Improved seed virus

Abstract

This disclosure relates to the field of influenza viruses. More specifically, this disclosure relates to donor or seed influenza viruses for use in the preparation of reassembled influenza viruses for vaccine production.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 508,946, filed on 19 June 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] This disclosure relates to the field of influenza viruses. More specifically, this disclosure relates to donor or seed influenza viruses for use in the preparation of reassembled influenza viruses for vaccine production. [Background technology]

[0003] Influenza is the leading respiratory disease in mammalian species, causing significant mortality, morbidity, and economic losses each year. Three broad types of influenza viruses are recognized: types A, B, and C, which are defined by the absence of serological cross-reactivity between their internal proteins. Influenza A viruses are further classified into subtypes based on antigenic and genetic differences in their glycoproteins, hemagglutinins (HA), and neuraminidase (NA) proteins.

[0004] More recent vaccine production methods may involve the generation and culture of reassembled influenza viruses in cell cultures such as Madin Darby Canine Kidney (MDCK) cells. However, certain influenza viruses appear to be less adaptable to growth and replication than others when cultured as reassembled viruses containing the skeletal viral segment of a donor influenza virus. This can lead to challenges in viral protein yield and vaccine production. Therefore, there remains a need for the development of donor influenza viruses in which the skeletal viral segment confers efficient growth and replication to reassembled viruses expressing the HA and / or NA proteins of vaccine virus candidates in cell culture. [Overview of the project]

[0005] This disclosure is based on the remarkable discovery of specific influenza viruses that can be used as donor or seed viruses in a reassembly method for improving the proliferation and / or yield of influenza viruses cultured on cells such as Madin Darby Canine Kidney (MDCK) cells.

[0006] In a first aspect, the disclosure provides an isolated influenza virus comprising one or more PA, PB1, PB2, NP, M, and NS viral segments encoding one or more viral proteins independently comprising, consisting of, or essentially comprising amino acid sequences selected from the group consisting of SEQ ID NOs: 9-17, 28-36, 47-55, 66-74, 85-93, and 104-112, or fragments, variants, or derivatives thereof.

[0007] Preferably, the present disclosure provides an isolated influenza virus comprising one or more PA, PB1, PB2, NP, M, and NS viral segments encoding one or more viral proteins independently comprising, consisting of, or essentially comprising amino acid sequences selected from SEQ ID NOs. 9-17, or fragments, variants, or derivatives thereof. In certain examples, the isolated influenza virus comprises PA, PB2, NP, M, and NS viral segments encoding viral proteins independently comprising, consisting of, or essentially comprising amino acid sequences selected from SEQ ID NOs. 9 and 12-17, or fragments, variants, or derivatives thereof. In such examples, the isolated influenza virus may or may not comprise a PB1 viral segment encoding a viral protein independently comprising, consisting of, or essentially comprising amino acid sequences of SEQ ID NOs. 10 and / or 11, or fragments, variants, or derivatives thereof. Therefore, in some cases, isolated influenza viruses contain PA, PB1, PB2, NP, M, and NS viral segments that independently contain, consist of, or are essentially derived from amino acid sequences selected from SEQ ID NOs. 9–17, or fragments, variants, or derivatives thereof, which constitute viral proteins.

[0008] Preferably, the isolated influenza virus of this embodiment includes one or more PB2, PB1, PA, M, NP, and NS viral segments that independently include, consist of, or are essentially derived from nucleotide sequences selected from the group consisting of SEQ ID NOs: 1-6, 20-25, 39-44, 58-63, 77-82, and 96-101, or their fragments, variants, or derivatives. In one example, the isolated influenza virus of this embodiment includes one or more PB2, PB1, PA, M, NP, and NS viral segments that independently include, consist of, or are essentially derived from nucleotide sequences selected from SEQ ID NOs: 1-6, or their fragments, variants, or derivatives. In a particular example, the isolated influenza virus includes PB2, PA, M, NP, and NS viral segments that independently include, consist of, or are essentially derived from nucleotide sequences selected from SEQ ID NOs: 1 and 3-6, or their fragments, variants, or derivatives. In such examples, the isolated influenza virus may or may not contain the PB1 viral segment, which contains, consists of, or is essentially derived from, the nucleotide sequence shown in SEQ ID NO: 2. Thus, in some examples, the isolated influenza virus contains the PA, PB1, PB2, NP, M, and NS viral segments, which independently contain, consist of, or are essentially derived from nucleotide sequences selected from SEQ ID NOs: 1-6, or fragments, variants, or derivatives thereof.

[0009] In certain cases, isolated influenza viruses are capable of enhanced replication when grown in cells compared to wild-type influenza virus isolates that do not contain one or more PA, PB1, PB2, NP, M, and NS viral segments.

[0010] Preferably, isolated influenza viruses can yield enhanced yields of viral proteins, such as hemagglutinin (HA) protein, when grown in cells, compared to wild-type influenza virus isolates that do not contain one or more PA, PB1, PB2, NP, M, and NS viral segments.

[0011] In certain cases, the cells are MDCK cells.

[0012] Preferably, the isolated influenza virus in this embodiment is (a) Sequence IDs 9-17, or fragments, variants, or derivatives thereof, (b) Sequence IDs 28-36, or fragments, variants, or derivatives thereof (c) Sequence IDs 47-55, or fragments, variants, or derivatives thereof, (d) Sequence IDs 66-74, or fragments, variants, or derivatives thereof, (e) Sequence IDs 85-93, or fragments, variants, or derivatives thereof, (f) comprising five or six of the PA, PB1, PB2, NP, M, and NS viral segments encoding one or more viral proteins that independently comprise, consist of, or are essentially derived from amino acid sequences selected from sequence numbers 104-112, or fragments, variants, or derivatives thereof.

[0013] Therefore, in one example, the isolated influenza virus of this embodiment comprises five or six of the PA, PB1, PB2, NP, M, and NS viral segments encoding one or more viral proteins that independently contain, consist of, or are essentially derived from amino acid sequences selected from those shown in SEQ ID NOs: 9-17, or fragments, variants, or derivatives thereof.

[0014] In various cases, the isolated influenza virus in this embodiment is (a) Sequence IDs 1-6, or fragments, variants, or derivatives thereof, (b) Sequence IDs 20-25, or fragments, variants, or derivatives thereof, (c) Sequence IDs 39-44, or fragments, variants, or derivatives thereof, (d) Sequence IDs 58-63, or fragments, variants, or derivatives thereof, (e) Sequence IDs 77-82, or fragments, variants, or derivatives thereof, (f) comprising five or six PA, PB1, PB2, NP, M, and NS viral segments, which independently comprise, consist of, or are essentially derived from nucleotide sequences selected from those shown in SEQ ID NOs. 96-101, or fragments, variants, or derivatives thereof.

[0015] Therefore, in one example, the isolated influenza virus of this embodiment comprises five or six of the PA, PB1, PB2, NP, M, and NS viral segments, each independently comprising, consisting of, or essentially comprising a nucleotide sequence selected from SEQ ID NOs: 1-6, or fragments, variants, or derivatives thereof.

[0016] In some examples, the isolated influenza virus of this embodiment further comprises heterologous or chimeric HA virus segments and / or heterologous or chimeric NA virus segments.

[0017] Preferably, the isolated influenza virus is of the N1, N2, N3, N7, or N9 subtype. More particularly, the isolated influenza virus may be of the N1 or N2 subtype.

[0018] Preferably, the isolated influenza virus is of the H1, H2, H3, H5, H7, or H9 subtype. More particularly, the isolated influenza virus may be of the H1 or H3 subtype.

[0019] In certain embodiments, the isolated influenza virus is of the H1N1 subtype or the H3N2 subtype.

[0020] In certain embodiments, the isolated influenza virus is a recombinant influenza virus.

[0021] In other embodiments, the isolated influenza virus is a reassortant influenza virus.

[0022] In a second aspect, the present disclosure provides a method for preparing an influenza virus, the method comprising contacting one or more gene constructs encoding one or more viral proteins independently comprising, consisting of, or consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-17, 28-36, 47-55, 66-74, 85-93, and 104-112, or fragments, variants, or derivatives thereof, with a cell.

[0023] Preferably, the one or more gene constructs independently comprise, consist of, or consist essentially of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-6, 20-25, 39-44, 58-63, 77-82, and 96-101, or a nucleotide sequence complementary thereto, or fragments, variants, or derivatives thereof, and comprise or encode one or more of the PB2, PB1, PA, M, NP, and NS viral segments.

[0024] In a third aspect, the Disclosure provides a method for preparing an influenza virus, the method comprising the step of contacting a cell with an isolated influenza virus comprising one or more PA, PB1, PB2, NP, M, and NS viral segments encoding one or more viral proteins independently comprising, consisting of, or essentially comprising amino acid sequences selected from the group consisting of SEQ ID NOs: 9-17, 28-36, 47-55, 66-74, 85-93, and 104-112, or fragments, variants, or derivatives thereof.

[0025] In the method of this embodiment, the isolated influenza virus is preferably of the first embodiment.

[0026] A method according to a second or third embodiment may further include the step of isolating or collecting one or more influenza viruses and / or viral proteins from cells.

[0027] In a fourth aspect, the disclosure provides isolated influenza viruses prepared by the method of the second or third aspect.

[0028] In a fifth aspect, the disclosure provides isolated cells infected with an isolated influenza virus according to the first or fourth aspect.

[0029] Preferably, the cells are MDCK cells.

[0030] In a sixth aspect, the disclosure provides a gene construct encoding one or more viral proteins that independently comprises, consist of, or are essentially derived from amino acid sequences selected from the group consisting of SEQ ID NOs: 9-17, 28-36, 47-55, 66-74, 85-93, and 104-112, or fragments, variants, or derivatives thereof.

[0031] In a seventh aspect, the disclosure provides a plurality of gene constructs encoding one or more viral proteins, each independently comprising, consisting of, or essentially comprising amino acid sequences selected from the group consisting of SEQ ID NOs: 9-17, 28-36, 47-55, 66-74, 85-93, and 104-112, or fragments, variants, or derivatives thereof.

[0032] Referring to the sixth and seventh aspects, a gene construct or a group of gene constructs preferably comprises, comprises, or encodes one or more PB2, PB1, PA, M, NP, and NS viral segments, independently comprising, comprising, or essentially comprising one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 1-6, 20-25, 39-44, 58-63, 77-82, and 96-101, or complementary nucleotide sequences, or fragments, variants, or derivatives thereof.

[0033] In the eighth aspect, the present disclosure relates to a method for preparing an immunogenic composition, (a) Providing an isolated influenza virus and / or viral protein derived therefrom according to the first or fourth embodiment, (b) A method is provided comprising the steps of combining isolated influenza virus and / or viral proteins with an adjuvant, and / or treating the isolated influenza virus with a virus-inactivating agent.

[0034] In some cases, the adjuvant includes immunostimulatory DNA sequences, bacterial components, aluminum salts (alum), or squalene oil-in-water emulsion systems.

[0035] In a ninth aspect, the present disclosure provides an immunogenic composition produced according to the method of the eighth aspect.

[0036] In a tenth aspect, the disclosure provides an immunogenic composition comprising an isolated influenza virus and / or viral protein derived therefrom according to the first or fourth aspect, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0037] Preferably, isolated influenza viruses and / or viral proteins derived therefrom according to the first or fourth embodiment, or immunogenic compositions according to the ninth or tenth embodiment, are for therapeutic use.

[0038] Preferably, isolated influenza viruses and / or viral proteins derived therefrom according to the first or fourth embodiment, or immunogenic compositions according to the ninth or tenth embodiment, are for use in methods of inducing an immune response in a subject.

[0039] Preferably, isolated influenza viruses and / or viral proteins derived therefrom according to the first or fourth embodiment, or immunogenic compositions according to the ninth or tenth embodiment, are for use in methods of preventing and / or treating influenza-related diseases, disorders, or conditions in a subject.

[0040] In an eleventh aspect, the present disclosure provides a method for inducing an immune response in a subject, the method comprising the step of administering a therapeutically effective amount of an isolated influenza virus and / or viral protein of the first or fourth aspect, or an immunogenic composition of the ninth or tenth aspect, to the subject, thereby inducing an immune response in the subject.

[0041] In a twelfth aspect, the Disclosure provides a method for preventing and / or treating an influenza-related illness, disorder, or condition in a subject, the method comprising the step of administering a therapeutically effective amount of an isolated influenza virus and / or viral protein of the first or fourth aspect, or an immunogenic composition of the ninth or tenth aspect, to the subject, thereby preventing and / or treating an influenza-related illness, disorder, or condition.

[0042] In a thirteenth aspect, the disclosure provides the use of isolated influenza viruses and / or viral proteins derived therefrom according to the first or fourth aspect, or immunogenic compositions according to the ninth or tenth aspect, in the manufacture of a pharmaceutical product for inducing an immune response in a subject.

[0043] In a fourteenth aspect, the Disclosure provides the use of isolated influenza viruses and / or viral proteins derived therefrom according to the first or fourth aspect, or immunogenic compositions according to the ninth or tenth aspect, in the manufacture of a pharmaceutical product for preventing and / or treating influenza-related illnesses, disorders, or conditions in a subject.

[0044] The following drawings form part of this specification and are included to further illustrate certain aspects of the disclosure. This disclosure can be better understood by referring to one or more of these drawings in conjunction with the detailed descriptions of the specific embodiments presented herein. Those skilled in the art will understand that numerous variations and / or modifications can be made to the above embodiments without departing from the broad and comprehensive scope of this disclosure. Thus, these embodiments should be considered in all respects as illustrative and not limiting. [Brief explanation of the drawing]

[0045] [Figure 1] Schematic diagram of candidate vaccine virus (CVV) transfection and rescue. [Figure 2]Individual HA yield results for wild-type and reaggregated strains, A / Idaho / 07 / 2018. [Figure 3] Individual HA yield results for wild-type and reaggregated strains from A / Nebraska / 14 / 2019. [Figure 4] Individual HA yield results for A / Iowa / 56 / 2019 wild-type and reaggregated strains. [Figure 5] Individual HA yield results for wild-type and reaggregated strains from A / Delaware / 55 / 2019. [Figure 6] Individual HA yield results for wild-type and reaggregated strains of A / Illinois / 02 / 2020. [Figure 7] Individual HA yield results for A / Canberra / 407 / 2019 wild type and reaggregated strains. [Figure 8] Individual HA yield results for wild-type and reaggregated strains of A / Tasmania / 503 / 2020. [Figure 9] Individual HA yield results for wild-type and reaggregated strains of A / Bangaldesh / 1002 / 2020. [Figure 10] Individual HA yield results for wild-type and reaggregated strains from A / Darwin / 94 / 2019. [Figure 11] Individual HA yield results for wild-type and reaggregated strains from A / Delaware / 39 / 2019. [Figure 12] Individual HA yield results for wild-type and reaggregated strains, A / Virginia / 03 / 2020. [Figure 13] Summary of HA yield results for donor strains, A / Ohio / 02 / 2019. [Figure 13-1] Same as above. [Figure 14] The rate of increase in HA yield of reassembled viruses derived from the skeletal or donor virus strain compared to manufactured or vaccine virus controls.

[0046] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Modes for carrying out the invention]

[0047] General Techniques and Definitions Unless specifically defined otherwise, all technical and scientific terms used herein shall be construed to have the same meaning as those generally understood by those skilled in the art (e.g., genomics, immunology, molecular biology, immunohistochemistry, biochemistry, oncology, and pharmacology).

[0048] This disclosure is carried out without excessive experimentation using conventional techniques of molecular biology, microbiology, recombinant DNA technology, and immunology, unless otherwise indicated. Such procedures are described in, for example, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Fourth Edition (2012), whole of Vols I, II, and III; DNA Cloning: A Practical Approach, Vols. I and II (DNGlover, Second Edition, 1995), IRL Press, Oxford, whole of text; Oligonucleotide Synthesis: A Practical Approach (MJ Gait, ed, 1984), IRL Press, Oxford, whole of text; and in particular, the articles within it by Gait, ppl-22, Atkinson et al, pp35-81, Sproat et al, pp83-115, and Wu et al, pp135-151; 4. Nucleic Acid Hybridization: A Practical Approach (B.D. Hames & S.J. Higgins, eds., 1985), IRL Press. This information is described in IRL Press, Oxford, whole of text, Immobilized Cells and Enzymes: A Practical Approach (1986), Perbal, B., A Practical Guide to Molecular Cloning (1984), and Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.), whole of series.

[0049] Those skilled in the art will understand that this disclosure is subject to variations and modifications other than those specifically described. It should be understood that this disclosure includes all such variations and modifications. This disclosure also includes, individually or collectively, all of the steps, features, compositions, and compounds referred to or indicated herein, as well as any and all combinations of any two or more of such steps or features.

[0050] This disclosure is intended for illustrative purposes only and should not be limited in scope by the specific embodiments described herein. Functionally equivalent products, compositions, and methods are clearly within the scope of this disclosure as described herein.

[0051] Any particular aspect, embodiment, or feature of this disclosure may be applied mutatis mutandis to any other aspect, embodiment, or feature of this disclosure.

[0052] Throughout this specification, unless otherwise specifically stated or the context requires, any reference to a single step, composition of a substance, group of steps, or group of compositions of a substance shall be construed as encompassing one or more (i.e., one or more) of those steps, compositions of a substance, groups of steps, or groups of compositions of a substance.

[0053] As used herein, the singular forms of “a,” “and,” and “the” include the plural forms of these words unless the context explicitly indicates otherwise. For example, a reference to “bacteria” includes multiple such bacteria, and a reference to “allergen” refers to one or more allergens.

[0054] The term "and / or," for example, "X and / or Y," is understood to mean "X and Y" or "X or Y," and is considered to provide explicit support for both meanings or either of them.

[0055] Throughout this specification, the word “comprise,” or variations such as “comprises” or “comprising,” shall be understood to imply the inclusion of the specified element, component, or step, or group of elements, components, or steps, but not to exclude any other element, component, or step, or group of elements, components, or steps.

[0056] In the following context, “essentially consisting of” means (a) an amino acid sequence is an amino acid sequence listed with one, two, or three additional amino acids at its N-terminus or C-terminus, and (b) a nucleotide sequence is a nucleotide sequence listed with one, two, three, four, five, or six additional nucleic acid bases at its 5' or 3' end.

[0057] The term "substantially" does not exclude "completely" (for example, a composition that "substantially" does not contain Y may not contain Y at all).

[0058] The term "approximately" in relation to a number x is optional and means any number within, for example, 0.5%, 1%, 5%, or 10% of the referenced number or value. In certain examples, the term "approximately" encompasses the exact number listed.

[0059] All computer programs, algorithms, patents, and scientific literature referenced herein are incorporated herein by reference.

[0060] In this disclosure, database accession numbers or proprietary identifiers provided herein for genes, proteins, or virus strains, and for any associated gene and / or protein sequences or sequences, are incorporated herein by reference.

[0061] Isolated influenza virus The inventors have surprisingly discovered a number of influenza viruses in which skeletal viral segments (e.g., PA, PB1, PB2, NP, M, and NS viral segments) can be utilized in reassembly techniques to improve the growth and yield of reassembled viruses containing said skeletal viral segments in cell culture, which may be advantageous for vaccine production.

[0062] Accordingly, in one embodiment, the present disclosure provides an isolated influenza virus comprising one or more PA, PB1, PB2, NP, M, and NS viral segments that encode one or more viral proteins provided herein (e.g., one or more of the PA, PB1, PB1-F2, PB2, NP, M1, M2, NEP, and NS1 skeletal viral proteins).

