to those skilled in the art from the disclosure herein, and it is therefore clearly understood that the foregoing description is to be interpreted merely as illustrative of the present disclosure, and not as limiting.

[0143] Technical advantages 1. Currently, almost all influenza vaccines produced use eggs as a host for the preparation of the virus pool. The use of eggs for the production of LAIV has certain drawbacks, which can be overcome by using cell culture as a substrate. The limitations of using eggs as a substrate are: · Limited supplies of vaccine quality eggs and specific pathogen-free eggs.

[0144] A minimum of four months advance order is required before eggs are available. Some of the candidate pandemic strains cause fatal infections in poultry, making the substrate (eggs) for vaccine production unavailable. Egg-based manufacturing requires specialized factories for egg incubation, harvesting, etc., which in turn limits the ability to scale up rapidly.

[0145] 2. Tissue culture-based production has the advantage of being a fully controlled system that is easy to scale up. 3. In the event of a pandemic, large-scale production of the vaccine could be easily accomplished using existing tissue culture manufacturing units for other viral vaccines.

[0146] 4. Cell culture derived viruses have a high degree of similarity to circulating strains compared to egg produced viruses which may have antigenic modifications. 5. A minimum number of components are included in the vaccine composition.

[0147] 6. Free of preservatives, polymers, and surfactants. 7. The purification process utilizes low concentrations of an endonuclease (benzonase). 8. The purification process is free of costly and cumbersome chromatography steps.

[0148] 9. Improved methods for producing stable compositions / formulations resulting in improved yields. 10. Intranasal delivery is the simplest route of immunization as it does not require a high level of expertise and is easy to administer multiple doses.

[0149] 11. No association with Guillain-Barre syndrome has been reported and delivery at the site of infection provides more protection. 12. A difficult to achieve liquid presentation of the vaccine helps overcome the problems of limited freeze-drying capacity, the need for a supply of diluent for reconstitution, and the additional step of reconstitution required prior to delivery of the vaccine.

[0150] 13 The MDV backbone used to generate the LAIV reassortants has a well-established safety profile and has been reported to confer a high level of protection. EXAMPLES

[0151] The following examples are included to demonstrate preferred embodiments of the present invention. It should be understood by those skilled in the art that the compositions and techniques disclosed in the examples, which function well in carrying out the present invention according to representative techniques discovered by the inventors, can be considered to constitute preferred modes for carrying out the invention. However, those skilled in the art should understand in light of this disclosure that many modifications can be made to the specific embodiments disclosed and still obtain similar or similar results without departing from the spirit and scope of the present invention.

[0152] Reassortant LAIV strains are obtained from WHO collaborating centers such as the Institute of Experimental Medicine (IEM) in St. Petersburg, Russia, or the Centers for Disease Control and Prevention (CDC) in Atlanta.

[0153] Example 1: Stability Data of Reassortant LAIV Viral Immunogenic Compositions Component 1 - Live Attenuated Influenza Vaccine Virus (LAIV) Influenza vaccine viruses are reassortant LAIV viruses derived by the classical method of reassortment, containing cold-adapted, temperature-sensitive and / or attenuated phenotype gene segments of Master Donor Virus (MDV) and hemagglutinin (HA) and / or neuraminidase (NA) gene segments of wild-type epidemic or seasonal influenza A, B or C virus strains in a ratio of 6:2 or 7:1.

[0154] [Table 1] LAIV immunogenic composition: 6~7logEID 50 / 0.5ml dose range for type A viruses; more preferably, 7 log EID 50 / 0.5ml dose 6~7logEID 50 / 0.5ml dose range for type B virus; more preferably, 6.5 logEID 50 / 0.5ml dose The LAIV compositions are either monovalent or multivalent (bivalent; trivalent; quadrivalent) with respect to the reassortant LAIV virus strains used in the immunological composition as disclosed in Table 1 in any combination thereof.

[0155] [Table 2]

[0156] [Table 3]

[0157] [Table 4]

[0158] [Table 5] interpretation: 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 Tricine + 1.6% w / v L-Arginine Hydrochloride): Unacceptably rapid degradation was observed for influenza A / H1N1 and B vaccine strains for both stress stability at 37°C and real-time stability at temperatures between 2 and 8°C (see Figures 4 and 5).