[0063] Preferably, the viral protein comprises, consists of, or is essentially composed of, an amino acid sequence selected from the group shown in SEQ ID NOs. 9-17, 28-36, 47-55, 66-74, 85-93, and 104-112, or fragments, variants, or derivatives thereof. For this purpose, the PB2 viral protein preferably contains, consists of, or is essentially composed of an amino acid sequence selected from the group shown in SEQ ID NOs: 9, 28, 47, 66, 85, and 104, or their fragments, variants, or derivatives; the PB1 viral protein preferably contains, consists of, or is essentially composed of an amino acid sequence selected from the group shown in SEQ ID NOs: 10, 29, 48, 67, 86, and 105, or their fragments, variants, or derivatives; the PB1-F2 viral protein preferably contains, consists of, or is essentially composed of an amino acid sequence selected from the group shown in SEQ ID NOs: 11, 30, 49, 68, 87, and 106, or their fragments, variants, or derivatives; and the PA viral protein preferably contains, consists of, or is essentially composed of an amino acid sequence selected from the group shown in SEQ ID NOs: 12, 31, 50, 69, 88, and 107, or their fragments, variants, or derivatives. NP viral proteins preferably contain, consist of, or essentially consist of amino acid sequences selected from the group shown in SEQ ID NOs: 13, 32, 51, 70, 89, and 108, or their fragments, variants, or derivatives; NS1 viral proteins preferably contain, consist of, or essentially consist of amino acid sequences selected from the group shown in SEQ ID NOs: 14, 33, 52, 71, 90, and 109, or their fragments, variants, or derivatives; NEP viral proteins preferably contain, consist of, or essentially consist of amino acid sequences selected from the group shown in SEQ ID NOs: 15, 34, 53, 72, 91, and 110, or their fragments, variants, or derivatives; M1 viral proteins preferably contain, consist of, or essentially consist of amino acid sequences selected from the group shown in SEQ ID NOs: 16, 35, 54, 73, 92, and 111, or their fragments, variants,Alternatively, the M2 viral protein may contain, consist of, or be essentially composed of an amino acid sequence selected from the group shown in the derivatives, and / or preferably contain, consist of, or be essentially composed of an amino acid sequence selected from the group shown in SEQ ID NOs: 17, 36, 55, 74, 93, and 112, or fragments, variants, or derivatives thereof.

[0064] In a related form, the present disclosure provides an isolated influenza virus comprising one or more of the PB2, PB1, PA, M, NP, and NS viral segments (i.e., skeletal viral segments) provided herein. Preferably, the PB2, PB1, PA, M, NP, and NS viral segments independently comprise, consist of, or essentially comprise of nucleotide sequences selected from those shown in SEQ ID NOs: 1-6, 20-25, 39-44, 58-63, 77-82, and 96-101, or fragments, variants, or derivatives thereof. In this regard, the PB2 virus segment preferably contains, consists of, or is essentially derived from, a nucleotide sequence selected from the group shown in SEQ ID NOs: 1, 20, 39, 58, 77, and 96, or their fragments, variants, or derivatives; the PB1 virus segment preferably contains, consists of, or is essentially derived from, a nucleotide sequence selected from the group shown in SEQ ID NOs: 2, 21, 40, 59, 78, and 97, or their fragments, variants, or derivatives; and the PA virus segment preferably contains, consists of, or is essentially derived from, a nucleotide sequence selected from the group shown in SEQ ID NOs: 3, 22, 41, 60, 79, and 98, or their fragments, variants, or derivatives. The NP virus segment preferably comprises, consists of, or is essentially derived from, a nucleotide sequence selected from the group shown in SEQ ID NOs: 4, 23, 42, 61, 80, and 99, or their fragments, variants, or derivatives; the NS virus segment preferably comprises, consists of, or is essentially derived from, a nucleotide sequence selected from the group shown in SEQ ID NOs: 5, 24, 43, 62, 81, and 100, or their fragments, variants, or derivatives; and / or the M virus segment preferably comprises, consists of, or is essentially derived from, a nucleotide sequence selected from the group shown in SEQ ID NOs: 6, 25, 44, 63, 82, and 101, or their fragments, variants, or derivatives.

[0065] Influenza viruses are enveloped RNA viruses belonging to the family Orthomyxoviridae (Palese and Shaw (2007) Orthomyxoviridae: The Viruses and Their Replication, 5th ed. Fields' Virology, edited by B.N. Fields, D.M. Knipe and P.M. Howley. Wolters Kluwer Health / Lippincott Williams & Wilkins, Philadelphia, USA, pp. 1647-1689). Influenza A and B viruses are major human pathogens, causing respiratory illness ranging in severity from asymptomatic infection to primary viral pneumonia, and can be fatal. The clinical effects of infection vary depending on the virulence of the influenza strain, as well as the host's exposure, medical history, age, and immune status. While the natural hosts of influenza viruses are primarily birds, influenza viruses, particularly influenza A viruses (including those of avian origin), can also infect and cause illness in humans and other animal hosts (e.g., bats, dogs, pigs, horses, marine mammals, and weasels).

[0066] The isolated influenza viruses in this disclosure may be influenza A viruses or influenza B viruses. According to some examples, the isolated influenza virus is an influenza A virus. In alternative examples, the isolated influenza virus is an influenza B virus. As an example, the influenza A viruses provided herein may include HA subtypes selected from H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, and H16. In some examples, the influenza A virus is of the H1, H2, H3, H5, H7, or H9 subtype. In various examples, the influenza A virus is of the H1 or H3 subtype. Furthermore, such viruses may include influenza A virus NA subtypes N1, N2, N3, N4, N5, N6, N7, N8, or N9. In some examples, the influenza A virus is of the N1, N2, N3, N7, or N9 subtype. In other examples, the influenza A virus is of the N1 or N2 subtype. Specifically, the influenza A virus can be a strain selected from the group consisting of H1N1, H1N2, H2N2, H3N1, H3N2, H3N8, H5N1, H7N1, H7N2, H7N3, H7N7, H9N2, and H10N7. According to some examples, the influenza A virus is an H3N2 strain. In alternative examples, the influenza A virus is an H1N1 strain.

[0067] Considering the above, the isolated influenza viruses referred to herein preferably include HA virus segments and / or NA virus segments derived at least partially from a seasonal influenza virus strain. As used herein, the term “seasonal influenza virus strain” refers to an influenza virus strain to which a target population is seasonally exposed. Thus, the isolated influenza viruses described herein may be suitable for use in vaccines or immunogenic compositions to protect against seasonal virus strains that are currently circulating in human populations or are endemic. In certain examples, the term seasonal influenza virus strain refers to a strain of influenza A virus. In other examples, the term seasonal influenza virus strain refers to a strain of influenza A virus belonging to the H1 or H3 subtype (i.e., two subtypes that are currently circulating in human populations or are endemic). In some examples, the term seasonal influenza virus strain refers to a strain of influenza B virus.

[0068] As other examples show, isolated influenza viruses referred to herein include HA virus segments and / or NA virus segments derived at least partially from pandemic influenza virus strains. As used herein, the term “pandemic influenza virus strain” refers to a strain of influenza virus that is associated with, or may be associated with, an outbreak of influenza disease. Generally, the characteristics that give an influenza strain the potential to cause a pandemic outbreak are: (a) it contains a novel hemagglutinin, i.e., one that has not been evident in human populations (e.g., H2) for more than 10 years or has never been previously found in human populations (e.g., H5, H6, or H9, generally found only in avian populations) compared to hemagglutinins in currently circulating human strains, and as a result, human populations are immunologically naive to the hemagglutinin of that strain; (b) it is capable of horizontal transmission in human populations; and (c) it is pathogenic to human populations. Therefore, isolated influenza viruses can spread from non-human animal populations to humans, or may be suitable for use in vaccines or immunogenic compositions to protect against spreading potential pandemic virus strains. Thus, in certain examples, the term pandemic influenza virus strain refers to strains of influenza A virus. Suitable pandemic strains include, but are not limited to, H5N1, H9N2, H7N7, H2N2, H2N3, H7N1, and H1N1. Other suitable pandemic strains found in humans are H7N3, H10N7, and H5N2.

[0069] It is further intended that isolated influenza viruses may contain HA viral segments encoding chimeric HA proteins (i.e., HA proteins containing amino acid sequences derived from two or more influenza strains). For example, a chimeric HA may contain the cytoplasmic portion, or the cytoplasmic and transmembrane portions, of HA derived from one influenza strain, as well as at least the extracellular antigenic portion of HA derived from different influenza strains. This approach has been previously described as a technique for producing influenza viruses containing the antigenic portion of HA proteins in situations where unmodified HA segments may be produced in low yields.

[0070] In other cases, isolated influenza viruses express non-chimeric HA proteins. In other words, the HA protein sequence contains cytoplasmic, transmembrane, and extracellular domains derived from the same influenza strain.

[0071] The influenza viruses referred to herein include, but are not limited to, any virus type, subtype, or strain, including naturally occurring strains, variants or variants, mutagenic or modified viruses, reassorted viruses, and / or genetically modified viruses.

[0072] The influenza viruses, nucleic acids, proteins, gene constructs, or cells described herein may be considered isolated. For the purposes of this disclosure, “isolated” means a substance that has been removed from its natural state or otherwise subjected to human manipulation. An isolated substance may substantially or essentially lack components that would normally accompany it in its natural state, or it may be manipulated to become artificial with components that would normally accompany it in its natural state. An isolated substance may be in natural, chemically synthesized, or recombinant form.

[0073] Isolated recombinant influenza viruses are intended for this disclosure. A “recombinant” virus is a virus that has been manipulated in vitro, for example, by using recombinant DNA technology to introduce changes into the viral genome.

[0074] Preferably, the isolated influenza virus is a reassortment virus, such as a recombinant reassortment virus. The term “reassortment virus” refers to a virus containing genetic material resulting from a combination of genetic material from at least two donor viruses (e.g., one or more genetic segments from a first parent influenza virus strain (donor, skeleton, or seed strain) and one or more genetic segments from a second parent influenza virus strain (vaccine strain)). When a reassortment virus is used to prepare a vaccine or immunogenic composition, its genetic material typically contains at least an HA viral segment and optionally an NA viral segment derived from a seasonal or pandemic influenza virus, but other viral segments (i.e., skeleton viral segments) are derived from one or more other donor or seed viruses selected for their ability to readily replicate on a substrate for production used to manufacture an influenza vaccine (e.g., the allanine cavity of hatched hen eggs or a permissive cell line) and / or are less pathogenic or non-pathogenic to humans. Non-limiting examples of donor or seed viruses that contribute as donors to skeletal viral segments include A / Puerto Rico / 8 / 1934(PR8), A / Texas / 1 / 1977, A / New York / 55 / 2004, A / Ann Arbor / 6 / 60, A / Leningrad / 134 / 17 / 57, B / Ann Arbor / 1 / 66, B / Florida / 4 / 2006, B / Panama / 45 / 1990, and B / Lee / 1940. Reassorted viruses may be produced by any method known in the art, including reverse genetics, classical reassortment, and hybrid versions thereof.

[0075] Based on the improvements in viral replication and yield observed in the cell cultures outlined in the following examples, the isolated influenza viruses described herein (e.g., A / Ohio / 02 / 2019, A / Singapore / TT1384 / 2016, A / South Carolina / 04 / 2017, A / Alaska / 06 / 2019, A / Darwin / 11 / 2021, and A / Tasmania / 503 / 2020) may function as donor virus strains with respect to the provision of skeletal viral segments in the generation of reassembled viruses. Influenza donor strains are typically strains that provide one or more skeletal viral segments in reassembled influenza viruses, but may sometimes also provide the NA segment of the virus. Vaccine strains are typically influenza strains that provide HA and / or NA segments. Generally, both HA and NA segments in reassembled influenza viruses may be derived from vaccine strains. Vaccine strains are typically circulating strains, such as seasonal or pandemic influenza virus strains. Preferably, the vaccine strain is different from or heterologous to the donor strain. The viral segments present in the reassembled virus can be described using locus ratios that indicate the number of segments provided by each parent influenza virus strain. For example, if the reassembled virus contains genomic segments from two parent influenza virus strains (such as a donor strain and a vaccine strain), it may have locus ratios of 1:7, 2:6, 3:5, 4:4, 5:3, 6:2, or 7:1.

[0076] Generally, the majority of the genetic segments of a reassembled virus are donor-derived, as it is desirable to utilize the characteristics of the donor strain (e.g., improved replication and / or yield in cell culture) by reassembling the donor strain segments with segments derived from the vaccine strain. In certain examples, the reassembled influenza viruses produced by the methods provided herein have locus ratios of 5:3, 6:2, or 7:1, where the first number in the ratio indicates the number of segments from the donor strain and the second number indicates the number of segments from the vaccine strain.

[0077] In certain cases, the reassembled influenza virus has a 6:2 locus ratio. In these cases, the reassembled influenza virus preferably comprises six skeletal segments from the donor strain (i.e., PB1, PB2, PA, NP, M, and NS) and two segments from the vaccine strain (i.e., HA and NA).

[0078] In other examples, reassembled influenza viruses have a locus ratio of 7:1. In such examples, the reassembled influenza virus may contain six skeletal segments from the donor strain, an HA segment from the vaccine strain, and an NA segment from the donor strain. In other words, the reassembled influenza virus contains the HA segment from the vaccine strain, and the remaining seven segments are derived from the donor strain. In alternative examples, the reassembled influenza virus contains six skeletal segments and an HA segment from the donor strain, as well as an NA segment from the vaccine strain. In other words, the reassembled influenza virus contains the NA segment from the vaccine strain and the remaining seven segments from the donor strain.

[0079] In further examples, reassembled influenza viruses have a 5:3 locus ratio. In these examples, the reassembled virus may contain five skeletal segments from the donor strain (i.e., five viral segments selected from the group consisting of PB1, PB2, PA, NP, M, and NS viral segments) and three segments from the vaccine strain. In such examples, the three segments from the vaccine strain are typically HA, NA, and one skeletal segment (i.e., one segment selected from the group consisting of PB1, PB2, PA, NP, M, and NS). In specific examples, the three segments from the vaccine strain are HA, NA, and PB1, and the remaining five skeletal segments (i.e., PB2, PA, NP, M, and NS viral segments) are derived from the donor strain.

[0080] In certain cases, isolated reassembled influenza viruses, (a) One or more PA, PB1, PB2, NP, NS, and M viral segments derived from the first influenza virus isolate (for example, one or more PA, PB1, PB2, NP, M, and NS viral segments encoding one or more viral proteins containing, consisting of, or essentially comprising amino acid sequences selected from the group consisting of SEQ ID NOs. 9-17, 28-36, 47-55, 66-74, 85-93, and 104-112), (b) HA virus segments, including their chimeric forms, derived from a second influenza virus isolate (e.g., vaccine virus or strain), and (c) optionally comprising an NA virus segment containing a chimeric form thereof derived from the first influenza virus isolate, the second influenza virus isolate, or the third influenza virus isolate.

[0081] For this purpose, the NA virus segment and HA virus segment may originate from the same influenza virus isolate, while the skeletal virus segment may be different from or derived from a different influenza virus isolate than those of the NA and HA virus segments. Alternatively, the NA virus segment may originate from the same influenza virus isolate (e.g., donor virus) as the skeletal virus segment (e.g., the NA virus segment contains, consists of, or is essentially derived from the nucleotide sequence shown in SEQ ID NOs: 8, 27, 46, 65, 84, or 103, and / or the NA virus segment encodes an NA virus protein containing, consisting of, or is essentially derived from the amino acid sequence shown in SEQ ID NOs: 19, 38, 57, 76, 95, 114). Furthermore, the NA virus segment may originate from an influenza virus isolate that is different from or is of a different species than those from which the skeletal virus segment and HA virus segment originated. In this regard, the HA viral segment may originate from the same influenza virus isolate (e.g., donor virus) as the skeletal viral segment (for example, the HA viral segment may contain, consist of, or be essentially derived from the nucleotide sequence shown in SEQ ID NOs: 7, 26, 45, 64, 83, or 102, and / or the HA viral segment may encode an HA viral protein containing, consisting of, or being essentially derived from the amino acid sequence shown in SEQ ID NOs: 18, 37, 56, 75, 94, or 113).

[0082] As used herein, “heterogeneous” influenza virus gene or viral segment is derived from an influenza virus source or isolate that is different from most of the other influenza virus genes or gene segments in a reassembled influenza virus or recombinant influenza virus, including recombinant reassembled influenza virus.

[0083] In certain examples, the isolated influenza virus includes a viral segment encoding two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or 9) of the skeletal viral proteins provided herein (i.e., PB2, PB1, PA, M, NP, and NEP viral proteins) (i.e., PB2, PB1, PA, M, NP, and NS viral segments) (e.g., SEQ ID NOs. 9-17, 28-36, 47-55, 66-74, 85-93, and 104-112, or fragments, variants, or derivatives thereof). In certain examples, the isolated influenza virus includes a viral segment encoding two or more, three or more, more particularly four or more, even more particularly five or more, even more particularly six or more, and even more particularly seven or more (i.e., 7, 8, or 9) of the skeletal viral proteins provided herein.

[0084] In a relevant example, the isolated influenza virus contains two or more (e.g., 2, 3, 4, 5, or 6) of the skeletal viral segments provided herein (i.e., PB2, PB1, PA, M, NP, and NS viral segments) (e.g., nucleotide sequences including, consisting of, or essentially derived from, those shown in SEQ ID NOs: 1-6, 20-25, 39-44, 58-63, 77-82, and 96-101, or their fragments, variants, or derivatives). In a particular example, the isolated influenza virus contains three or more, more particularly four or more, or even more particularly five or more (i.e., five or six) of the skeletal viral segments provided herein.

[0085] Preferably, the isolated influenza virus comprises viral segments encoding nine of the skeletal viral proteins provided herein (i.e., each of the PB2, PB1, PA, M, NP, and NS viral segments). In these examples, the isolated influenza virus preferably comprises viral segments encoding the PB2, PB1, PB1-F2, PA, M1, M2, NP, NS1, and NEP viral proteins, independently comprising, consisting of, or essentially comprising amino acid sequences selected from those shown in SEQ ID NOs. 9-17, 28-36, 47-55, 66-74, 85-93, and 104-112, or their fragments, variants, or derivatives. For this purpose, the isolated influenza virus is intended to contain any combination of nine of those skeletal viral proteins provided herein.

[0086] Therefore, in certain cases, isolated influenza viruses, (a) A PB2 viral segment encoding a PB2 viral protein comprising, consisting of, or essentially comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 28, 47, 66, 85, and 104, or fragments, variants, or derivatives thereof, (b) PB1 viral proteins comprising, comprising, or essentially comprising amino acid sequences selected from the group consisting of SEQ ID NOs. 10, 29, 48, 67, 86, and 105, or fragments, variants, or derivatives thereof, and / or PB1 viral segments encoding PB1-F2 viral proteins comprising, comprising, or essentially comprising amino acid sequences selected from the group consisting of SEQ ID NOs. 11, 30, 49, 68, 87, and 106, or fragments, variants, or derivatives thereof, (b) PA virus segments encoding a PA virus protein comprising, consisting of, or essentially comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 31, 50, 69, 88, and 107, or fragments, variants, or derivatives thereof, (c) An NP viral segment encoding an NP viral protein comprising, consisting of, or essentially comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 32, 51, 70, 89, and 108, or fragments, variants, or derivatives thereof. (d) NS1 viral proteins comprising, comprising, or essentially comprising amino acid sequences selected from the group consisting of SEQ ID NOs. 14, 33, 52, 71, 90, and 109, or their fragments, variants, or derivatives, and / or NS viral segments encoding NEP viral proteins comprising, comprising, or essentially comprising amino acid sequences selected from the group consisting of SEQ ID NOs. 15, 34, 53, 72, 91, and 110, or their fragments, variants, or derivatives, (e) An M1 viral protein comprising, consisting of, or essentially derived from, an amino acid sequence selected from the group consisting of SEQ ID NOs. 16, 35, 54, 73, 92, and 111, or fragments, variants, or derivatives thereof, and / or an M viral segment encoding an M2 viral protein comprising, consisting of, or essentially derived from, an amino acid sequence selected from the group consisting of SEQ ID NOs. 17, 36, 55, 74, 93, and 112, or fragments, variants, or derivatives thereof.

[0087] Preferably, the isolated influenza virus comprises six of the skeletal viral segments provided herein. In these examples, the isolated influenza virus comprises PB2, PB1, PA, M, NP, and NS viral segments, each independently comprising, consisting of, or essentially comprising nucleotide sequences selected from those shown in SEQ ID NOs: 1-6, 20-25, 39-44, 58-63, 77-82, and 96-101, or their fragments, variants, or derivatives. For this purpose, the isolated influenza virus is intended to contain any combination of six of these skeletal viral segments provided herein.

[0088] Furthermore, in related cases, isolated influenza viruses, (a) A PB2 viral segment comprising, consisting of, or essentially comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 20, 39, 58, 77, and 96, or fragments, variants, or derivatives thereof, (a) A PB1 viral segment comprising, consisting of, or essentially comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 21, 40, 59, 78, and 97, or fragments, variants, or derivatives thereof, (b) PA virus segments comprising, consisting of, or essentially comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3, 22, 41, 60, 79, and 98, or fragments, variants, or derivatives thereof, (c) NP viral segments comprising, consisting of, or essentially comprising nucleotide sequences selected from the group consisting of SEQ ID NOs: 4, 23, 42, 61, 80, and 99, or fragments, variants, or derivatives thereof, (d) NS virus segments comprising, consisting of, or essentially comprising nucleotide sequences selected from the group consisting of SEQ ID NOs. 5, 24, 43, 62, 81, and 100, or fragments, variants, or derivatives thereof, and (e) A M virus segment comprising, consisting of, or essentially comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs. 6, 25, 44, 63, 82, and 101, or fragments, variants, or derivatives thereof.

[0089] In some examples, the isolated influenza virus includes a viral segment encoding seven of the skeletal viral proteins provided herein. In other examples, the isolated influenza virus includes a viral segment encoding eight of the skeletal viral proteins provided herein. In such examples, the isolated influenza virus preferably includes a viral segment encoding seven or eight skeletal viral proteins (i.e., seven or eight of the PB2, PB1, PB1-F2, PA, M1, M2, NP, NS1, and NEP viral proteins) independently comprising, consisting of, or essentially comprising amino acid sequences selected from those shown in SEQ ID NOs. 9-17, 28-36, 47-55, 66-74, 85-93, and 104-112, or their fragments, variants, or derivatives. Herein too, it is assumed that the isolated influenza virus may express any combination of seven or eight of these skeletal viral proteins provided herein (i.e., seven or eight viral proteins selected from PB2, PB1, PB1-F2, PA, M1, M2, NP, NS1, and NEP viral proteins).

[0090] Preferably, the isolated influenza virus comprises the PB2, PA, M1, M2, NP, NS1, and NEP viral proteins provided herein, and optionally, viral segments encoding the PB1 and / or PB1-F2 viral proteins provided herein. Thus, in a particular example, the isolated influenza virus is (a) A PB2 viral segment encoding a PB2 viral protein comprising, consisting of, or essentially comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 28, 47, 66, 85, and 104, or fragments, variants, or derivatives thereof, (b) PA virus segments encoding a PA virus protein comprising, consisting of, or essentially comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 31, 50, 69, 88, and 107, or fragments, variants, or derivatives thereof, (c) An NP viral segment encoding an NP viral protein comprising, consisting of, or essentially comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 32, 51, 70, 89, and 108, or fragments, variants, or derivatives thereof. (d) NS1 viral proteins comprising, consisting of, or essentially comprising amino acid sequences selected from the group consisting of SEQ ID NOs. 14, 33, 52, 71, 90, and 109, or fragments, variants, or derivatives thereof, and / or NS viral segments encoding NEP viral proteins comprising, consisting of, or essentially comprising amino acid sequences selected from the group consisting of SEQ ID NOs. 15, 34, 53, 72, 91, and 110, or fragments, variants, or derivatives thereof, and (e) An M1 viral protein comprising, consisting of, or essentially derived from, an amino acid sequence selected from the group consisting of SEQ ID NOs. 16, 35, 54, 73, 92, and 111, or fragments, variants, or derivatives thereof, and / or an M viral segment encoding an M2 viral protein comprising, consisting of, or essentially derived from, an amino acid sequence selected from the group consisting of SEQ ID NOs. 17, 36, 55, 74, 93, and 112, or fragments, variants, or derivatives thereof.

[0091] In other examples, the isolated influenza virus comprises five of the skeletal viral segments provided herein. In such examples, the isolated influenza virus comprises five viral segments that independently contain, consist of, or are essentially composed of nucleotide sequences selected from those shown in SEQ ID NOs: 1-6, 20-25, 39-44, 58-63, 77-82, and 96-101, or their fragments, variants, or derivatives. Here again, it is assumed that the isolated influenza virus may contain any combination of five of these skeletal viral segments provided herein (i.e., five viral segments selected from the PB2, PB1, PA, M, NP, and NS viral segments).

[0092] Preferably, the isolated influenza virus comprises viral segments encoding the PB2, PA, M, NP, and NS viral segments provided herein, and optionally, the PB1 viral segment provided herein. Furthermore, in relevant examples, the isolated influenza virus is (a) A PB2 viral segment comprising, consisting of, or essentially comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 20, 39, 58, 77, and 96, or fragments, variants, or derivatives thereof, (b) PA virus segments comprising, consisting of, or essentially comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3, 22, 41, 60, 79, and 98, or fragments, variants, or derivatives thereof, (c) NP viral segments comprising, consisting of, or essentially comprising nucleotide sequences selected from the group consisting of SEQ ID NOs: 4, 23, 42, 61, 80, and 99, or fragments, variants, or derivatives thereof, (d) NS virus segments comprising, consisting of, or essentially comprising nucleotide sequences selected from the group consisting of SEQ ID NOs. 5, 24, 43, 62, 81, and 100, or fragments, variants, or derivatives thereof, and (e) A M virus segment comprising, consisting of, or essentially comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs. 6, 25, 44, 63, 82, and 101, or fragments, variants, or derivatives thereof.

[0093] In certain cases, isolated influenza viruses contain one or more PB2, PB1, PA, M, NP, and NS viral segments encoding one or more viral proteins that contain, consist of, or are essentially derived from amino acid sequences selected from those shown in SEQ ID NOs. 9–17, or their fragments, variants, or derivatives. More specifically, isolated influenza viruses may contain PB2, PB1, PA, M, NP, and NS viral segments encoding seven or more viral proteins that contain, consist of, or are essentially derived from amino acid sequences selected from those shown in SEQ ID NOs. 9–17, or their fragments, variants, or derivatives. In certain cases, isolated influenza viruses contain PB2, PA, M, NP, and NS viral segments encoding PB2, PA, M1, M2, NP, NS1, and NEP viral proteins, respectively, that contain, consist of, or are essentially derived from amino acid sequences shown in SEQ ID NOs. 9 and 12–17, or their fragments, variants, or derivatives. In such examples, the isolated influenza virus may optionally further contain a PB1 viral segment encoding the PB1 and / or PB1-F2 viral proteins, independently containing, consisting of, or essentially comprising the amino acid sequences shown in SEQ ID NOs. 10 and / or 11, or their fragments, variants, or derivatives. Preferably, the isolated influenza virus contains PB2, PB1, PA, M, NP, and NS viral segments encoding the PB2, PB1, PB1-F2, PA, M1, M2, NP, NS1, and NEP viral proteins, each independently containing, consisting of, or essentially comprising the amino acid sequences shown in SEQ ID NOs. 9-17, or their fragments, variants, or derivatives. With respect to the above examples, the isolated influenza viruses, such as reassembled influenza viruses, are preferably of the H1N1 or H3N2 subtype.

[0094] According to relevant examples, an isolated influenza virus contains one or more of the PB2, PB1, PA, M, NP, and NS viral segments that contain, consist of, or are essentially derived from the nucleotide sequences shown in SEQ ID NOs. 1-6, or their fragments, variants, or derivatives. More specifically, an isolated influenza virus may contain five or more of the PB2, PB1, PA, M, NP, and NS viral segments that contain, consist of, or are essentially derived from the nucleotide sequences shown in SEQ ID NOs. 1-6, or their fragments, variants, or derivatives. For example, an isolated influenza virus may contain the PB2, PA, M, NP, and NS viral segments that contain, consist of, or are essentially derived from the nucleotide sequences shown in SEQ ID NOs. 1 and 3-6, or their fragments, variants, or derivatives, respectively. In such examples, the isolated influenza virus may optionally further contain a PB1 viral segment that contains, consists of, or is essentially derived from the nucleotide sequence shown in SEQ ID NO. 2 or its fragments, variants, or derivatives. Preferably, the isolated influenza virus comprises PB2, PB1, PA, M, NP, and NS viral segments, each containing, consisting of, or essentially comprising the nucleotide sequences shown in SEQ ID NOs: 1-6, or their fragments, variants, or derivatives. In the above example, the isolated influenza virus, such as a reassembled influenza virus, is preferably of the H1N1 or H3N2 subtype.

[0095] In certain cases, isolated influenza viruses contain one or more PB2, PB1, PA, M, NP, and NS viral segments encoding one or more viral proteins that contain, consist of, or are essentially derived from amino acid sequences selected from those shown in SEQ ID NOs. 28-36, or their fragments, variants, or derivatives. More specifically, isolated influenza viruses may contain PB2, PB1, PA, M, NP, and NS viral segments encoding seven or more viral proteins that contain, consist of, or are essentially derived from amino acid sequences selected from those shown in SEQ ID NOs. 28-36, or their fragments, variants, or derivatives. In certain cases, isolated influenza viruses contain PB2, PA, M, NP, and NS viral segments encoding PB2, PA, M1, M2, NP, NS1, and NEP viral proteins, respectively, that contain, consist of, or are essentially derived from amino acid sequences shown in SEQ ID NOs. 28 and 31-36, or their fragments, variants, or derivatives. In such examples, the isolated influenza virus may optionally further contain PB1 viral segments encoding PB1 and / or PB1-F2 viral proteins, each comprising, consisting of, or essentially comprising the amino acid sequences shown in SEQ ID NOs. 29 and / or 30, or their fragments, variants, or derivatives, respectively. Preferably, the isolated influenza virus contains PB2, PB1, PA, M, NP, and NS viral segments encoding PB2, PB1, PB1-F2, PA, M1, M2, NP, NS1, and NEP viral proteins, each independently comprising, consisting of, or essentially comprising the amino acid sequences shown in SEQ ID NOs. 28-36, or their fragments, variants, or derivatives, respectively. With respect to the above examples, the isolated influenza viruses, such as reassembled influenza viruses, are preferably of the H1N1 subtype.

[0096] In other examples, isolated influenza viruses may contain one or more of the PB2, PB1, PA, M, NP, and NS viral segments that contain, consist of, or are essentially derived from the nucleotide sequences shown in SEQ ID NOs. 20-25, or their fragments, variants, or derivatives. More specifically, isolated influenza viruses may contain five or more of the PB2, PB1, PA, M, NP, and NS viral segments that contain, consist of, or are essentially derived from the nucleotide sequences shown in SEQ ID NOs. 20-25, or their fragments, variants, or derivatives. For example, isolated influenza viruses may contain the PB2, PA, M, NP, and NS viral segments that contain, consist of, or are essentially derived from the nucleotide sequences shown in SEQ ID NOs. 20 and 22-25, or their fragments, variants, or derivatives, respectively. In such examples, isolated influenza viruses may optionally further contain a PB1 viral segment that contains, consists of, or is essentially derived from the nucleotide sequence shown in SEQ ID NO. 21 or its fragments, variants, or derivatives. Preferably, the isolated influenza virus comprises PB2, PB1, PA, M, NP, and NS viral segments, each containing, consisting of, or essentially derived from, the nucleotide sequences shown in SEQ ID NOs. 20-25, or fragments, variants, or derivatives thereof. In the above example, the isolated influenza virus, such as a reassembled influenza virus, is preferably of the H1N1 subtype.

[0097] In various examples, isolated influenza viruses contain one or more PB2, PB1, PA, M, NP, and NS viral segments encoding one or more viral proteins that contain, consist of, or are essentially derived from amino acid sequences selected from those shown in SEQ ID NOs. 47–55, or their fragments, variants, or derivatives. More specifically, isolated influenza viruses may contain PB2, PB1, PA, M, NP, and NS viral segments encoding seven or more viral proteins that contain, consist of, or are essentially derived from amino acid sequences selected from those shown in SEQ ID NOs. 47–55, or their fragments, variants, or derivatives. In certain examples, isolated influenza viruses contain PB2, PA, M, NP, and NS viral segments encoding PB2, PA, M1, M2, NP, NS1, and NEP viral proteins, respectively, that contain, consist of, or are essentially derived from amino acid sequences shown in SEQ ID NOs. 47 and 50–55, or their fragments, variants, or derivatives. In such examples, the isolated influenza virus may optionally further include a PB1 viral segment encoding the PB1 and / or PB1-F2 viral proteins, comprising, consisting of, or essentially comprising the amino acid sequences shown in SEQ ID NOs. 48 and / or 49, or their fragments, variants, or derivatives. Preferably, the isolated influenza virus includes PB2, PB1, PA, M, NP, and NS viral segments encoding the PB2, PB1, PB1-F2, PA, M1, M2, NP, NS1, and NEP viral proteins, each independently comprising, consisting of, or essentially comprising the amino acid sequences shown in SEQ ID NOs. 47-55, or their fragments, variants, or derivatives. With respect to the above examples, the isolated influenza viruses, such as reassembled influenza viruses, are preferably of the H1N1 or H3N2 subtype.

[0098] According to relevant examples, an isolated influenza virus contains one or more of the PB2, PB1, PA, M, NP, and NS viral segments that contain, consist of, or are essentially derived from the nucleotide sequences shown in SEQ ID NOs. 39-44, or their fragments, variants, or derivatives. More specifically, an isolated influenza virus may contain five or more of the PB2, PB1, PA, M, NP, and NS viral segments that contain, consist of, or are essentially derived from the nucleotide sequences shown in SEQ ID NOs. 39-44, or their fragments, variants, or derivatives. For example, an isolated influenza virus may contain the PB2, PA, M, NP, and NS viral segments that contain, consist of, or are essentially derived from the nucleotide sequences shown in SEQ ID NOs. 39 and 41-44, or their fragments, variants, or derivatives, respectively. In such examples, the isolated influenza virus may optionally further contain a PB1 viral segment that contains, consists of, or is essentially derived from the nucleotide sequence shown in SEQ ID NOs. 40 or its fragments, variants, or derivatives. Preferably, the isolated influenza virus comprises PB2, PB1, PA, M, NP, and NS viral segments, each containing, consisting of, or essentially derived from, the nucleotide sequences shown in SEQ ID NOs. 39-44, or fragments, variants, or derivatives thereof. In the above example, the isolated influenza virus, such as a reassembled influenza virus, is preferably of the H1N1 or H3N2 subtype.

[0099] In certain cases, isolated influenza viruses contain one or more PB2, PB1, PA, M, NP, and NS viral segments encoding one or more viral proteins that contain, consist of, or are essentially derived from amino acid sequences selected from those shown in SEQ ID NOs. 66–74, or their fragments, variants, or derivatives. More specifically, isolated influenza viruses may contain PB2, PB1, PA, M, NP, and NS viral segments encoding seven or more viral proteins that contain, consist of, or are essentially derived from amino acid sequences selected from those shown in SEQ ID NOs. 66–74, or their fragments, variants, or derivatives. In certain cases, isolated influenza viruses contain PB2, PA, M, NP, and NS viral segments encoding PB2, PA, M1, M2, NP, NS1, and NEP viral proteins, respectively, that contain, consist of, or are essentially derived from amino acid sequences shown in SEQ ID NOs. 66 and 69–74, or their fragments, variants, or derivatives. In such examples, the isolated influenza virus may optionally further include a PB1 viral segment encoding the PB1 and / or PB1-F2 viral proteins, comprising, consisting of, or essentially comprising the amino acid sequences shown in SEQ ID NOs. 67 and / or 68, or their fragments, variants, or derivatives. Preferably, the isolated influenza virus includes PB2, PB1, PA, M, NP, and NS viral segments encoding the PB2, PB1, PB1-F2, PA, M1, M2, NP, NS1, and NEP viral proteins, each independently comprising, consisting of, or essentially comprising the amino acid sequences shown in SEQ ID NOs. 66-74, or their fragments, variants, or derivatives. With respect to the above examples, the isolated influenza viruses, such as reassembled influenza viruses, are preferably of the H1N1 subtype.

[0100] In various examples, isolated influenza viruses may contain one or more of the PB2, PB1, PA, M, NP, and NS viral segments that contain, consist of, or are essentially derived from the nucleotide sequences shown in SEQ ID NOs. 58-63, or their fragments, variants, or derivatives. More specifically, isolated influenza viruses may contain five or more of the PB2, PB1, PA, M, NP, and NS viral segments that contain, consist of, or are essentially derived from the nucleotide sequences shown in SEQ ID NOs. 58-63, or their fragments, variants, or derivatives. For example, isolated influenza viruses may contain the PB2, PA, M, NP, and NS viral segments that contain, consist of, or are essentially derived from the nucleotide sequences shown in SEQ ID NOs. 58 and 60-63, or their fragments, variants, or derivatives, respectively. In such examples, isolated influenza viruses may optionally further contain a PB1 viral segment that contains, consists of, or is essentially derived from the nucleotide sequence shown in SEQ ID NO. 59 or its fragments, variants, or derivatives. Preferably, the isolated influenza virus comprises PB2, PB1, PA, M, NP, and NS viral segments, each containing, consisting of, or essentially comprising the nucleotide sequences shown in SEQ ID NOs. 58-63, or fragments, variants, or derivatives thereof. In the above example, the isolated influenza virus, such as a reassembled influenza virus, is preferably of the H1N1 subtype.