[0159] 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 Tricine + 1.6% w / v L-Arginine Hydrochloride): Unacceptably rapid degradation was observed for influenza A / H1N1 and B vaccine strains for both stress stability at 37° C. and real-time stability at temperatures between 2 and 8° C. (see FIGS. 6 and 7).

[0160] 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 tricine + 2.1% w / v L-arginine hydrochloride): Unacceptably rapid degradation was observed for both stress stability at 37° C. and real-time stability at temperatures between 2 and 8° C. (see FIGS. 8 and 9).

[0161] 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 tricine + 2.1% w / v L-arginine hydrochloride): Acceptable rates of degradation (values ​​within acceptable ranges) were observed for both stress stability at 37°C and real-time stability at temperatures between 2 and 8°C (see Figures 2, 3, 10 and 11).

[0162] Example 2: MDCK cell-based LAIV virus production process The process of preparing an MDCK cell culture-based LAIV composition may include any subset or all of the following steps: k) LAIV candidate vaccine viruses are first passaged in embryonated SPF chicken eggs to produce egg-based master seed virus (MSV).

[0163] l) Adapt the egg-based master seed virus to grow in an MDCK cell culture (ATCC CCL-34) host to prepare a cell-based working seed virus (WSV). Use this cell-based WSV to grow in a 175 cm2 surface area MDCK cell culture (ATCC CCL-34) host. 2 Tissue culture flask (TCS), surface area 850 cm 2 Roller bottle (RB), surface area 6320 cm 2MDCK cell cultures are infected at an MOI between 1:100 and 1:10000 in different cell culture vessels / systems, such as Cell Factories (CF) from Pall Life Sciences, Port Washington, NY, and fixed-bed bioreactors (e.g., iCELLis® bioreactors, e.g., Nano and 500 / 100 bioreactors from Pall® Life Sciences, Port Washington, NY). (MDCK cells were grown using MEM containing FBS; washed with MEM containing 5-25 U / ml trypsin prior to inoculation; WSV was inoculated into the cells at an MOI between 1:10 and 1:10000 and incubated for 48-72 hours at 31-33° C.) m) Harvest the cultured virus.

[0164] n) Filtering the virus harvest by direct flow filtration (DFF) through at least one clarification filter to obtain a clarified virus pool (CVP). o) treating the CVP with a non-specific endonuclease (e.g., benzonase) at a temperature ranging between 30-34° C. for 2-6 hours, followed by a temperature ranging between 2-8° C. for 5-15 hours; p) concentrating 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; q) stabilizing 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; r) Sterilizing the stabilized TFF concentrate by DFF through at least one sterilizing grade filter having a pore size of about 0.2 micrometers to obtain a sterile CMVP (clarified monovalent virus pool).

[0165] s) Store the sterilized CMVP in a polycarbonate bottle at or below -60°C. t) Fill the sterilized formulation into vials and store at 2-8°C.

[0166] [Table 6]

[0167] [Table 7] Cell culture medium: MEM containing 10% FBS (pH adjusted to 7.0-7.4 with 1N HCl) (additional glutamine 350mg / L) Viral medium: MEM without FBS (pH adjusted to 7.2-7.6 with 1N HCl) (additional glucose - 500 mg / L, glutamine - 350 mg / L) An additional 0.4% glucose is added to the CM and VM used for the bioreactor system.

[0168] [Table 8]

[0169] [Table 9]

[0170] [Table 10]

[0171] [Table 11]

[0172] [Table 12] Example 3: Effect of virus input (MOI) and post-inoculation incubation period on yield

[0173] [Table 13] inference: A. Time of infection: Based on optimization studies, specific cell number limits of 120-180 million cells per roller bottle and 7-10 billion cells in the bioreactor system were selected for infection with influenza working seed virus from MDCK cells. Microscopic observations of roller bottles containing MDCK cells were performed at monolayer density prior to the infection procedure.

[0174] B. MOI and incubation period after inoculation: Based on all the observations from the MOI optimization studies, the selected MOI ranges for influenza A(H1N1), A(H3N2), and B viruses were between 1:100 and 1:10,000, and the post-inoculation incubation periods ranged from 48 to 72 hours.

[0175] Example 4: Effect of different concentrations of trypsin on yield

[0176] [Table 14] inference: Trypsin is required for activation of influenza virus for inoculation of MDCK cells. From the results above, it is observed that 2000-3000 units of trypsin per roller bottle produces virus of maximum potency.

[0177] Example 5: Effect of Benzonase concentration and temperature on cellular DNA content and viral titer Different concentrations of benzonase were tested to degrade host cell DNA at different temperatures.

[0178] The clarified virus pool (CVP) was subjected to benzonase treatment at concentrations of 500 (containing 2 mM MgCl2), 500, 1000, 2500 and 5000 U / L, and the treated CVP was kept at 32°C for 3 hours and further continued overnight at 2-8°C. Sampling was done at each stage, and the results of DNA content at each stage are as follows:

[0179] [Table 15]

[0180] [Table 16] inference: From the results, it was observed that CVP treated with 500 U / L benzonase in the presence of 2 mM MgCl2 showed higher DNA degradation than CVP treated with 500 U / L benzonase without 2 mM MgCl2. It can also be seen that higher benzonase concentrations of 1000 U / L, 2500 U / L, and 5000 U / L showed comparable DNA degradation to 500 U / L benzonase (containing 2 mM MgCl2).

[0181] Example 6: Viral yield at different stages of production

[0182] [Table 17] 1. CVP: Clarified virus pool (harvest after filtration); 2. BCVP: CVP treated with benzonase; 3. CMVP: Clarified monovalent virus pool (TFF, after addition of stabilizers, and after 0.2μ filtration) inference: The viral concentrations at each stage were checked for each A(H1N1), A(H3N2), and B seasonal influenza virus and it was observed that the initial viral concentrations at harvest level were maintained throughout the process up to the last stage, i.e., preparation of vaccine bulk (CMVP).

[0183] [Table 18] inference: Viral recovery can be calculated as the percentage of virus retention during production, with harvest being the starting point of production and CMVP being the end point of production. The results showed that the average virus recovery was 44.67%, which is 0.34 LogEID 50 It can be concluded that this is equivalent to a titer reduction of 0.5 ml / mL. It is also observed that the final virus recovery (virus titer) at CMVP levels is within acceptable limits, indicating that CMVP can be used to manufacture final product batches of MDCK-based LAIV viruses.

[0184] Example 7: Comparative data of host cell DNA concentration at each stage during the production of CMVP Clarified virus pools (CVP) of different strains were subjected to benzonase treatment and sampled at each stage for DNA content. The results of DNA content at each stage are shown below.

[0185] [Table 19] inference: From the results, it was observed that CVP showed a significant reduction in DNA when treated with benzonase. Furthermore, it was observed that the residual DNA was effectively removed again during the TFF process (diafiltration / concentration) and the CMVP preparation process. The DNA content in the final CMVP level is within the desired and acceptable limits.

[0186] Example 8: Various tests of TFF experiments Various TFF experiments were performed considering parameters such as the dilution media (virus medium and PBS) used in the TFF process, diafiltration, and concentration procedures. TFF concentrated samples were tested for virus concentration.

[0187] [Table 20] interpretation: From the above set of experiments, a TFF process was developed and followed by 2x DF and 4x concentration stages was selected to obtain the desired TFF concentration with optimal virus yield. Compared to VM, PBS provides more stability to the virus.

[0188] Example 9: Immunogenicity Results A study was conducted to evaluate the performance of egg- and MDCK cell-based trivalent and quadrivalent seasonal influenza vaccines for vaccine immune responses and efficacy in a ferret model. All animals were immunized intranasally on day 0 and challenged 4 weeks later (day 28) with egg- and MDCK cell culture-based trivalent or quadrivalent preparations containing strains similar to A / Michigan / 45 / 2015 (H1N1), A / Hong Kong / 4801 / 2015 (H3N2), B / Brisbane / 60 / 2008, and B / Phuket / 3073 / 2013.

[0189] [Table 21] Inference (see Figures 12 and 13) It was concluded that vaccination with egg- and MDCK-based trivalent and quadrivalent vaccines containing A-H1N1 could protect animals against A-H1N1 infection when challenged with the homologous A-H1N1 virus.