[0101] In certain cases, isolated influenza viruses contain one or more PB2, PB1, PA, M, NP, and NS viral segments encoding one or more viral proteins that contain, consist of, or are essentially derived from amino acid sequences selected from those shown in SEQ ID NOs. 85–93, or their fragments, variants, or derivatives. More specifically, isolated influenza viruses may contain PB2, PB1, PA, M, NP, and NS viral segments encoding seven or more viral proteins that contain, consist of, or are essentially derived from amino acid sequences selected from those shown in SEQ ID NOs. 85–93, or their fragments, variants, or derivatives. In certain cases, isolated influenza viruses contain PB2, PA, M, NP, and NS viral segments encoding PB2, PA, M1, M2, NP, NS1, and NEP viral proteins, respectively, that contain, consist of, or are essentially derived from amino acid sequences shown in SEQ ID NOs. 85 and 88–93, or their fragments, variants, or derivatives. In such examples, the isolated influenza virus may optionally further include a PB1 viral segment encoding the PB1 and / or PB1-F2 viral proteins, comprising, consisting of, or essentially comprising the amino acid sequences shown in SEQ ID NOs. 86 and / or 87, or their fragments, variants, or derivatives. Preferably, the isolated influenza virus includes PB2, PB1, PA, M, NP, and NS viral segments encoding the PB2, PB1, PB1-F2, PA, M1, M2, NP, NS1, and NEP viral proteins, each independently comprising, consisting of, or essentially comprising the amino acid sequences shown in SEQ ID NOs. 85-93, or their fragments, variants, or derivatives. With respect to the above examples, the isolated influenza viruses, such as reassembled influenza viruses, are preferably of the H1N1 or H3N2 subtype.

[0102] In various examples, isolated influenza viruses may contain one or more of the PB2, PB1, PA, M, NP, and NS viral segments that contain, consist of, or are essentially derived from the nucleotide sequences shown in SEQ ID NOs. 77-82, or their fragments, variants, or derivatives. More specifically, isolated influenza viruses may contain five or more of the PB2, PB1, PA, M, NP, and NS viral segments that contain, consist of, or are essentially derived from the nucleotide sequences shown in SEQ ID NOs. 77-82, or their fragments, variants, or derivatives. For example, isolated influenza viruses may contain PB2, PA, M, NP, and NS viral segments that contain, consist of, or are essentially derived from the nucleotide sequences shown in SEQ ID NOs. 77 and 79-82, or their fragments, variants, or derivatives, respectively. In such examples, isolated influenza viruses may optionally further contain a PB1 viral segment that contains, consists of, or is essentially derived from the nucleotide sequence shown in SEQ ID NO. 78 or its fragments, variants, or derivatives. Preferably, the isolated influenza virus comprises PB2, PB1, PA, M, NP, and NS viral segments, each containing, consisting of, or essentially derived from the nucleotide sequences shown in SEQ ID NOs. 77-82, or fragments, variants, or derivatives thereof. In the above example, the isolated influenza virus, such as a reassembled influenza virus, is preferably of the H1N1 or H3N2 subtype.

[0103] In certain cases, isolated influenza viruses contain one or more PB2, PB1, PA, M, NP, and NS viral segments encoding one or more viral proteins that contain, consist of, or are essentially derived from amino acid sequences selected from those shown in SEQ ID NOs. 104-112, or their fragments, variants, or derivatives. More specifically, isolated influenza viruses may contain PB2, PB1, PA, M, NP, and NS viral segments encoding seven or more viral proteins that contain, consist of, or are essentially derived from amino acid sequences selected from those shown in SEQ ID NOs. 104-112, or their fragments, variants, or derivatives. In certain cases, isolated influenza viruses contain PB2, PA, M, NP, and NS viral segments encoding PB2, PA, M1, M2, NP, NS1, and NEP viral proteins, respectively, that contain, consist of, or are essentially derived from amino acid sequences shown in SEQ ID NOs. 104 and 107-112, or their fragments, variants, or derivatives. In such examples, the isolated influenza virus may optionally further include a PB1 viral segment encoding the PB1 and / or PB1-F2 viral proteins, comprising, consisting of, or essentially comprising the amino acid sequences shown in SEQ ID NOs. 105 and / or 106, or their fragments, variants, or derivatives. Preferably, the isolated influenza virus includes PB2, PB1, PA, M, NP, and NS viral segments encoding the PB2, PB1, PB1-F2, PA, M1, M2, NP, NS1, and NEP viral proteins, each independently comprising, consisting of, or essentially comprising the amino acid sequences shown in SEQ ID NOs. 104-112, or their fragments, variants, or derivatives. With respect to the above examples, the isolated influenza viruses, such as reassembled influenza viruses, are preferably of the H1N1 or H3N2 subtype.

[0104] In various examples, isolated influenza viruses may contain one or more of the PB2, PB1, PA, M, NP, and NS viral segments that contain, consist of, or are essentially derived from the nucleotide sequences shown in SEQ ID NOs. 96-101, or their fragments, variants, or derivatives. More specifically, isolated influenza viruses may contain five or more of the PB2, PB1, PA, M, NP, and NS viral segments that contain, consist of, or are essentially derived from the nucleotide sequences shown in SEQ ID NOs. 96-101, or their fragments, variants, or derivatives. For example, isolated influenza viruses may contain the PB2, PA, M, NP, and NS viral segments that contain, consist of, or are essentially derived from the nucleotide sequences shown in SEQ ID NOs. 96 and 98-101, respectively. In such examples, isolated influenza viruses may optionally further contain a PB1 viral segment that contains, consists of, or is essentially derived from the nucleotide sequence shown in SEQ ID NOs. 97 or its fragments, variants, or derivatives. Preferably, the isolated influenza virus comprises PB2, PB1, PA, M, NP, and NS viral segments, each containing, consisting of, or essentially derived from, the nucleotide sequences shown in SEQ ID NOs. 96-101, or fragments, variants, or derivatives thereof. In the above example, the isolated influenza virus, such as a reassembled influenza virus, is preferably of the H1N1 or H3N2 subtype.

[0105] Suitablely, isolated influenza viruses provided herein can grow or replicate in cells, more particularly mammalian cells such as MDCK cells. For this purpose, isolated influenza viruses can preferably form virions when cultured in cells. More particularly, isolated influenza viruses containing the skeletal viral segments described herein can preferably have improved growth or replication in cells, such as when cultured under standard culture conditions, including those described herein. In this regard, when expressing the unmodified, wild-type, or reference form of one or more skeletal viral segments provided herein, the influenza virus preferably has limited or reduced ability to grow or replicate in cells. More particularly, with respect to reassembled influenza viruses, isolated reassembled influenza viruses derived from a candidate vaccine virus strain containing one or more of the skeletal viral segments described herein are preferably able to grow or replicate in cells (e.g., MDCK cells) with improved growth or replication in cells (e.g., MDCK cells) compared to wild-type or reference influenza viruses (e.g., wild-type versions of the corresponding candidate vaccine virus strain) that do not contain one or more skeletal viral segments described herein (e.g., wild-type versions of the skeletal viral segments). However, isolated influenza viruses and wild-type influenza viruses may contain or express the same HA and / or NA viral segments. In other examples, the isolated influenza viruses provided herein may have improved growth or replication in cells, more particularly in mammalian cells (e.g., MDCK cells), compared to donor influenza viruses known in the art and described herein (e.g., A / Puerto Rico / 8 / 1934(PR8), A / Texas / 1 / 1977, A / New York / 55 / 2004, A / Ann Arbor / 6 / 60, A / Leningrad / 134 / 17 / 57, B / Ann Arbor / 1 / 66, B / Florida / 4 / 2006, B / Panama / 45 / 1990, and B / Lee / 1940).More specifically, the isolated influenza viruses provided herein can preferably improve growth or replication in cells, more particularly in MDCK cells, compared to the A / Puerto Rico / 8 / 1934 strain, including its cell-adapted form (e.g., PR8x). Therefore, one or more skeletal viral segments described herein preferably provide isolated influenza viruses containing such viral segments with improved ability to grow and replicate in cells such as MDCK cells.

[0106] The level of growth or replication of isolated influenza viruses can be assessed by any means in the art, for example, indirectly by viral yield or viral protein yield (e.g., viral titer, HA, and / or NA protein yield at the end of infection), or directly by their rescue ability, growth ability, and / or replication ability when grown in a cell culture, such as mammalian cells like MDCK cells. Therefore, phrases such as "increase the growth ability of isolated influenza viruses" or "enhance the growth ability of isolated influenza viruses" may mean that when a virus having one or more of the skeletal viral segments according to this disclosure grows in a cell culture (such as mammalian cells like MDCK cells), it has improved growth ability and / or replication ability compared to a virus having the wild-type version of the skeletal viral segment (e.g., wild-type viruses that have not been modified to express one or more of the skeletal viral segments of SEQ ID NOs: 1-6, 20-25, 39-44, 58-63, 77-82, and 96-101, or their fragments, variants, or derivatives). Preferably, the isolated influenza virus in question comprises one or both of the HA and NA viral segments (or their chimeric versions) of a reference or wild-type influenza virus (e.g., a vaccine virus strain).

[0107] Thanks to this increased or enhanced proliferation capacity or other factors, isolated influenza viruses may exhibit enhanced yields of viral proteins (e.g., one or more of HA, NA, PA, PB1, PB1-F2, PB2, NP, M1, M2, NEP, and NS1) when they grow in cells. The yield of influenza viral proteins such as HA or NA can be measured by any means of protein quantification known in the art, such as gel electrophoresis (e.g., Western blotting), ELISA, or chromatography, e.g., HPLC (high-performance liquid chromatography), HPLC-UV, or mass spectrometry, e.g., LC-MS (liquid chromatography-mass spectrometry). In certain cases, isolated influenza viruses exhibit enhanced yields of HA protein when they grow in cells.

[0108] As used herein, terms such as “higher,” “enhanced,” “increased,” and “greater” refer to an increased level of growth or yield of isolated influenza virus and / or an increased level of yield of viral proteins produced by isolated influenza virus, such as compared to a control or reference level or amount thereof. The level of growth or yield of isolated influenza virus or viral proteins may be relative or absolute (i.e., relatively or absolutely higher, increased, or greater). In specific examples, enhanced or increased growth capacity or yield of isolated influenza virus (or viral proteins produced thereby) refers to an increased level of growth, replication, or yield compared to a control, wild-type, or reference influenza virus (e.g., an influenza virus strain containing wild-type or unmodified versions of the PA, PB1, PB2, NP, M, and NS viral segments typical of that virus strain, rather than those provided in SEQ ID NOs: 1-6, 20-25, 39-44, 58-63, 77-82, and 96-101).

[0109] Viruses and / or viral proteins are intended to be present in cell culture supernatants. Therefore, the yield of isolated influenza viruses and / or their viral proteins can be evaluated after their collection, isolation, purification, or separation from cells and / or cell culture media. To do this, for example, cells or cell residues can be separated from the culture media by methods known to those skilled in the art, such as centrifugation, separators, or filters. The influenza viruses, including their viral proteins, present in the test culture media can then be isolated, concentrated, or collected by methods known to those skilled in the art, such as those described herein (e.g., gradient centrifugation, filtration, precipitation, etc.). In certain examples, influenza viruses and / or viral proteins produced therefrom are isolated or collected from the culture media at least partially by gradient ultracentrifugation. Therefore, the yield level can be evaluated in relation to the isolated influenza viruses or their viral proteins, at least partially purified or separated from the test culture media by gradient ultracentrifugation, etc.

[0110] To evaluate the yield of isolated influenza virus and / or its viral protein, the obtained or measured yield level may be compared to one or more threshold levels. The nature and numerical value of the yield threshold levels typically vary based on the influenza virus and / or influenza viral protein in question, and / or the method or means chosen to determine the yield level of that virus or viral protein. In various examples, the improved growth and / or replication capacity of isolated influenza virus can be determined by achieving a threshold level of viral yield or viral protein yield (e.g., HA protein yield) when the isolated influenza virus grows on cells (e.g., MDCK cells). Preferably, the threshold level of viral yield or viral protein yield is such that it allows for the production of an amount of virus and / or viral protein suitable for use in the preparation of vaccines or immunogenic compositions on a commercial scale.

[0111] As described above, it is assumed that the assessment of the yield of isolated influenza virus may be at least partially based on specific influenza virus proteins, such as the HA protein. Referring to HA, a preferred threshold level for the yield of this virus protein may be at least about 10 mg / L (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 mg / L or any range therein) in the culture medium at the end of infection or a preferred incubation period. Therefore, at the end of the infection or incubation period (e.g., approximately 24, 36, 48, 60, 72, 84, 96, 108, 120, 132, 144, 156, 168, 180, 192, 204, 216, 228, 240 hours, or any range within those), at least approximately 10 mg / L (e.g., approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) in the test culture medium. Isolated influenza viruses that produce HA yields of 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 mg / L, or any range therein, at least about 15 mg / L, at least about 20 mg / L, or at least about 25 mg / L may be considered to exhibit improved or enhanced growth or replication ability.

[0112] The degree of improvement in the yield and / or proliferation ability of isolated influenza viruses may vary. For example, the degree of improvement may be greater than about 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, or at least about 500% (or any range within that range) of the levels observed in control, reference, or wild-type influenza viruses. For example, the degree of improvement in the proliferation or replication ability or yield of isolated influenza viruses may be greater than about 50% compared to viruses that do not have one or more of the skeletal viral segments provided herein, such as when cultured under standard culture conditions known in the art, including those described herein.

[0113] Based on the foregoing, in another form, the present disclosure provides a method for improving the proliferation and / or yield of influenza viruses in cells, the method comprising the step of modifying an influenza virus such that the method comprises one or more PA, PB1, PB2, NP, M, and NS viral segments provided herein (e.g., SEQ ID NOs: 1-6, 20-25, 39-44, 58-63, 77-82, and 96-101, or fragments, variants, or derivatives thereof) and / or one or more skeletal viral proteins described herein (e.g., one or more viral proteins comprising, comprising, or essentially being therea, an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-17, 28-36, 47-55, 66-74, 85-93, and 104-112, or fragments, variants, or derivatives thereof). Such influenza viruses may be prepared or modified by the methods described herein, including classical recombination, reverse genetics, or a combination thereof.

[0114] "Protein" means amino acid polymer. The amino acids may be natural or unnatural amino acids, D- or L-amino acids, as is well understood in the art.

[0115] The term "protein" typically includes and encompasses "peptides," which are used to describe 50 or fewer amino acids (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 or fewer amino acids and any range therein), and "polypeptides," which are typically used to describe proteins that have more than 50 amino acids.

[0116] Fragments, variants, and derivatives of skeletal viral proteins (i.e., PB2, PB1, PB1-F2, PA, NP, NS1, NEP, M1, and M2) encoded by the skeletal viral segments described herein are assumed to exist in this disclosure.

[0117] As used herein, a protein, polypeptide, or peptide "variant" shares a definable amino acid sequence relationship with a reference amino acid sequence. The reference amino acid sequence may be, for example, one of the amino acid sequences of SEQ ID NOs: 9-19, 28-38, 47-57, 66-76, 85-95, and 104-114. A "variant" protein, polypeptide, or peptide may have one or more amino acids of the reference or wild-type amino acid sequence that are deleted or substituted by different amino acids. Such a protein variant may include, for example, amino acid residues and / or amino acid sequences of naturally occurring variants and orthologues (e.g., from different influenza strains or different host animals) of a viral protein, including its consensus sequence. Thus, a variant backbone viral HA protein may have at least about 2, 3, 4, 5, 6, 7, or more different residues at other positions compared to its respective wild-type or reference backbone viral protein. As will be understood by those skilled in the art, the number of additional positions that may have amino acid substitutions will depend on the wild-type viral protein or coding nucleic acid used to generate the variant. For this purpose, the modified viral proteins provided herein may be derived from either known skeletal protein sequences or encoding nucleotide sequences from influenza isolates known in the art. For example, the National Center for Biotechnology Information (NCBI) maintains a database of known skeletal viral protein sequences and encoding nucleic acids (https: / / www.ncbi.nlm.nih.gov / genomes / FLU / Database / ).

[0118] It is further intended that some amino acid residues of viral proteins can be substituted or deleted without altering the activity of the variant protein (i.e., conservative substitutions). Typical conservative substitutions include the substitution of aliphatic amino acids Ala, Val, Leu, and lle with each other, the exchange of hydroxyl residues Ser and Thr, the exchange of acidic residues Asp and Glu, the substitution of amide residues Asn and Gln, the exchange of basic residues Lys and Arg, and the substitution of aromatic residues Phe and Tyr. Guidance on which amino acid changes are likely to be phenotypic silent can be found, for example, in Bowie et al., Science 247:1306-1310 (1990). Accordingly, one or more residues of the viral backbone proteins described herein (e.g., PB2, PB1, PB1-F2, PA, NP, NS1, NEP, M1, or M2 proteins), such as those defined by SEQ ID NOs. 9-17, 28-36, 47-55, 66-74, 85-93, and 104-112, can be conservatively modified (e.g., by amino acid substitution or deletion) in such a manner that substantially retains the functionality and / or immunogenicity of the backbone viral protein. Furthermore, variant backbone proteins may include additional amino acid mutations known in the art to further increase or enhance, for example, the proliferation capacity or yield of isolated influenza viruses. Such additional amino acid mutations are described in WO2015009743, WO2015196150, WO2017007839, WO2017143236, and WO2020223699, which are incorporated herein in their entirety.

[0119] The protein or peptide variant is one of the reference amino acid sequences shown in SEQ ID NOs: 9-19, 28-38, 47-57, 66-76, 85-95, and 104-114, or more particularly, one of the reference amino acid sequences shown in SEQ ID NOs: 9-17, 28-36, 47-55, 66-74, 85-93, and 104-112, and is at least about 70% or 75%, more particularly at least about 80% or 85%, or even more particularly at least about 90%, 90.5%, 91%, 91.5%, 92%, 9 It is assumed that the sequences share sequence identities of 2.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%.

[0120] Terms commonly used herein to describe the sequence relationship between each protein and nucleic acid include “comparison window,” “sequence identity,” “percentage of sequence identity,” and “substantial identity.” Since each nucleic acid / protein may contain (1) only one or more portions of the complete nucleic acid / protein sequence shared by the nucleic acid / protein, and (2) one or more portions that differ between the nucleic acids / proteins, sequence comparison is typically performed by comparing sequences across a “comparison window” to identify and compare local regions of sequence similarity. A “comparison window” refers, for example, to a conceptual segment of 6, 9, 12, or 20 consecutive residues compared to a reference sequence. For optimal alignment of each sequence, the comparison window may contain approximately 20% or less of additions or deletions (i.e., gaps) compared to the reference sequence. The optimal alignment of sequences for aligning the comparison window may be achieved by a computerized implementation of the algorithm (Geneworks program by Intelligenetics; incorporated herein by reference, GAP, BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by an examination and optimal alignment (i.e., producing the highest homology percentage across the comparison window) generated by any of the various selected methods. Alternatively, the BLAST family of programs, such as those disclosed in Altschul et al., 1991, Nucl. Acids Res. 25 3389, which is incorporated herein by reference, may also be consulted. A detailed discussion of sequence analysis can be found in Unit 19.3 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al. (John Wiley & Sons Inc NY, 1995-1999).

[0121] The term “sequence identity” is used herein in its broadest sense to include the number of exact nucleotide or amino acid matches, taking into account the degree to which sequences are identical across a comparison window, considering proper alignment using a standard algorithm. Thus, “percentage of sequence identity” is calculated by comparing two optimally aligned sequences across a comparison window, determining the number of positions in which identical nucleic acid bases (e.g., A, T, C, G, U) or amino acid residues occur in both sequences, obtaining the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity. For example, “sequence identity” may be understood to mean the “percentage of matches” calculated by the DNASIS computer program (version 2.5 for Windows; available from Hitachi Software Engineering Co., Ltd., South San Francisco, California, USA).

[0122] As used herein, “fragment” refers to a segment, domain, portion, or region of a protein or peptide (such as those shown in SEQ ID NOs: 9–17, 28–36, 47–55, 66–74, 85–93, and 104–112) that constitutes less than 100% of the amino acid sequence of the protein or peptide. It will be understood that a fragment may be a single fragment, or may be repeated alone or in conjunction with other fragments. Generally, fragments may contain, essentially be, or be of approximately 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 710, 720, 730, 740, 750, or up to 755 consecutive amino acids (such as SEQ ID NOs. 9-17, 28-36, 47-55, 66-74, 85-93, and 104-112). Preferably, the fragment is a functional fragment that retains at least partially the function of the corresponding full-length peptide or protein.

[0123] As used herein, “derivative” refers to a molecule such as a protein, fragment, or variant that has been modified, for example, by conjugation or complexation with other chemical moieties, post-translational modification (e.g., phosphorylation, acetylation, etc.), glycosylation modification (e.g., addition, removal, or alteration of glycosylation), lipidization, and / or inclusion of additional amino acid sequences as understood in the art.

[0124] In certain examples, a derivative or variant protein or peptide may contain one or more amino acid residues at its N-terminus and / or C-terminus. Additional amino acid sequences may also include fusion partner amino acid sequences that generate a fusion protein. Other derivatives disclosed herein include, but are not limited to, modifications of side chains, incorporation of unnatural amino acids and / or their derivatives in peptides, or protein synthesis, as well as the use of crosslinking agents and other methods for imposing conformational constraints on the isolated proteins of this disclosure. Those skilled in the art will refer to Chapter 15 of *CURRENT PROTOCOLS IN PROTEIN SCIENCE*, Eds. Coligan et al. (John Wiley & Sons NY 1995–2008) for a broader methodology relating to the chemical modification of proteins.

[0125] This specification also intends to include fragments, variants, and derivatives of nucleic acid molecules, including the skeletal viral segments described herein. A variant is any nucleotide sequence encoding the viral protein of this disclosure (e.g., SEQ ID NOs: 1-8, 20-27, 39-46, 58-65, 77-84, or 96-103), or more particularly, the skeletal viral protein of this disclosure (e.g., SEQ ID NOs: 1-6, 20-25, 39-44, 58-63, 77-82, or 96-101) and at least about 70%, at least about 75%, preferably at least about 80%, at least about 85%, more preferably at least about 90%, 90.5%, 91%, 91.5%, 93% The nucleotide sequences may contain nucleotide sequences with nucleotide sequence identity of %, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%. Nucleic acid derivatives may include chemically modified nucleic acids, modified nucleotide bonds, nucleic acid analogs, artificial nucleic acids, and combinations thereof, as are known in the art.

[0126] The isolated nucleic acid fragments may contain, or consist of, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95–99% of the consecutive nucleotides present in any nucleotide sequence encoding a viral protein, including the viral backbone protein of the Disclosure, so as to encode at least a portion of the viral protein. Generally, the fragments encode parts of the viral proteins described herein (e.g., SEQ ID NOs: 1-8, 20-27, 39-46, 58-65, 77-84, or 96-103, or complementary nucleotide sequences), specifically 150, 165, 180, 195, 210, 225, 240, 255, 270, 285, 300, 315, 330, 345, 360, 375, 390, 405, 420, 435, 450, 465, 480, 495, 510, 525, 540, 555, 570, 585, 60 It may contain, essentially be, or be able to contain, a sequence of up to 0, 615, 630, 645, 660, 675, 690, 705, 720, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, and 2250 consecutive nucleic acids.

[0127] In certain cases, the isolated nucleic acids described herein may be modified to include, as known in the art, the 5' non-coding region of the influenza virus (e.g., AGC[A / G]AAAGCAGG (SEQ ID NO: 115)) (wherein [A / G] indicates a nucleotide change of A or G at this position) and / or the 3' non-coding region (e.g., CCTTGTTTCTACT (SEQ ID NO: 116)), or alternatively, to omit it.

[0128] This disclosure also provides nucleic acids, such as skeletal viral segments, that are modified, for example, by utilizing codon sequence redundancy. In more specific examples, the use of codons may be modified to optimize the expression of nucleic acids in a particular organism or cell type.

[0129] Variant backbone viral segments and viral proteins can be produced by any means known in the art, including but not limited to chemical synthesis, recombinant DNA techniques including site-directed mutagenesis, or by replacing a portion of the coding sequence with a portion containing characteristic residues, and proteolytic cleavage to produce peptide fragments.

[0130] Chemical synthesis includes solid-phase and solution-phase synthesis. Such methods are well known in the art; see examples of chemical synthesis techniques provided in Chapter 9 of Synthetic Vaccines Ed., Nicholson (Blackwell Scientific Publications) and Chapter 15 of Current Protocols in Protein Science Eds, Coligan et al. (John Wiley & Sons, Inc. NY USA 1995-2008). In this regard, see also International Publications 99 / 02550 and 97 / 45444.

[0131] Recombinant nucleic acids and proteins can be readily prepared by those skilled in the art using standard protocols such as those described in Sambrook et al, Molecular Cloning. A Laboratory Manual (Cold Spring Harbor Press, 1989), particularly sections 16 and 17; Current Protocols in Molecular Biology, Eds. Ausubel et al, (John Wiley & Sons, Inc. NY USA 1995-2008), particularly chapters 10 and 16; and Current Protocols in Protein Science, Eds. Coligan et al, (John Wiley & Sons, Inc. NY USA 1995-2008), particularly chapters 1, 5, and 6. Typically, recombinant protein preparations involve the expression of a nucleic acid encoding a protein in a suitable host cell. Modified proteins can be obtained, for example, by mutating one or more genes (i.e., viral gene segments) encoding the protein of interest by site-directed or random mutagenesis. Such mutations may include point mutations, deletion mutations, and insertion mutations. For example, one or more point mutations (e.g., substitution of one or more amino acids with one or more different amino acids) may be used to construct the modified proteins described herein.

[0132] Nucleic acid encoding This disclosure also provides isolated nucleic acids encoding skeletal viral proteins described herein.

[0133] As used herein, the term “nucleic acid” refers to single-stranded or double-stranded DNA and RNA. DNA includes genomic DNA and cDNA. RNA includes mRNA, RNA, RNAi, siRNA, cRNA, and autocatalytic RNA. Nucleic acids can also be DNA-RNA hybrids. Nucleic acids typically contain nucleotide sequences, which include nucleotides containing A, G, C, T, or U bases. However, nucleotide sequences may also contain other bases, such as modified purines (e.g., inosine, methylinosine, and methyladenosine) and modified pyrimidines (e.g., thiouridine and methylcytosine).

[0134] In certain examples, the isolated nucleic acid is or includes a viral segment encoding a viral protein (i.e., an influenza RNA segment), including a skeletal viral protein provided herein. Preferably, the viral segment includes, consists of, or is essentially composed of, the nucleotide sequence shown in any one of SEQ ID NOs: 1-8, 20-27, 39-46, 58-65, 77-84, and 96-103, or fragments, derivatives, or variants thereof. More particularly, the viral segment may include, consist of, or is essentially composed of, the nucleotide sequence shown in any one of SEQ ID NOs: 1-6, 20-25, 39-44, 58-63, 77-82, and 96-101, or nucleotide sequences complementary thereto, or fragments, derivatives, or variants thereof. In some examples, the isolated nucleic acid is or includes a nucleotide sequence complementary to a viral segment encoding a viral protein, more particularly a skeletal viral protein, provided herein. Accordingly, this disclosure further intends that the isolated nucleic acid is preferably a nucleotide sequence complementary to any one of SEQ ID NOs: 1-8, 20-27, 39-46, 58-65, 77-84, and 96-103, or a fragment, derivative, or variant thereof, or comprises such sequence. In alternative examples, the isolated nucleic acid is a DNA or cDNA sequence encoding a viral segment (i.e., viral RNA) encoding a viral protein, more particularly a skeletal viral protein, provided herein, or comprises such sequence. In various examples, the isolated nucleic acid is a viral mRNA sequence encoding a viral protein, more particularly a skeletal viral protein, provided herein, or comprises such sequence.

[0135] As used herein, “polynucleotide” generally refers to a nucleic acid having 80 or more consecutive nucleotides, while “oligonucleotide” generally refers to an oligonucleotide having fewer than 80 consecutive nucleotides. “Primer” is typically a single-stranded oligonucleotide, preferably having 15 to 50 consecutive nucleotides, which can anneal to a complementary nucleic acid “template” and is template-dependently extended by the action of a DNA polymerase such as Taq polymerase, RNA-dependent DNA polymerase, or Sequenase®. “Probe” may be a single-stranded or double-stranded oligonucleotide or polynucleotide, preferably labeled for the purpose of detecting complementary sequences in Northern or Southern blotting.

[0136] The isolated nucleic acids disclosed herein can be readily prepared using standard protocols, such as those described in Chapters 2 and 3 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Eds. Ausubel et al. John Wiley & Sons NY, 1995-2008).

[0137] The nucleic acids of this disclosure may be produced, isolated, detected, and / or subjected to recombinant DNA technology using nucleic acid sequence amplification techniques.

[0138] Suitable nucleic acid amplification techniques covering both thermal and isothermal methods are well known to those skilled in the art and include, but are not limited to, polymerase chain reaction (PCR), strand displacement amplification (SDA), rolling circle replication (RCR), nucleic acid sequence-based amplification (NASBA), Q-β replicase amplification, recombinase polymerase amplification (RPA), and helicase-dependent amplification.

[0139] Genetic constructs This disclosure also provides a gene construct comprising and / or encoding the aforementioned isolated nucleic acid. The gene construct may be a vector.

[0140] In certain examples, a gene construct comprises an isolated nucleic acid operably ligated or linked to one or more other genetic components. The gene construct may be suitable for the therapeutic delivery of isolated nucleic acids (e.g., DNA or RNA vaccines) or for the recombinant production of the viral proteins of this disclosure in host cells. In addition, the gene construct may be used for the production or generation of influenza viruses (e.g., variants or modified strains of influenza virus isolates or reassembled influenza virus isolates) expressing the viral proteins, more particularly, the viral backbone proteins, provided herein.

[0141] In general, gene constructs may be in the form of plasmids, bacteriophages, cosmids, yeast or bacterial artificial chromosomes, or may contain their genetic components, as is well understood in the art. Gene constructs may be suitable for the maintenance and proliferation of isolated nucleic acids in bacteria or other host cells, manipulation by recombinant DNA technology, and / or expression of nucleic acids or encoded proteins of this disclosure. Vectors may also be naked RNA polynucleotides, naked DNA polynucleotides, polynucleotides composed of both DNA and RNA in the same strand, poly-lysine conjugate DNA or RNA, peptide conjugate DNA or RNA, liposomal conjugate DNA, etc. Such vectors may or may not be autonomously replicated.

[0142] For the purpose of host cell expression, a gene construct is an expression construct. Preferably, an expression construct comprises nucleic acids of the present disclosure operably ligated to one or more additional sequences in an expression vector. The “expression vector” may be either a self-replicating extrachromosomal vector such as a plasmid, or a vector incorporated into the host genome. Alternatively, an expression construct may be a linear expression construct. Such a linear expression construct typically does not contain any amplification and / or selection sequences. However, a linear construct containing such amplification and / or selection sequences is also within the scope of the present disclosure. A linear expression construct may, for example, comprise an individual linear expression construct for each viral segment. It is also possible to include two or more viral segments, such as two, three, four, five, or six, on the same linear expression construct.

[0143] Expression constructs suitable for use in the methods of this disclosure may be unidirectional or bidirectional expression constructs. Since influenza viruses require proteins for infectivity, it is generally preferred to use bidirectional expression constructs to reduce the total number of expression constructs required by the host cell. A bidirectional expression construct contains at least two promoters that drive expression in different directions from the same construct (i.e., both 5' to 3' and 3' to 5'). The two promoters may be operably ligated to different strands of the same double-stranded DNA. Preferably, one of the promoters is a pol I promoter and at least one of the other promoters is a pol II promoter. Thus, the methods of this disclosure may utilize at least one bidirectional expression construct in which at least one gene or cDNA is located between an upstream pol II promoter and a downstream non-endogenous pol I promoter. Transcription of the gene or cDNA from the pol II promoter produces capped positive-strand viral mRNA that can be translated into a protein, while transcription from the non-endogenous pol I promoter produces negative-strand viral RNA (vRNA).

[0144] "Operatively linked" means that the additional nucleotide sequence(s) is positioned to the nucleic acid of the Disclosure, preferably to initiate, regulate, or otherwise control its transcription.

[0145] The regulatory nucleotide sequences are generally suitable for the host cells used for expression. As described herein, numerous types of suitable expression vectors and suitable regulatory sequences are known in the art for various host cells. Expression vectors may be designed for the expression of viral proteins described herein using prokaryotes (e.g., E. coli) or eukaryotic cells (e.g., insect cells (using baculovirus expression vectors, e.g., see Treenor et al., 2007, JAMA, 297(14):1577-1582, the whole of which is incorporated herein by reference)).

[0146] Typically, the one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosome binding sites, polyadenylation sequences, transcription start and termination sequences, translation start and termination sequences, and enhancer or activator sequences. Constitutive, repressible, or inducible promoters known in the art are contemplated in this disclosure.

[0147] In some examples, the gene construct includes one or more untranslated 5' and / or 3' regions operably linked or attached to a viral segment encoding a corresponding viral protein. For this purpose, the UTR may be derived from the same viral isolate or a different influenza virus isolate from which the viral segment and / or viral protein originate.

[0148] The expression construct may also include additional nucleotide sequences encoding a fusion partner (typically provided by the expression vector) so that the recombinant protein is expressed as a fusion protein.

[0149] Expression constructs also amp R neo R , or Kan R This may include, but is not limited to, additional nucleotide sequences encoding selection markers such as, etc.

[0150] Preferably, the gene constructs encoding viral proteins provided herein are suitable or suitable for use in the production or generation of reassembled influenza viruses expressing the viral proteins by reverse genetic methods or hybrid reverse genetics-classical reassembly methods. Therefore, one or more gene constructs provided herein can be introduced into host cells using any method for introducing expression constructs from known reverse genetic techniques. For example, gene constructs can be introduced into host cells by electroporation, DEAE-dextran, calcium phosphate precipitation, liposomes, microinjection, or microparticle bombardment. In some examples, the gene constructs may be in the form of naked nucleic acids, which may be purified from influenza viruses. In other examples, the gene constructs may be in the form of transcription RNA (e.g., viral mRNA). In other examples, the gene constructs may be in the form of one or more shuttle vectors. Examples of shuttle vectors include non-influenza viruses and replicons, such as alphavirus-based replicons.

[0151] The gene constructs provided herein may include RNA transcription termination sequences. These termination sequences may be endogenous or non-endogenous for the host cell. Suitable termination sequences, as will be apparent to those skilled in the art, include, but are not limited to, RNA polymerase I transcription termination sequences, RNA polymerase II transcription termination sequences, and ribozymes. Furthermore, the expression constructs may contain, in particular, one or more polyadenylation signals for mRNA at the terminal of a gene whose expression is controlled by a pol II promoter.

[0152] In another form, the Disclosure also provides a plurality of gene constructs comprising: (a) encoding one or more of the viral proteins, or more particularly, skeletal viral proteins, described herein, and / or (b) comprising one or more of the viral segments, or more particularly, skeletal viral segments, described herein, comprising a nucleotide sequence complementary thereto; and / or encoding. The plurality of gene constructs may further comprise one or more further gene constructs that can be used to prepare reassembled viruses, including 6:1:1 reassembled, 6:2 reassembled, 5:3 reassembled, and 7:1 reassembled. The further gene constructs may comprise one or both of the HA and NA viral segments and / or encode (i.e., encoding one or more of the NA and HA viral proteins).

[0153] In a related form, the disclosure also relates to a method for producing a viral protein provided herein, the method comprising (i) culturing previously transformed host cells as described herein, and (ii) isolating the viral protein from the host cells cultured in step (i).

[0154] cell This disclosure also provides host cells that are (a) transformed with isolated nucleic acids and / or gene constructs described herein, or (b) infected with isolated influenza viruses provided herein.

[0155] The host cell can be any known in the art. One well-known method for influenza virus propagation is to use specific pathogen-free (SPF) hatched hens eggs, inoculate the virus into the egg contents (i.e., allantoalanthin fluid), allow it to grow, and then purify it. Influenza viruses may also be propagated in animal cell cultures, and this culture method is preferred for reasons of replication accuracy, rate, and patient allergies.

[0156] Referring to the cells described herein, this method typically uses cell lines, but primary cells may be used as an alternative. Such cells or cell lines may be bacteria, insect cells, yeast cells, plant cells, algae, or mammalian cells. Examples of yeast host cells include, but are not limited to, S. pombe and S. cerevisiae. Examples of mammalian host cells include, but are not limited to, Crucell Per. C6 cells, Vero cells, CHO cells, VERY cells, BHK cells, HeLa cells, COS cells, MDCK cells, 293 cells, 3T3 cells, or WI-38 cells. In certain cases, the host cells are myeloma cells such as NSO cells, 45.6 TG1.7 cells, AF-2 clone 9B5 cells, AF-2 clone 9B5 cells, J558L cells, MOPC 315 cells, MPC-11 cells, NCI-H929 cells, NP cells, NSO / 1 cells, P3 NS1 Ag4 cells, P3 / NS1 / 1-Ag4-1 cells, P3U1 cells, P3X63Ag8 cells, P3X63Ag8.653 cells, P3X63Ag8U.1 cells, RPMI 8226 cells, Sp20-Ag14 cells, U266B1 cells, X63AG8.653 cells, Y3.Ag.1.2.3 cells, and YO cells. Non-specific examples of insect cells include SJ9, SJ21, Trichoplusia ni, Spodoptera fugiperda, and Bombyx mori. Exemplary plant cell lines for viral protein expression are provided in U.S. Patents Nos. 7,504,560, 6,770,799, 6,551,820, 6,136,320, 6,034,298, 5,914,935, 5,612,487, and 5,484,719, as well as U.S. Patent Publications Nos. 2009 / 0208477, 2009 / 0082548, 2009 / 0053762, 2008 / 0038232, 2007 / 0275014, and 2006 / 0204487.

[0157] In certain cases, the host cells are mammalian. Suitable mammalian cells include, but are not limited to, hamster, cattle, primate (including human and monkey) and canine cells. Various cell types can be used, as is known in the art, such as kidney cells, fibroblasts, retinal cells, and lung cells. An example of a suitable hamster cell is the cell line named BHK21 or HKCC. Suitable monkey cells include African green monkey cells such as kidney cells in the Vero cell line (Kistner et al. (1998) Vaccine 16:960-8, Kistner et al. (1999) Dev Biol Stand 98:101-110, Bruhl et al. (2000) Vaccine 19:1149-58). Suitable canine cells include canine kidney cells such as the CLDK and MDCK cell lines (W097 / 37000, Brands et al. (1999) Dev Biol Stand 98:93-100, Halperin et al. (2002) Vaccine 20:1240-7, Tree et al. (2001) Vaccine 19:3444-50). Therefore, suitable cell lines include, but are not limited to, MDCK, CHO, 293T, BHK, Vero, MRC-5, PER.C6, and WI-38 cell lines.