[0190] Another study was conducted to evaluate the performance of both egg- and MDCK cell-based monovalent LAIVs (A-H5N2 and A-H7N9) for immune response and efficacy in a ferret model. Different groups of animals were immunized with egg- and MDCK cell culture-based A-H5N1 and A-H7N9 monovalent LAIVs and challenged with homologous A-H5N1 and A-H7N9 viruses, respectively. The results showed that animals immunized with monovalent H5N2 LAIVs were protected against homologous challenge with A-H5N1 viruses. Animals immunized with monovalent A-H7N9 LAIVs were protected against homologous challenge with A-H7N9 viruses.

Claims

1. a) 6-7 LogEID per 0.5 ml ５０ and b) a stabilizer composition comprising: i. sucrose 1-10% (w / v); ii. Histidine 0.1% to 2% (w / v); iii. Alanine 0.01% to 1% (w / v); iv. Tricine 0.1% to 2% (w / v); v. arginine 0.1-5% (w / v); and vi. Gelatin 0.1-5% (w / v); 20. An immunogenic composition comprising:

2. 2. The immunogenic composition of claim 1, wherein the influenza virus comprises a pandemic or seasonal influenza virus, a live attenuated influenza vaccine (LAIV) virus, an inactivated influenza virus, a chimeric influenza virus, or a recombinant influenza virus.

3. 2. The immunogenic composition of claim 1, wherein the composition is monovalent with respect to influenza virus from influenza types A or B or C or subtypes thereof.

4. 2. The immunogenic composition of claim 1, wherein the composition is multivalent with respect to influenza viruses from influenza types A or B or C or subtypes thereof.

5. The immunogenic composition of claim 1, wherein the influenza virus is a reassortant LAIV virus comprising cold-adapted, temperature-sensitive and / or attenuated phenotype gene segments of Master Donor Virus (MDV) and hemagglutinin (HA) and / or neuraminidase (NA) gene segments of a wild-type epidemic or seasonal influenza A or B or C virus strain in a ratio of 1:7, 2:6, 3:5, 4:4, 5:3, 6:2 or 7:

1.

6. 6. The immunogenic composition of claim 5, wherein the Master Donor Virus (MDV) is selected from the group comprising: A / Leningrad / 134 / 17 / 57 (H2N2) influenza A strain, B / USSR / 60 / 69 influenza B strain.

7. 6. The immunogenic composition of claim 5, wherein the reassortant LAIV virus comprises a hemagglutinin (HA) gene and / or a neuraminidase (NA) gene from an influenza A virus, or H1-H18 and N1-N11, and its subtypes H1N1, H2N2, H3N2, H5N1, H5N3, H9N2, H7N1, H7N3, H7N7, H6N1, H7N9, and H10N8.

8. 6. The immunogenic composition of claim 5, wherein the reassortant LAIV virus comprises the hemagglutinin (HA) gene and / or the neuraminidase (NA) gene from an influenza A virus strain pdmH1N1-A / California / 07 / 2009 (designated A / Cal)-like strain, or an A / Cal, an A / Michigan / 45 / 2015-like strain, an A / South-Africa / 3626 / 2013-like strain, or an H3N2-A / Hong Kong / 4801 / 2014-like strain.

9. The immunogenic composition of claim 5, wherein the reassortant LAIV virus comprises a hemagglutinin (HA) gene and / or a neuraminidase (NA) gene from an influenza B virus strain, Victoria lineage-B / Brisbane / 60 / 2008-like strain or Yamagata lineage-B / Phuket / 3073 / 2013-like strain.

10. 2. The immunogenic composition of claim 1, wherein the composition further comprises an adjuvant selected from the group of aluminum hydroxide, aluminum phosphate, aluminum hydroxide phosphate, and aluminum potassium sulfate, or a mixture thereof.

11. 2. The immunogenic composition of claim 1, wherein the composition further comprises an immunostimulatory component selected from the group of: oil and water emulsion, MF-59, liposome, lipopolysaccharide, saponin, lipid A, lipid A derivative, monophosphoryl lipid A, 3-desacylated monophosphoryl lipid A, AS01, AS03, oligonucleotide, oligonucleotide comprising at least one unmethylated CpG and / or liposome, Freund's adjuvant, Freund's complete adjuvant, Freund's incomplete adjuvant, CRL-8300 adjuvant, muramyl dipeptide, TLR-4 agonist, flagellin, flagellin from gram-negative bacteria, TLR-5 agonist, fragment of flagellin capable of binding to the TLR-5 receptor, QS-21, ISCOMS, chitosan, saponin, saponin in combination with a sterol and lipid.

12. 2. The immunogenic composition of claim 1, wherein the single dose composition is preservative-free and the multi-dose composition comprises one or more preservatives selected from the group of 2-phenoxyethanol, benzethonium chloride (femerol), phenol, thiomersal, formaldehyde, methylparaben, propylparaben, benzyl alcohol, or combinations thereof.

13. 2. The immunogenic composition of claim 1, wherein the composition comprises a buffer selected from sodium chloride, carbonic acid, citric acid, lactic acid, gluconic acid, tartaric acid, phosphate buffered saline, HEPES, citrate-phosphate, or TRIS.

14. The immunogenic composition of claim 1, wherein the composition comprises a pharma- ceutically acceptable transporter, excipient, binder, carrier, tonicity agent, emulsifier, or wetting agent.

15. The composition comprises sugar, polyol, NaCl, KCl, KH ２ P.O. ４ , Na ２ H.P.O. ４ . 2H ２ O, CaCl ２ , or MgCl ２ 15. The immunogenic composition of claim 14, comprising a pharma- ceutically acceptable excipient selected from the group consisting of salts comprising:

16. 2. The immunogenic composition of claim 1, wherein the composition is formulated as a single or multi-dose vial or multi-dose kit, or as a pre-filled syringe or nasal spray, for use in a method of reducing the incidence of or preventing a health condition involving influenza A virus infection or subtypes thereof, influenza B virus infection or subtypes thereof, or influenza C virus infection or subtypes thereof.

17. The immunogenic composition of claim 1, wherein the final pH of the immunogenic composition is comprised between pH 6.5 and 8.

18. 2. The immunogenic composition of claim 1, wherein the influenza virus is Madin-Darby canine kidney (MDCK) (NBL2) (ATCC CCL34) grown in MDCK cells.

19. The influenza B virus has a concentration of 6 to 7 Log EID ５０ ; more preferably NLT 6.5 Log EID per 0.5 ml ５０ The immunogenic composition of claim 1, wherein the immunogenic composition is present in a dose of

20. The influenza A virus has a concentration of 6 to 7 Log EID ５０ ; more preferably NLT 7 Log EID per 0.5 ml ５０ The immunogenic composition of claim 1, wherein the immunogenic composition is present in a dose of

21. a) infecting an MDCK cell culture host with influenza virus at an MOI of between 1:100 and 1:10,000, wherein the MDCK cells are MDCK(NBL2) (ATCC CCL34); b) harvesting the influenza virus-containing supernatant after an incubation period of 40-70 hours in MEM containing trypsin in the range of 5-25 U / ml; c) filtering the viral harvest by direct flow filtration (DFF) through at least one clarification filter having a pore size between about 6 micrometers and about 0.45 micrometers; d) treating the CVP with a non-specific endonuclease at a temperature ranging between 30-34° C. for 2-6 hours, followed by 5-15 hours at a temperature of 2-8° C.; e) concentrating 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; f) stabilizing the TFF concentrate with the stabilizer composition of claim 1 to form a stabilized virus harvest; g) sterilizing the stabilized TFF concentrate by DFF through at least one sterilizing grade filter having a pore size between about 0.8 micrometers and about 0.2 micrometers to form a sterile CMVP. Including, The overall recovery of purified virus is 40% or more. A method for preparing an immunogenic composition according to any one of claims 1 to 20.

22. Step (d) is to subject the viral harvest to a non-specific endonuclease, more specifically, benzonase having a concentration ranging from 0.5 units / ml to 5 units / ml, in an amount between 0.1 mM and 100 mM Ca ２＋ , Mg ２＋ , Mn ２＋ , and Cu ２＋ 22. A method for producing the immunogenic composition of claim 21, comprising treating in the presence of a divalent cation selected from the group consisting of:

23. Step (d) is to subject the viral harvest to a non-specific endonuclease, more specifically, benzonase having a concentration ranging from 0.5 units / ml to 5 units / ml, in the presence of a divalent cation Mg at a concentration of 1 mM to 3 mM. ２＋ 22. A method of producing the immunogenic composition of claim 21, comprising treating in the presence of a salt.