[0158] In certain cases, the cells or cell lines described herein are suitable for growing influenza viruses. Such cell lines may include MDCK cells derived from Madin Darby canine kidney, Vero cells derived from African green monkey (Cercopithecus aethiops) kidney, or PER.C6 cells derived from human embryonic retinoblasts (Pau et al. (2001) Vaccine 19:2716-21). These cell lines are widely available from collections such as the American Type Cell Culture (ATCC) collection, Coriell Cell Repositories, and the European Collection of Cell Cultures (ECACC). Alternative cell lines may include avian cell lines, including those derived from ducks (e.g., duck retinal cells) or hens (e.g., chicken embryonic fibroblasts (CEF)) (see, e.g., WO2003 / 076601, WO2005 / 042728, WO2003 / 043415). Examples include avian embryonic stem cells, including the EBx cell lines derived from chicken embryonic stem cells, EB45, EB14, EB14-074, and EB66.

[0159] Preferably, the cells or cell line are MDCK cells derived from Madin Darby canine kidney. The original MDCK cells are available from ATCC as CCL-34. Derivatives of MDCK cells may also be used. For example, the MDCK cell line may be adapted for growth in suspension culture (e.g., “MDCK 33016” deposited as DSM ACC 2219). Similarly, WO2001 / 064846 discloses an MDCK-derived cell line (“B-702” deposited as FERM BP-7449) that grows in suspension in serum-free culture. WO2006 / 071563 discloses non-tumor-forming MDCK cells, including “MDCK-S” (ATCC PTA-6500), “MDCK-SF101” (ATCC PTA-6501), “MDCK-SF102” (ATCC PTA-6502), and “MDCK-SF103” (PTA-6503). WO2005 / 113758 discloses MDCK cell lines highly susceptible to infection, including “MDCK.5F1” cells (ATCC CRL-12042). Any MDCK cell line, including those provided herein, can be used in the methods of this disclosure.

[0160] For viral replication or propagation on cell lines such as MDCK cells, influenza viruses can be grown on cells in suspension or adherent cultures. Furthermore, the cells described herein can be cultured in a variety of serum-free media or cultures that are substantially free of serum (e.g., Iscove's medium, Ultra CHO medium (Bio Whittaker), EX-CELL (JRH Biosciences)), as is known to those skilled in the art. In other cases, cells for replication can be cultured in serum-containing media (e.g., MEM or DMEM medium containing about 0.5% to about 10%, more particularly about 1.5% to about 5%, of fetal bovine serum), or protein-free media (e.g., PF-CHO (JRH Biosciences)). Suitable culture vessels that can be used in the process of the methods described herein may be vessels known to those skilled in the art, such as spinner bottles, roller bottles, or fermenters.

[0161] When cells are used as culture hosts for influenza viruses as described herein, it is known that cell culture conditions (e.g., temperature, MOI, cell density, pH value, trypsin, etc.) can vary widely depending on the cell line and influenza virus strain used and can be adapted to the requirements of this application. Therefore, the following information is merely a guideline for standard culture conditions.

[0162] In certain cases, cells grow favorably in serum-free and / or protein-free media, for example, to support cell proliferation and / or influenza virus replication. A medium is referred to as a serum-free medium in the context of this disclosure if it does not contain, or substantially contains (e.g., less than 0.5% by weight, 0.25% by weight, or 0.1% by weight) adducts from human or animal serum. Protein-free means a culture medium in which cell proliferation occurs excluding proteins, growth factors, other protein adducts, and serum-free proteins, but which can optionally contain proteins such as trypsin or other proteases that may be necessary for viral replication. Cells growing in such cultures naturally contain proteins themselves.

[0163] Cell proliferation can be carried out according to methods known to those skilled in the art. For example, cells can be cultured in a perfusion system using conventional support methods such as centrifugation or filtration. Furthermore, cells can be grown in a supply batch system according to the present invention before infection. In this context, the culture system may be referred to as a supply batch system in which cells are first cultured in a batch system, and depletion of nutrients (or a portion of nutrients) in the culture medium is compensated by a controlled supply of concentrated nutrients. The pH value of the culture medium can be adjusted to a value of pH 6.6 to pH 7.8, particularly pH 7.2 to pH 7.3, during cell proliferation before infection. After infection with the influenza virus, cells are preferably cultured at a pH of about 6.7 to about 7.7.

[0164] Cell lines that support influenza virus replication are preferably cultured at a temperature below 37°C (e.g., about 30°C to about 36°C, or about 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, or any range therein) during virus replication. When the virus grows on the cell line, the culture medium, and also the virus inoculation source used to initiate the culture, are preferably free of contaminating viruses such as herpes simplex virus, respiratory syncytial virus, parainfluenza virus 3, SARS coronavirus, adenovirus, rhinovirus, reovirus, polyomavirus, bima virus, sarcoma virus, and / or parvovirus (e.g., tested for negative results of contamination and given such).

[0165] Methods for growing influenza virus in cultured cells generally include the steps of inoculating the cultured cells with the strain to be cultured, culturing the infected cells for a desired period for virus growth (e.g., about 24 to about 168 hours after inoculation) as determined by virus titer or antigen expression, and collecting the grown virus. The cultured cells can be inoculated at a virus (e.g., measured by PFU or TCID 50 to cell ratio of 1:500 to 1:1, more particularly 1:100 to 1:5, or even more particularly 1:50 to 1:10. Then the virus can be added to the cell suspension or applied to the cell monolayer, and the virus is absorbed by the cells for 90 to 240 minutes at least 60 minutes but usually less than 300 minutes, preferably at 25°C to 40°C, or more particularly 28°C to 37°C. The infected cell culture (e.g., monolayer) may be removed by freeze-thawing or enzymatic action to increase the virus content of the collected culture supernatant. Then the collected fluid can be inactivated or cryopreserved.

[0166] The cultured cells are about 10 -8 to about 10 (e.g., about 10 -8 、10 -7 、10 -6 、10 -5Infection can occur with multiples of infection ("moi") of approximately 0.002 to approximately 5, and even more particularly, approximately 0.001 to approximately 2, among other MOIs of 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, and any range within these MOIs. Infected cells can be collected 30 to 96 hours post-infection, or more particularly, 34 to 48 hours post-infection. Determining the optimal collection time is within the range of the usual ability of those skilled in the art. Proteases such as trypsin are generally added during cell culture to enable viral release, and proteases can be added at any suitable stage during culture.

[0167] Method for preparing influenza virus Furthermore, this specification provides a method for preparing or producing influenza viruses within cells.

[0168] Such a method preferably comprises the step of contacting cells with a gene construct comprising: (a) encoding a skeletal viral protein as described herein and / or (b) encoding a skeletal viral segment as described herein and / or comprising. Thus, in one embodiment, the present disclosure provides a method for preparing an influenza virus, the method comprising the step of contacting cells such as MDCK cells with one or more gene constructs encoding one or more viral proteins independently comprising, comprising, or essentially comprising amino acid sequences selected from the group consisting of SEQ ID NOs: 9-17, 28-36, 47-55, 66-74, 85-93, and 104-112, or fragments, variants, or derivatives thereof.

[0169] Furthermore, this disclosure assumes that classical reassembly (or part thereof) methods may be used to prepare or produce influenza viruses in cells such as MDCK cells. In other words, this disclosure provides a method for preparing influenza viruses, the method comprising the step of contacting cells with an influenza virus expressing one or more of the skeletal viral proteins described herein (for example, the influenza virus isolate comprises one or more PB2, PB1, PA, M, NP, and NS viral segments encoding one or more skeletal viral proteins comprising, consisting of, or essentially comprising amino acid sequences selected from the group consisting of SEQ ID NOs: 9-17, 28-36, 47-55, 66-74, 85-93, and 104-112, or fragments, variants, or derivatives thereof).

[0170] Taking the above into consideration, the present method can be used to prepare or produce modified or variant strains of influenza virus, such as those provided herein, which include one or more skeletal viral segments encoding the skeletal viral proteins described herein. For this purpose, the present method can be used to prepare or produce reassembled influenza viruses, such as those provided herein, which include one or more skeletal viral segments encoding the skeletal viral proteins described herein. In certain cases, seven, eight, or all of the nine skeletal viral proteins (i.e., PA, PB1, PB1-F2, PB2, NP, NS1, NEP, M1, and M2 viral proteins) may originate from a single influenza virus isolate (e.g., SEQ ID NOs. 9-17 from A / Ohio / 02 / 2019, SEQ ID NOs. 28-36 from A / Singapore / TT1384 / 2016, SEQ ID NOs. 47-55 from A / South Carolina / 04 / 2017, SEQ ID NOs. 66-74 from A / Alaska / 06 / 2019, SEQ ID NOs. 85-93 from A / Darwin / 11 / 2021, SEQ ID NOs. 104-112 from A / Tasmania / 503 / 2020, including fragments, variants, or derivatives thereof). In certain cases, five or all of the six skeletal viral segments (i.e., PA, PB1, PB2, NP, NS, and M viral segments) may originate from a single influenza virus isolate (e.g., SEQ ID NOs. 1-6 from A / Ohio / 02 / 2019, SEQ ID NOs. 20-25 from A / Singapore / TT1384 / 2016, SEQ ID NOs. 39-44 from A / South Carolina / 04 / 2017, SEQ ID NOs. 58-63 from A / Alaska / 06 / 2019, SEQ ID NOs. 77-82 from A / Darwin / 11 / 2021, SEQ ID NOs. 96-101 from A / Tasmania / 503 / 2020, including fragments, variants, or derivatives thereof).

[0171] In other examples, all eight viral segments (i.e., PA, PB1, PB2, NP, NS, M, NA, and HA) may originate from a single influenza virus isolate. In such examples, the influenza virus isolate is preferably a skeleton or donor influenza virus isolate, such as those described above. In addition, such isolated influenza virus strains are preferably not considered to be reassembled influenza viruses.

[0172] Preferably, the method may be a reverse genetic method for generating reassembled viruses, or may at least partially include one. In reverse genetics, the genetic information necessary to produce a desired influenza virus is delivered to a cell, which can then produce the influenza virus. Reverse genetics initially required in vitro assembly and transfection of viral ribonucleoprotein (RNP) into cells infected with a helper virus (Luytjes et al. (1989) Cell 59(6):1107-1113, Enami et al. (1990) PNAS 87(10):3802-3805). Subsequent techniques involved transfection of an RNA polymerase I plasmid encoding all of the viral RNA (vRNA), along with protein expression constructs for polymerase and NP genes (Fodor et al. (1999) J Virol. 73(11):9679-9682). More recently, reverse genetic methods have involved the use of a modified RNA polymerase I system that allows for the expression of both negative-sense vRNA and positive-sense mRNA from the same template (Hoffmann et al. (2000) PNAS 97(11):6108-6113). In such a method, each of the desired genes is inserted between the RNA polymerase I promoter and termination sequence and cloned into a pHW2000 plasmid consisting of viral cDNA flanked by the CMV promoter and polyadenylation signal. After transfection of the eight plasmids into cells, synthesis of both vRNA and mRNA occurs, resulting in viral production. Further improvements have led to the development of systems in which linear DNA expression constructs are used instead of plasmids (see, e.g., WO2009 / 000891) and the use of single expression constructs (see, e.g., WO2011 / 012999).

[0173] Therefore, the method may further include the step of contacting a cell with one or more further gene constructs, the one or more further gene constructs comprising one or more further nucleic acids encoding one or more of the following: HA protein, PB1 protein, PB1-F2 protein, and NA protein (e.g., 5:3 reassembled virus), more particularly, HA protein and NA protein (e.g., 6:2 reassembled virus), even more particularly, HA protein and optionally NA protein (e.g., 6:2 or 7:1 reassembled virus), or even more particularly, NA protein and optionally HA protein (e.g., 6:2 or 7:1 reassembled virus).

[0174] In a specific example, this method involves the following steps: (a) The step of contacting cells with one or more expression constructs, such as those described above, which include and / or encode one or more nucleic acid molecules comprising one or more PA virus segments, PB1 virus segments, PB2 virus segments, NP virus segments, M virus segments, and NS virus segments derived from a first influenza virus (for example, independently encoding one or more viral proteins comprising, comprising, or essentially comprising one or more amino acid sequences selected from the group consisting of (i) SEQ ID NOs: 9-17, 28-36, 47-55, 66-74, 85-93, and 104-112, or fragments, variants, or derivatives thereof, and / or encoding one or more skeletal viral segments comprising, comprising, or essentially comprising one or more nucleic acid molecules comprising one or more of the above, (b) The step of contacting the cells with one or more expression constructs, such as those described above, which include an HA virus segment derived from a second influenza virus (e.g., vaccine influenza virus) and, optionally, one or more nucleic acid molecules including or encoding an NA virus segment, (c) A step of culturing cells in order to produce reassembled viruses, and (d) The step of selecting a reassembled virus that includes an HA virus segment derived from a second influenza virus and, optionally, an NA virus segment.

[0175] In other examples, the method may include a hybrid method of classical recombination and reverse genetics. For example, a method in which host cells are infected with a first influenza strain (e.g., a donor strain) and transfected (e.g., before, after, or simultaneously) with one or more expression constructs encoding at least one viral segment (e.g., HA from the vaccine strain and, optionally, an NA viral segment) from a second influenza strain. An example of such a method is outlined in WO2021 / 099419, which is incorporated herein by reference.

[0176] Therefore, in a specific example, this method involves the following steps: (a) cells, (i) Encoding one or more viral proteins that independently include, consist of, or are essentially thereof an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-17, 28-36, 47-55, 66-74, 85-93, and 104-112, or fragments, variants, or derivatives thereof, and / or independently include, consist of, or are essentially thereof one or more PA, PB1, PB2, NP, M, and NS viral segments, and (ii) Contacting a donor influenza virus strain containing a first HA virus segment and a first NA virus segment (for example, HA and NA virus segments encoding HA and NA viral proteins, respectively, which independently contain, consist of, or are essentially derived from an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 19, 37, 38, 56, 57, 75, 76, 94, 95, 113, and 114, or fragments, variants, or derivatives thereof, and / or independently contain, consist of, or are essentially derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs: 7, 8, 26, 27, 45, 46, 64, 65, 83, 84, 102, and 103, or fragments, variants, or derivatives thereof), (b) The step of contacting cells with one or more expression constructs comprising a second HA virus segment derived from a vaccine influenza virus strain and, optionally, one or more nucleic acid molecules comprising or encoding a second NA virus segment, (c) A step of culturing cells in order to produce reassembled viruses, and (d) The step of selecting a reassembled virus which includes a second HA virus segment and, optionally, a second NA virus segment.

[0177] As used herein, the term “vaccine influenza virus strain” refers to an influenza virus strain suitable for use in immunogenic compositions or immunogenic viruses (e.g., reassorted viruses). Vaccine influenza virus strains include, but are not limited to, pathogenic strains, non-pathogenic or relatively non-pathogenic strains, toxic strains, and / or attenuated strains. In certain examples, the vaccine influenza virus strain is a pandemic influenza virus strain. In other examples, the vaccine influenza virus strain is a seasonal influenza virus strain.

[0178] The vectors or expression constructs used in this method may be as known in the art, including those described above. Accordingly, this disclosure assumes the use of isolated and purified vectors or plasmids that express or encode influenza virus proteins, or both natural and recombinant influenza vRNAs. The vectors may contain influenza cDNA (see, for example, Fields Virology (Fields et al. (eds.), Lippincott, Williams and Wickens (2013)), incorporated herein by reference). Any suitable promoter or termination sequence can be used to express proteins or peptides, such as viral proteins, including the skeletal viral proteins described herein. As an example, one or more expression constructs can be adapted for (a) vRNA production, comprising a promoter operably linked to an influenza virus DNA molecule linked to a termination sequence, and / or (b) mRNA production, comprising a promoter operably linked to a DNA segment encoding an influenza virus segment.

[0179] As described above, additional selection steps for enhancing the production of reassembled viruses containing a second HA viral segment and, optionally, a second NA viral segment are also contemplated by this disclosure. The selection steps may include any method for enhancing the selection of reassembled viruses containing an HA viral segment encoding an HA protein derived from a vaccine virus strain. Preferably, the selection steps are performed after culturing host cells to produce reassembled influenza viruses capable of expressing HA proteins and / or NA proteins derived from a vaccine virus strain.

[0180] Preferably, the methods provided herein include a step of collecting, isolating, or separating the reassembled virus from host cells prior to the selection step. For example, the cell culture supernatant containing the reassembled virus is separated from the host cells, and the selection step is performed on the supernatant containing the reassembled virus.

[0181] In some examples, the selection step includes negative selection against reassembled viruses containing HA proteins from a donor virus strain. Negative selection may include, for example, contacting host cells, reassembled viruses isolated therefrom, and / or cell culture supernatants with one or more antibodies that specifically bind to or evoke HA proteins from the donor virus strain. Negative selection may also include exposure of host cells to inhibitors (e.g., small interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), or small interfering DNA (siDNA)) that preferentially or specifically reduce the transcription and / or translation of the HA virus segment or protein of the donor strain compared to the HA virus segment or protein of the vaccine strain. In addition to the above, the selection step may include negative selection against reassembled viruses containing NA proteins from the donor strain, for example, by utilizing one or more antibodies that specifically bind to or evoke NA proteins from the donor strain.

[0182] In other examples, the selection step is or includes a positive selection step. The positive selection step may include contacting host cells, reassembled viruses isolated therefrom, and / or cell culture supernatant with one or more antibodies specific to the HA protein derived from the vaccine virus strain. In this way, reassembled viruses containing the HA virus segment derived from the vaccine virus strain can be positively selected from the host cells or cell culture supernatant. Preferably, one or more antibodies used for positive selection are labeled (e.g., with magnetic beads). The labeling assists in subsequent isolation of the reassembled viruses containing the HA gene encoding the HA protein from the vaccine virus strain, such as by affinity chromatography.

[0183] The methods described herein may include one or more positive and / or negative selection steps. For example, the reassembled virus may be passaged multiple times in the presence of one or more of the antibodies described above for positive and / or negative selection. Multiple selection steps may be performed to enhance the selection of reassembled influenza viruses containing HA virus segments encoding HA proteins derived from vaccine virus strains.

[0184] The method preferably produces a pool of reassembled viruses from which a specific class of reassembled viruses can be isolated. For example, reassembled viruses having high growth characteristics and expressing the HA protein of a seasonal or pandemic influenza strain and, optionally, the NA protein, can be isolated for use in vaccine production. Thus, the method may further include the step of isolating a reassembled influenza virus containing an HA virus segment encoding the HA protein of a vaccine virus strain. In other examples, the method may further include the step of isolating a reassembled influenza virus containing an NA virus segment encoding the NA protein of a vaccine virus strain.

[0185] Preferably, the method comprises contacting cells with an influenza virus isolate and / or one or more genetic constructs in an amount sufficient to produce infectious influenza virus. For this purpose, the method may comprise collecting or isolating intact or complete virions from cell culture media. Alternatively or additionally, the method may comprise collecting or isolating split virions from culture media. In such an example, the collecting or isolating step preferably comprises contacting the influenza virus with a splitting agent such as detergent. Alternatively or additionally, the method may comprise harvesting or isolating one or more specific influenza virus proteins, such as HA protein, from culture media or from the collected influenza virus, for example, by affinity chromatography.

[0186] During the collection or isolation of influenza viruses and / or influenza viral proteins, cells can be separated from the culture medium by standard methods such as centrifugation, separation, filtration, or ultrafiltration. The influenza virus or viral proteins produced therefrom can then be concentrated and / or purified according to methods known to those skilled in the art, such as gradient centrifugation (e.g., gradient ultracentrifugation (GUC)), filtration, precipitation, chromatography, and any combination thereof. In certain examples, the influenza virus and / or one or more proteins produced therefrom are isolated or collected from the culture medium by gradient ultracentrifugation. Preferably, the influenza virus is inactivated during or after purification. Viral inactivation can be achieved, for example, by contacting the influenza virus with an inactivator at any point in the purification process (e.g., by adding β-propiolactone or formaldehyde).