24. 22. The method of producing the immunogenic composition of claim 21, wherein step (e) comprises concentrating the viral harvest by tangential flow filtration (TFF) to result in a viral harvest that is at least 4x concentrated.

25. 22. The method of producing an immunogenic composition according to claim 21, wherein step (f) comprises stabilising the virus harvest with a stabilizer composition comprising sucrose at a concentration of 1-10% (w / v), histidine at a concentration of 0.1%-2% (w / v), alanine at a concentration of 0.01%-1% (w / v), tricine at a concentration of 0.1%-2% (w / v), arginine at a concentration of 0.1-5% (w / v) and gelatin at a concentration of 0.1-5% (w / v).

26. 22. The method of producing an immunogenic composition of claim 21, wherein step (f) comprises stabilizing the virus harvest with a stabilizer composition comprising sucrose at a concentration of 3-6% (w / v), histidine at a concentration of 0.1%-1% (w / v), alanine at a concentration of 0.05%-0.5% (w / v), tricine at a concentration of 0.1%-0.5% (w / v), arginine at a concentration of 0.1-3% (w / v), and gelatin at a concentration of 0.1-3% (w / v).

27. 22. The method of producing the immunogenic composition of claim 21, wherein step (f) comprises stabilizing the virus harvest with a stabilizer composition comprising sucrose 4% (w / v), histidine 0.21% (w / v), alanine 0.1% (w / v), tricine 0.3% (w / v), arginine 2.1% (w / v), and gelatin 0.85% (w / v).

28. 22. The method of producing the immunogenic composition of claim 21, wherein step (f) comprises stabilizing the virus harvest with a stabilizer composition comprising sucrose 4% (w / v), histidine 0.21% (w / v), alanine 0.1% (w / v), tricine 0.3% (w / v), arginine 2.1% (w / v), and gelatin 1.0% (w / v).

29. 22. The method of producing the immunogenic composition of claim 21, wherein step (f) comprises stabilizing the virus harvest with a stabilizer composition comprising sucrose 4% (w / v), histidine 0.21% (w / v), alanine 0.1% (w / v), tricine 0.3% (w / v), arginine 1.6% (w / v), and gelatin 1.0% (w / v).

30. The immunogenic composition of claim 1, wherein the method of administration of the immunogenic composition to a human subject includes intranasal, intramuscular, intravenous, subcutaneous, transdermal, or intradermal routes.

31. The composition comprising: a) 6-7 LogEID per 0.5 ml ５０ a dose of one or more live attenuated influenza vaccine (LAIV) viruses; b) sucrose 3-6% (w / v); c) histidine 0.1% to 1% (w / v); d) alanine 0.05% to 0.5% (w / v); e) Tricine 0.1% to 0.5% (w / v); f) arginine 0.1-3% (w / v); and g) Gelatin 0.1-3% (w / v) The immunogenic composition of claim 1 , comprising:

32. The composition comprising: a) 6-7 LogEID per 0.5 ml ５０ a dose of one or more live attenuated influenza vaccine (LAIV) viruses; b) sucrose 4% (w / v); c) Histidine 0.21% (w / v); d) Alanine 0.1% (w / v); e) Tricine 0.3% (w / v); f) arginine 2.1% (w / v); and g) Gelatin 0.85% (w / v) The immunogenic composition of claim 1 , comprising:

33. The composition comprising: a) 6-7 LogEID per 0.5 ml ５０ one or more live attenuated influenza vaccine (LAIV) viruses NLT; b) sucrose 4% (w / v); c) Histidine 0.21% (w / v); d) Alanine 0.1% (w / v); e) Tricine 0.3% (w / v); f) arginine 2.1% (w / v); and g) Gelatin 1% (w / v) The immunogenic composition of claim 1 , comprising:

34. The composition comprising: a) 6-7 LogEID per 0.5 ml ５０ a dose of one or more live attenuated influenza vaccine (LAIV) viruses; b) sucrose 4% (w / v); c) Histidine 0.21% (w / v); d) Alanine 0.1% (w / v); e) Tricine 0.3% (w / v); f) arginine 1.6% (w / v); and g) Gelatin 1% (w / v) The immunogenic composition of claim 1 , comprising:

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