[0187] Preferably, the method may further include the step of selecting an influenza virus, such as a reassorted influenza virus, that can enhance the growth and / or yield of cells, more particularly mammalian cells such as MDCK cells. In a particular example, the method includes the step of determining whether enhanced growth and / or yield is possible when the influenza virus is cultured in cells. Thus, in one broad form, the disclosure provides a method for preparing an influenza virus that is capable of enhanced growth and / or yield when grown in cells.

[0188] Based on the foregoing, this disclosure further provides isolated influenza viruses prepared by the methods described herein. In addition, this disclosure relates to isolated influenza viruses and isolated viral proteins (e.g., HA and / or NA viral proteins) produced by the methods provided herein.

[0189] immunogenic composition This disclosure assumes that isolated influenza viruses, more particularly isolated reassembled influenza viruses, or one or more viral proteins isolated therefrom, may be used in immunogenic compositions such as vaccine compositions.

[0190] Therefore, in one embodiment, the present disclosure is a method for preparing an immunogenic composition, (a) the step of providing an isolated influenza virus provided herein, (b) A method is provided comprising the steps of combining an isolated influenza virus with an adjuvant and / or treating the isolated influenza virus with a drug that inactivates or attenuates the virus.

[0191] In another form, the present disclosure relates to a method for preparing an immunogenic composition, (a) Providing one or more viral proteins (e.g., HA and / or NA viral proteins) isolated from the isolated influenza virus provided herein, (b) A method is provided comprising the step of combining one or more viral proteins with an adjuvant.

[0192] In a related form, this disclosure relates to immunogenic compositions, which are produced according to methods provided herein.

[0193] In another related form, the disclosure relates to an immunogenic composition comprising an isolated influenza virus as described herein and a pharmaceutically acceptable carrier, diluent, or excipient.

[0194] In a further relevant form, the disclosure relates to immunogenic compositions comprising one or more isolated viral proteins described herein (e.g., HA and / or NA viral proteins) and a pharmaceutically acceptable carrier, diluent, or excipient.

[0195] Influenza vaccines are generally based on either attenuated live viruses or inactivated viruses. Inactivated vaccines may be based on complete virions, "split" virions, or purified surface antigens. Viral antigens can also be presented in the form of virosomals. This method can be used to produce either of these types of vaccines or immunogenic compositions. When inactivated influenza viruses are used, the immunogenic composition may contain complete virions, split virions, or purified surface antigens (e.g., hemagglutinin, and optionally, neuraminidase). Chemical means for inactivating viruses include treatment with one or more effective amounts of the following inactivators: detergents, formaldehyde, β-propiolactone, methylene blue, psoralen, carboxyfullerene (C60), binary ethylamine, acetylethyleneimine, or combinations thereof. Non-chemical methods of viral inactivation are also known in the art, such as UV light or gamma ray irradiation. Subunit vaccines are also being considered.

[0196] Virions can be collected from virus-containing fluids, such as cell culture supernatants, by various methods. For example, the purification process may involve zone centrifugation using a linear sucrose gradient solution (optionally containing a detergent to destroy virions) or affinity chromatography. The antigen can then be purified by diafiltration after optional dilution.

[0197] The immunogenic compositions disclosed herein may contain HA and / or NA proteins from two or more influenza viruses (or viral proteins isolated from two or more influenza viruses) derived from different influenza strains or subtypes (e.g., 2, 3, 4, 5, etc.). Therefore, the immunogenic compositions disclosed herein may be polyvalent compositions. For example, the immunogenic compositions disclosed herein may be trivalent or tetravalent compositions. In one example, the immunogenic composition is a tetravalent composition containing at least one virus containing HA and / or NA proteins derived from the H1N1 strain, at least one virus containing HA and / or NA proteins derived from the H3N2 strain, and one or two viruses containing HA and / or NA proteins derived from strain B. In another example, the immunogenic composition is a tetravalent composition containing one or more viral proteins derived from at least one virus containing HA and / or NA proteins derived from the H1N1 strain, at least one virus containing HA and / or NA proteins derived from the H3N2 strain, and one or two viruses containing HA and / or NA proteins derived from strain B.

[0198] The amount of each virus or viral protein contained in the compositions disclosed herein may vary depending on the intended use of those compositions. For example, depending on the intended use of the composition (e.g., the intended patient population), standard doses, low doses, or high doses of HA and / or NA may be included. Typically, a “standard dose” influenza composition contains about 15 μg of HA per strain in each dosage form or dosing unit. Therefore, a “low dose” influenza composition may contain less than about 15 μg of HA per strain in each dosage form or dosing unit, e.g., about 12 μg, about 9 μg, about 7.5 μg, about 5 μg, or about 3.75 μg. A “high dose” influenza composition may contain more than about 15 μg of HA per strain in each dosage form or dosing unit, e.g., about 30 μg, about 45 μg, or about 60 μg.

[0199] Immunogenic compositions may contain pharmaceutically acceptable carriers, diluents, or excipients. “Pharmaceutically acceptable carriers, diluents, or excipients” means solid or liquid fillers, diluents, or encapsulating materials that can be safely used in systemic administration. Depending on the specific route of administration, a variety of carriers, diluents, and excipients well known in the art may be used. These may be selected from the group including sugars, starches, cellulose and their derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffer solutions, emulsifiers, isotonic salines, and salts such as mineral salts including hydrochlorides, bromides, and sulfates, organic acids such as acetates, propionates, and malonates, water, and pyrogen-free water.

[0200] A useful reference describing acceptable carriers, diluents, and excipients is Remington's Pharmaceutical Sciences (Mack Publishing Co., NJUSA, 1991), which is incorporated herein by reference.

[0201] Preferably, certain immunotherapeutic agents or immunogenic agents may be used in combination with the isolated influenza viruses described herein for the purpose of inducing an immune response. The term “immunogenic agent” includes, as is well known in the art, carriers, delivery agents, immunostimulants, and / or adjuvants. As understood in the art, immunostimulants and adjuvants refer to or include one or more substances that enhance the immunogenicity and / or efficacy of a composition. Non-limiting examples of suitable immunostimulants and adjuvants include squalane and squalene (or other oils of plant or animal origin), including squalene oil-in-water emulsions (e.g., MF59, AS03, and AF03); block copolymers; TLR agonists such as pathogen-derived compounds, including bacterial components such as lipopeptides, glycolipids, nucleotides, small molecule inhibitors, and flagellins; detergents such as Tween®-80; mineral oils such as Quil® A, Drakeol, or Marcol; vegetable oils such as peanut oil; Corynebacterium-derived adjuvants such as Corynebacterium parvum; Propionibacterium-derived adjuvants such as Propionibacterium acne; Mycobacterium bovis (Bacille Calmette and Guerin or BCG); and Bordetella Pertussis antigen; tetanus toxoid; diphtheria toxoid; surfactants such as hexadecylamine, octadecylamine, octadecyl amino acid esters, lysolecithin, dimethyldioctadecylammonium bromide, N,N-dicoctadecyl-N',N'bis(2-hydroxyethyl-propanediamine), methoxyhexadecylglycerol, and pluronic polyols; polyamines such as pyran, dextransulfate, and polyIC carbopole; peptides such as muramyl dipeptides and derivatives, dimethylglycine, and tuftosine; oil emulsions; and mineral gels such as aluminum phosphate, aluminum hydroxide, or alum; interleukins such as interleukin-2 and interleukin-12; monokines such as interleukin-1; tumor necrosis factor;Examples include interferons such as gamma interferon; immunostimulatory DNA such as CpG DNA, combinations such as saponin-aluminum hydroxide or Quil-A aluminum hydroxide; saponins such as matrix-M; liposomes; ISCOM® and ISCOMATRIX® adjuvants; mycobacterial cell wall extracts; synthetic glycopeptides such as muramyl dipeptide or other derivatives; abridine; lipid A derivatives; dextransphosphate; DEAE-dextran alone or with aluminum phosphate; carboxypolymethylenes such as carbopol EMA; acrylic copolymer emulsions such as Neocryl A640 (e.g., U.S. Patent No. 5,047,238); water-in-oil emulsifiers such as Montanide ISA720; poliovirus, vaccinia, or animal poxvirus; or mixtures thereof. Preferably, the adjuvant included in the immunogenic compositions of this disclosure is or contains squalene. In certain examples, the adjuvant is or contains squalene oil-in-water emulsion. In a preferred example, the isolated influenza virus described herein, and / or one or more viral proteins isolated therefrom, are provided together with MF59.

[0202] Examples of immunogenic agents include carriers such as thyroglobulin; albumins such as human serum albumin; toxins, toxoids, or any variant cross-reactive substances (CRMs) of toxins from tetanus, diphtheria, pertussis, Pseudomonas, E. coli, Staphylococcus, and Streptococcus; polyamino acids such as poly(lysine:glutamic acid); influenza; rotavirus VP6, parvovirus VP1 and VP2; hepatitis B virus core protein; and recombinant hepatitis B virus vaccine. Alternatively, fragments or epitopes of carrier proteins or other immunogenic proteins may be used. For example, T-cell epitopes of bacterial toxins, toxoids, or CRMs may be used. In this regard, U.S. Patent No. 5,785,973, incorporated herein by reference, can be seen. Carrier proteins or other immunogenic proteins are intended to be directly or indirectly linked to the viral proteins described herein (e.g., by linkers known in the art).

[0203] Oil-in-water emulsions have been found to be particularly suitable for use in the adjuvantization of influenza virus vaccines or immunogenic compositions. Various such emulsions are known, which typically contain at least one oil and at least one surfactant, and these oils and surfactants are biodegradable (metabolizable) and biocompatible. Oil droplets in the emulsion generally have a diameter of less than 5 μm, and may even be submicron in diameter; these small sizes are achieved using microfluidizers to provide a stable emulsion. Droplets with an average size of less than 220 nm are preferred because they can be filtered and sterilized.

[0204] In various examples, oil-in-water emulsions are homogeneous. A homogeneous emulsion is characterized in that the majority of droplets (particles) dispersed therein fall within a specified size range (e.g., diameter). Preferred specified size ranges may be, for example, about 50–220 nm (e.g., about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220 nm, or any range within these), about 50–180 nm, about 80–180 nm, about 100–175 nm, about 120–185 nm, about 130–190 nm, about 135–175 nm, or about 150–175 nm. In some examples, a homogeneous emulsion contains less than 10% of droplets (particles) outside the specified diameter range. In a particular example, the average particle size of oil droplets in an oil-in-water emulsion preparation is approximately 135–175 nm, for example, approximately 155 nm ± 20 nm, as measured by dynamic light scattering. Such preparations have a particle size of 1 × 10⁶ per mL of preparation, as measured by optical particle detection. 7 It contains no more than 100% large particles. As used herein, “large particles” means particles with a diameter of 1.2 μm or more, typically between approximately 1.2 and 400 μm. In certain examples, a uniform emulsion contains less than 10%, less than 5%, or less than 3% of droplets outside the preferred size range. In some examples, the average droplet size of particles in an oil-in-water emulsion preparation is approximately 125–185 nm, e.g., approximately 130 nm, 140 nm, 150 nm, 155 nm, 160 nm, 170 nm, or 180 nm, and the oil-in-water emulsion is uniform in that less than 5% of the number of droplets in the preparation are outside the 125–185 nm range.

[0205] The immunogenic compositions described herein can be used with oils derived from animal (such as fish) or plant sources. Sources of vegetable oils include nuts, seeds, and grains. The most commonly available peanut oil, soybean oil, coconut oil, and olive oil are examples of nut oils. Jojoba oil, obtained from jojoba beans, may also be used. Seed oils include safflower oil, cottonseed oil, sunflower seed oil, and sesame oil. Among grains, corn oil is the most readily available, but oils from other grains such as wheat, oats, rye, rice, teff, and rye may also be used. Glycerol and 6- to 10-carbon fatty acid esters of 1,2-propanediol are not naturally present in seed oils, but can be prepared by hydrolysis, separation, and esterification of suitable materials starting from nuts and seed oils. Fats and oils derived from mammalian milk are metabolizable and therefore can be used in the immunogenic compositions described herein. Procedures for separation, purification, saponification, and other means necessary to obtain pure oils of animal origin are well known in the art. Most fish contain readily recoverable metabolizable oils. For example, cod liver oil, shark liver oil, and whale oil such as whale fat are some examples of fish oils that may be used herein.

[0206] Some branched-chain oils are biochemically synthesized using 5-carbon isoprene units and are generally referred to as terpenoids. Shark liver oil contains branched unsaturated terpenoids known as squalene and 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene. Squalane, a saturated analogue of squalene, can also be used in this immunogenic composition. Fish oils containing squalene and squalane are readily available from commercial sources or can be obtained by methods known in the art. Other suitable oils are tocopherols. Mixtures of oils are also conceivable.

[0207] Surfactants can be classified according to their "HLB" (hydrophilic / lipophilic balance). Preferably, the surfactants described herein have an HLB of at least 10, more particularly at least 15, and even more particularly at least 16. Immunogenic compositions include polyoxyethylene sorbitan ester surfactants (commonly referred to as Tweens), particularly polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO), such as linear EO / PO block copolymers, marketed under the trademark name DOWFAX®; octoxynol (Triton X-100 or t-octylphenoxypolyethoxyethanol) with octoxynol-9, in which the number of repeating ethoxy(oxy-1,2-ethanediyl) groups can vary; (octylphenoxy)polyethoxyethanol (IGEPAL CA-630 / NP-40); phospholipids such as phosphatidylcholine (lecithin); and polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl, and oleyl alcohols (known as Brij surfactants), such as triethylene glycol monolauryl ether (Brij). 30); and may include one or more surfactants, including but not limited to sorbitan esters (commonly known as SPAN), such as sorbitan trioleate (Span 85) and sorbitan monolaurate. Nonionic surfactants are preferred. Exemplary surfactants to be included in the emulsion are Tween 80 (polyoxyethylene sorbitan monooleate), Span 85 (sorbitan trioleate), lecithin, and Triton X-100.

[0208] A mixture of surfactants can also be used (e.g., a Tween 80 / Span 85 mixture). A combination of a polyoxyethylene sorbitan ester, such as polyoxyethylene sorbitan monooleate (Tween 80), and an octoxynol, such as t-octylphenoxypolyethoxyethanol (Triton X-100), is also preferred. Another possible combination includes laureth-9 + polyoxyethylene sorbitan ester and / or octoxynol.

[0209] Exemplary amounts (by weight) of surfactants are: polyoxyethylene sorbitan ester (e.g., Tween 80) 0.01% to 1%, especially about 0.1%; octyl or nonylphenoxy polyoxyethanol (e.g., Triton X-100, or other detergents in the Triton series) 0.001% to 0.1%, especially 0.005% to 0.02%; polyoxyethylene ether (e.g., Laureth 9) 0.1% to 20%, more particularly 0.1% to 10%, and even more particularly 0.1% to 1% or about 0.5%.

[0210] In certain cases, oil-in-water emulsions are squalene emulsions in water, and more specifically, submicron squalene emulsions in water.

[0211] Any preferred procedure is intended to produce an immunogenic composition or a vaccine composition. An exemplary procedure is, for example, that described in *New Generation Vaccines* (1997, Levine et al., Marcel Dekker, Inc., New York, Basel, Hong Kong), which is incorporated herein by reference.

[0212] Any safe route of administration may be used, including but not limited to oral, rectal, parenteral, sublingual, buccal, intravenous, intra-articular, intramuscular, intracutaneous, subcutaneous, inhalation, intranasal, intraocular, intraperitoneal, intraventricular, topical, mucosal, and transdermal administration.

[0213] Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, lozenges, capsules, nasal sprays, suppositories, aerosols, and transdermal patches. These dosage forms may also involve injecting or implanting controlled-release devices specifically designed for this purpose, or other forms of implants modified to act further in this manner. Controlled release can be achieved by coating with hydrophobic polymers containing acrylic resins, waxes, higher aliphatic alcohols, polylactic acid, and polyglycolic acid, as well as certain cellulose derivatives such as hydroxypropyl methylcellulose. In addition, controlled release can be achieved by using other polymer matrices, liposomes, and / or microspheres.

[0214] The compositions may be presented as separate units such as capsules, sachets, functional foods / feeds or tablets, each containing a predetermined amount of one or more therapeutic agents of the Disclosure, either as powders or granules in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil emulsion, or as a solution or suspension. Such compositions may be prepared by any of the methods of compounding, all of which involve the step of associating one or more of the agents described above with a carrier that may constitute one or more required components. Generally, compositions are prepared by homogeneously and closely mixing the agents of the Disclosure with a liquid carrier or a finely divided solid carrier, or both, and then, if necessary, shaping the product into a desired presentation.

[0215] The above compositions may be administered in a manner compatible with the drug formulation and in such amounts as effective. In the context of this disclosure, the dose administered to a subject should be sufficient to produce a beneficial response in the subject over an appropriate period of time (e.g., to produce a protective immune response). The amount of the drug(s) administered may depend on the subject being treated, including factors that depend on the subject's age, sex, weight, and overall health, as well as the physician's judgment.

[0216] Containers containing immunogenic or vaccine compositions disclosed herein are also disclosed herein. Any suitable container known in the art may be used. For example, the container may be selected from the group consisting of vials, syringes, ampoules, flasks, fermenters, bioreactors, bags, jars, ampoules, cartridges, and disposable pens. In one example, the container is a vial, ampoule, or syringe.

[0217] The container may be made from glass, metal (e.g., steel, stainless steel, aluminum, etc.) and / or polymer (e.g., thermoplastic resin, elastomer, thermoplastic elastomer). The container may be at least partially siliconized.

[0218] The immunogenic compositions disclosed herein may further comprise a buffer. The buffer may be any suitable buffer known in the art. For example, the buffer may be TRIS, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate, glycinate, histidine, glycine, succinate, and triethanolamine buffer or phosphate buffer. In one example, the buffer is a phosphate buffer. In another example, the buffer is a succinate buffer. In yet another example, the buffer is a histidine buffer. In yet another example, the buffer is a citrate buffer.

[0219] The buffer may be selected from USP-compliant buffers for parenteral use, especially if the pharmaceutical formulation is intended for parenteral use. For example, the buffer may be selected from the group consisting of monobasic acids such as acetic acid, benzoic acid, gluconic acid, glyceric acid, and lactic acid; dibasic acids such as aconitic acid, adipic acid, ascorbic acid, carbonic acid, glutamic acid, malic acid, succinic acid, and tartaric acid; polybasic acids such as citric acid and phosphoric acid; and bases such as ammonia, diethanolamine, glycine, triethanolamine, and TRIS.

[0220] Methods for inducing immune responses and therapies The isolated influenza viruses and immunogenic compositions described herein may be suitable for administration to human or non-human animal subjects, thereby providing a method for increasing the immune response in a subject and / or preventing and / or treating influenza-related diseases, disorders, or conditions. The disclosure also provides compositions for pharmaceutically acceptable use and provides the use of the compositions of the disclosure for the manufacture of pharmaceuticals for increasing the immune response in a subject and / or preventing and / or treating influenza-related diseases, disorders, or conditions.

[0221] Accordingly, in one embodiment, the present disclosure provides a method for inducing an immune response in a subject, the method comprising the step of administering a therapeutically effective amount of an isolated influenza virus or immunogenic composition provided herein to the subject, thereby inducing an immune response in the subject.

[0222] In a related form, the Disclosure provides a method for preventing and / or treating an influenza-related illness, disorder, or condition in a subject, the method comprising the step of administering a therapeutically effective amount of an isolated influenza virus or immunogenic composition described herein to the subject, thereby preventing and / or treating an influenza-related illness, disorder, or condition.

[0223] In another related form, the Disclosure provides a method for immunizing or vaccinating a subject against an influenza-related disease, disorder, or condition, the method comprising the step of administering a therapeutically effective amount of an isolated influenza virus or immunogenic composition described herein to the subject, thereby immunizing or vaccinating it against an influenza-related disease, disorder, or condition.

[0224] In another form, this disclosure relates to the use of isolated influenza viruses and / or viral proteins derived herein, or immunogenic compositions herein, in the manufacture of pharmaceuticals for inducing an immune response in a subject.

[0225] In a further form, the Disclosure provides the use of the isolated influenza virus and / or viral proteins derived herein, or the immunogenic compositions herein, in the manufacture of a pharmaceutical product for preventing and / or treating an influenza-related disease, disorder, or condition in a subject.

[0226] In another related form, the Disclosure provides the use of isolated influenza viruses and / or viral proteins derived herein, or immunogenic compositions herein, in the manufacture of pharmaceuticals for immunizing or vaccinating subjects against influenza-related diseases, disorders, or conditions.

[0227] In one broader form, this disclosure also relates to isolated influenza viruses and / or viral proteins derived herein, or immunogenic compositions herein, for use in therapy.

[0228] In a related form, the disclosure also provides isolated influenza viruses and / or viral proteins derived herein, or immunogenic compositions herein, for use in methods of inducing an immune response in a subject.

[0229] In a more relevant form, the Disclosure provides isolated influenza viruses and / or viral proteins derived herein, or immunogenic compositions herein, for use in methods of preventing and / or treating influenza-related illnesses, disorders, or conditions in a subject.

[0230] In yet another related form, the Disclosure provides isolated influenza viruses and / or viral proteins derived herein, or immunogenic compositions herein, for use in methods of immunizing or vaccinating subjects against influenza-related diseases, disorders, or conditions.

[0231] In relation to the embodiments described herein, the terms “subject,” “patient,” and “individual” include, but are not limited to, mammals such as humans, performance animals (horses, camels, greyhounds, etc.), livestock (cattle, sheep, horses, pigs, chickens, ducks, etc.), and companion animals (cats and dogs, etc.). Preferably, the subject is human.

[0232] "To induce an immune response" means to generate or stimulate the production or activity of one or more elements of the immune system, including the cellular immune system, the humoral immune system (i.e., antibodies), and / or the innate immune system. Preferably, the immune response described herein includes one or more elements of the immune system, such as T lymphocytes, B lymphocytes, antibodies, neutrophils, dendritic cells including plasmacytoid dendritic cells, cytokines, and / or chemokines. Non-limiting examples of cytokines include inflammatory cytokines such as TNF-α, IL-2, IL-6, IL-8, IL-17A, and IL-1 (e.g., IL-1β). Non-limiting examples of chemokines include the neutrophil chemoattractant IL-8. In certain cases, the immune response induced by the immunogenic compositions described herein is protective.

[0233] As commonly used herein, the terms “immunization,” “vaccination,” and “vaccine” refer to methods and / or compositions that induce a protective immune response against the influenza virus, thereby preventing or minimizing, at least partially, subsequent infection by the influenza virus or associated serotype, strain, or variant.

[0234] "Protective immunity" means a level of immunity sufficient to at least partially improve or prevent a subsequent influenza virus infection in a subject, resulting in a response to the antigen(s) that leads to rapid binding and / or removal of the antigen(s).

[0235] "Protective immune response" means a level of immune response sufficient to prevent or reduce the severity, symptoms, manner, or characteristics of the current and / or influenza virus infection in the subject.

[0236] As used herein, the terms “to treat,” “to cure,” or “to treat” refer to a therapeutic intervention that at least partially improves, eliminates, or reduces the symptoms or pathological signs of an influenza-related illness, disorder, or condition, such as influenza infection, after they have begun to develop. A treatment does not need to be absolute in order to be beneficial to the subject. Beneficial effects can be determined using any method or standard known to those skilled in the art.

[0237] As used herein, “prevention,” “to prevent,” or “prevention” refers to a process of action initiated to prevent infection and / or reduce symptoms or pathological signs of a disease, disorder, or condition, prior to infection or exposure to the influenza virus or its molecular components, and / or prior to the onset of symptoms or pathological signs of a disease, disorder, or condition. It should be understood that such prevention does not need to be absolute for it to be beneficial to the subject. “Prophylactic” treatment is a treatment administered to a subject who is not showing signs of a disease, disorder, or condition, or who is showing only early signs, for the purpose of reducing the risk of developing symptoms or pathological signs of a disease, disorder, or condition.

[0238] The vaccines and immunogenic compositions described herein may be used to treat both children and adults. Influenza vaccines are currently recommended for use in immunization of children and adults from 6 months of age. Therefore, human subjects may be under 1 year of age, 1–5 years, 5–15 years, 15–55 years, or at least 55 years. Preferred subjects for vaccination include the elderly (e.g., 50 years and older, 60 years and older, and preferably 65 years and older), young people (e.g., under 5 years of age), hospitalized subjects, healthcare workers, military personnel, pregnant women, subjects with chronic illnesses, immunocompromised subjects, subjects who have taken antiviral compounds in the 7 days prior to vaccination, people with egg allergies, and people traveling abroad. However, vaccines are not only suitable for these groups and may be more commonly used in the population. For pandemic strains, administration to all age groups is preferred.

[0239] Treatment can be administered via a single-dose schedule or a multi-dose schedule. Multi-dose schedules may be used in primary and / or booster immunization schedules. In multi-dose schedules, various doses may be administered via the same or different routes (e.g., parenteral prime and mucosal boost, mucosal prime and parenteral boost). Administration of two or more doses (typically two doses) is particularly useful in immunologically unsensitized patients (e.g., those who have never received the influenza vaccine before) or in vaccination against new HA subtypes (e.g., in pandemic outbreaks). Multi-dose schedules are typically administered at least one-week intervals (e.g., approximately 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 16 weeks, etc.).

[0240] Please refer to the following non-limiting embodiments so that preferred embodiments of this disclosure may be fully understood and put into practical use. [Examples]

[0241] Example 1. The objective of this embodiment was to identify donor or seed influenza viruses in which a skeletal gene confers improved proliferation and / or yield characteristics to reassembled viruses containing such skeletal genes. Influenza viruses were prepared using known methods for synthetic seed virus production (see, e.g., PCT / US2000 / 009021, PCT / US2001 / 013656, Dormitzer et al., Sci Transl Med, 15 May 2013 Vol 5, Issue 185, p.185).

[0242] Methods and results: Synthetic seed process and procedure ● MDCK cells were treated with expression constructs for the HA and NA sequences of various vaccine virus strains (i.e., A / Idaho / 07 / 2019, A / Nebraska / 14 / 2019, A / Iowa / 56 / 2019, A / Delaware / 55 / 2019, A / Illinois / 02 / 2019, A / Canberra / 407 / 2019, A / Darwin / 94 / 2019, A / Delaware / 39 / 2019, A / Virginia / 03 / 2020, A / Tasmania / 503 / 2020, and A / Bangladesh / 1002 / 2020), as well as various candidate high-growth donor strains (A / Singapore / TT1384 / 2016, A / South) selected based on their relatively high viral yield when grown in cells with wild-type viruses. Skeletal viral segments for PA, PB1, PB2, NP, NS, and M were transfected from (including Carolina / 04 / 2017, A / Alaska / 06 / 2019, and A / Ohio / 02 / 2019). ●The experiment also included the cell-adapted A / Puerto Rico / 8 / 1934(PR8X) as a control donor virus to generate reassembled viruses under identical conditions, compared to those based on candidate high-growth donor strains. Next, the reassembled viruses were rescued and characterized using standard methods based on their HA yield at the end of infection. ●A schematic diagram of the transfection and rescue procedures is provided in Figure 1. ●The HA yields (determined by standard HPLC methods) for each of the tested vaccine virus strains and their respective reassembled viruses are shown in Figures 2-12. ●A summary of the HA yield results for the donor strain from A / Ohio / 02 / 2019 is provided in Figure 13, which shows a consistent improvement in HA yield of this skeletal strain across various HA and NA sequences derived from the vaccine virus strain. ●Figure 14 shows the yield increase rate for each candidate donor strain with high proliferation compared to the control vaccine virus strain.

[0243] In certain aspects of this disclosure, the skeletal sequence and / or HA and / or NA sequences may include any of the virus strains disclosed in this embodiment.

[0244] conclusion This embodiment identified numerous donor virus strains that can support the rescue of vaccine virus strains in MDCK cells, as well as improved proliferation and yield. Strains A / Ohio / 02 / 2019 and A / South Carolina / 04 / 2017 demonstrated that they can consistently and significantly increase the proliferation and yield of reassembled viruses (both H1N1 and H3N2) compared to wild-type virus controls and reassembled viruses derived from corresponding PRXs. Strains A / Singapore / TT1384 / 2016 and A / Alaska / 06 / 2019 demonstrated that they can significantly increase the proliferation and yield of H1N1 subtype reassembled viruses compared to wild-type virus controls and reassembled viruses derived from corresponding PRXs. Other strains that show promise from preliminary studies by the inventors in showing increased proliferation and yield when expressed as reassembled viruses include A / Darwin / 11 / 2021 and A / Tasmania / 503 / 2020.

[0245] Itemized list of embodiments 1. An isolated influenza virus comprising one or more PA, PB1, PB2, NP, M, and NS viral segments encoding one or more viral proteins independently comprising, consisting of, or essentially comprising amino acid sequences selected from the group consisting of SEQ ID NOs. 9-17, 28-36, 47-55, 66-74, 85-93, and 104-112, or fragments, variants, or derivatives thereof.

[0246] 2. An isolated influenza virus according to claim 1, comprising one or more of the PB2, PB1, PA, M, NP, and NS viral segments, independently comprising, comprising, or essentially comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-6, 20-25, 39-44, 58-63, 77-82, and 96-101, or fragments, variants, or derivatives thereof.

[0247] 3. The isolated influenza virus according to claim 1 or 2, wherein the isolated influenza virus is capable of enhanced replication when grown in cells compared to a wild-type influenza virus isolate that does not contain one or more PA, PB1, PB2, NP, M, and NS virus segments.

[0248] 4. The isolated influenza virus according to any one of the prior claims, wherein the isolated influenza virus allows for an enhanced yield of viral protein when grown in cells compared to a wild-type influenza virus isolate that does not contain one or more PA, PB1, PB2, NP, M, and NS viral segments.

[0249] 5. The isolated influenza virus according to claim 4, wherein the viral protein is hemagglutinin (HA) protein.

[0250] 6. The isolated influenza virus according to any one of claims 3 to 5, wherein the cells are MDCK cells.

[0251] 7. (a) SEQ ID NO: 9 - 17, or a fragment, variant, or derivative thereof, (b) SEQ ID NO: 28 - 36, or a fragment, variant, or derivative thereof, (c) SEQ ID NO: 47 - 55, or a fragment, variant, or derivative thereof, (d) SEQ ID NO: 66 - 74, or a fragment, variant, or derivative thereof, (e) SEQ ID NO: 85 - 93, or a fragment, variant, or derivative thereof, or (f) an amino acid sequence selected from those shown in SEQ ID NO: 104 - 112, or a fragment, variant, or derivative thereof, independently containing, consisting of, or essentially consisting of one or more viral proteins encoding five or six of the PA, PB1, PB2, NP, M, and NS viral segments, the isolated influenza virus according to any one of the preceding claims.

[0252] 8. (a) SEQ ID NO: 1 - 6, or a fragment, variant, or derivative thereof, (b) SEQ ID NO: 20 - 25, or a fragment, variant, or derivative thereof, (c) SEQ ID NO: 39 - 44, or a fragment, variant, or derivative thereof, (d) SEQ ID NO: 58 - 63, or a fragment, variant, or derivative thereof, (e) SEQ ID NO: 77 - 82, or a fragment, variant, or derivative thereof, or (f) a nucleotide sequence selected from those shown in SEQ ID NO: 96 - 101, or a fragment, variant, or derivative thereof, independently containing, consisting of, or essentially consisting of five or six of the PA, PB, NP, M, and NS viral segments, the isolated influenza virus according to any one of the preceding claims.

[0253] 9. An isolated influenza virus according to any one of the prior claims, further comprising a heterogeneous or chimeric HA virus segment and a heterogeneous or chimeric NA virus segment.

[0254] 10. The isolated influenza virus according to any one of the prior claims, wherein the isolated influenza virus is of subtype N1, N2, N3, N7, or N9.

[0255] 11. The isolated influenza virus according to claim 10, wherein the isolated influenza virus is of the N1 or N2 subtype.

[0256] 12. The isolated influenza virus according to any one of the prior claims, wherein the isolated influenza virus is of the H1, H2, H3, H5, H7, or H9 subtype.

[0257] 13. The isolated influenza virus according to claim 12, wherein the isolated influenza virus is of the H1 or H3 subtype.

[0258] 14. The isolated influenza virus according to any one of the prior claims, wherein the isolated influenza virus is of the H1N1 subtype or the H3N2 subtype.

[0259] 15. An isolated influenza virus according to any one of the prior claims, which is a recombinant influenza virus.

[0260] 16. An isolated influenza virus according to any one of the prior claims, which is a reassembled influenza virus.

[0261] 17. A method for preparing an influenza virus, the method comprising the step of contacting a cell with one or more gene constructs encoding one or more viral proteins, which independently contain, consist of, or are essentially derived from amino acid sequences selected from the group consisting of SEQ ID NOs. 9-17, 28-36, 47-55, 66-74, 85-93, and 104-112, or fragments, variants, or derivatives thereof.

[0262] 18. The method according to claim 17, wherein one or more gene constructs comprises, comprises, or encodes one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 1-6, 20-25, 39-44, 58-63, 77-82, and 96-101, or fragments, variants, or derivatives thereof, from among the PB2, PB1, PA, M, NP, and NS viral segments.

[0263] 19. A method for preparing an influenza virus, comprising the step of contacting a cell with an isolated influenza virus comprising one or more PA, PB1, PB2, NP, M, and NS viral segments encoding one or more viral proteins independently comprising, consisting of, or essentially comprising amino acid sequences selected from the group consisting of SEQ ID NOs: 9-17, 28-36, 47-55, 66-74, 85-93, and 104-112, or fragments, variants, or derivatives thereof.

[0264] 20. The method according to claim 19, wherein the isolated influenza virus is one of the claims described in any one of claims 1 to 16.

[0265] 21. The method according to any one of claims 17 to 20, further comprising the step of isolating or collecting the influenza virus from the cells.

[0266] 22. An isolated influenza virus prepared by the method described in any one of claims 17 to 21.

[0267] 23. Isolated cells infected with the isolated influenza virus according to any one of claims 1 to 16 or 22.

[0268] 24. The isolated cells according to claim 23, wherein the cells are MDCK cells.

[0269] 25. A gene construct encoding one or more viral proteins that independently contain, consist of, or are essentially derived from amino acid sequences selected from the group consisting of SEQ ID NOs. 9-17, 28-36, 47-55, 66-74, 85-93, and 104-112, or fragments, variants, or derivatives thereof.

[0270] 26. Multiple gene constructs encoding one or more viral proteins, comprising, consisting of, or essentially comprising amino acid sequences selected from the group consisting of SEQ ID NOs. 9-17, 28-36, 47-55, 66-74, 85-93, and 104-112, or fragments, variants, or derivatives thereof.

[0271] 27. The gene construct or the plurality of gene constructs according to claim 25, comprising, comprising, or encoding one or more PB2, PB1, PA, M, NP, and NS viral segments, independently comprising, comprising, or essentially comprising, one or more of the PB2, PB1, PA, M, NP, and NS viral segments, which are nucleotide sequences selected from the group consisting of SEQ ID NOs: 1-6, 20-25, 39-44, 58-63, 77-82, and 96-101, or nucleotide sequences complementary thereto, or fragments, variants, or derivatives thereof.

[0272] 28. A method for preparing an immunogenic composition, (a) providing an isolated influenza virus and / or a viral protein derived therefrom according to any one of claims 1 to 16 or 22; (b) combining the isolated influenza virus and / or the viral protein with an adjuvant, and / or treating the isolated influenza virus with an agent that inactivates the virus, the method comprising.

[0273] 29. The method according to claim 28, wherein the adjuvant comprises an immunostimulatory DNA sequence, a bacterium-derived component, an aluminum salt (alum), or a squalene water-in-oil emulsion system.

[0274] 30. An immunogenic composition produced according to the method of claim 28 or claim 29.

[0275] 31. An immunogenic composition comprising an isolated influenza virus and / or a viral protein derived therefrom according to any one of claims 1 to 16 or 22, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0276] 32. A method of inducing an immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of an isolated influenza virus according to any one of claims 1 to 16 or 22, and / or a viral protein derived therefrom, or an immunogenic composition according to claim 30 or claim 31, thereby inducing the immune response in the subject.

[0277] 33. A method for preventing and / or treating an influenza-related disease, disorder, or condition in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of an isolated influenza virus and / or viral protein derived therefrom according to any one of claims 1 to 16 or 22, or an immunogenic composition according to claim 30 or claim 31, thereby preventing and / or treating the influenza-related disease, disorder, or condition.

[0278] 34. Use of an isolated influenza virus and / or viral protein derived therefrom according to any one of claims 1 to 16 or 22, or an immunogenic composition according to claim 30 or 31, in the manufacture of a pharmaceutical product for inducing an immune response in a subject.

[0279] 35. Use of an isolated influenza virus and / or viral protein derived therefrom according to any one of claims 1 to 16 or 22, or an immunogenic composition according to claim 30 or 31, in the manufacture of a medicament for preventing and / or treating influenza-related diseases, disorders, or conditions in a subject.

[0280] 36. An isolated influenza virus according to any one of claims 1 to 16 or 22, and / or a viral protein derived therefrom, or an immunogenic composition according to claim 30 or 31, for use in therapy.

[0281] 37. An isolated influenza virus and / or viral protein derived therefrom according to any one of claims 1 to 16 or 22, or an immunogenic composition according to claim 30 or claim 31, for use in a method of inducing an immune response in a subject.

[0282] 38. An isolated influenza virus and / or viral protein derived therefrom according to any one of claims 1 to 16 or 22, or an immunogenic composition according to claim 30 or claim 31, for use in a method of preventing and / or treating influenza-related diseases, disorders, or conditions in a subject.

[0283] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11] [Table 2-12] [Table 2-13] [Table 2-14] Table 2-15 Table 2-16 Table 2-17 Table 2-18 Table 2-19 Table 2-20 Table 2-21 Table 2-22 Table 2-23 Table 2-24 Table 2-25 Table 2-26 Table 2-27 Table 2-28 Table 2-29 Table 2-30 Table 2-31 Table 2-32 Table 2-33 Table 2-34 Table 2-35 Table 2-36 Table 2-37 Table 2-38 Table 2-39 Table 2-40 Table 2-41 Table 2-42 Table 2-43 Table 2-44 Table 2-45 Table 2-46 Table 2-47 Table 2-48

Claims

1. An isolated influenza virus comprising one or more PA, PB1, PB2, NP, M, and NS viral segments encoding one or more viral proteins independently comprising, consisting of, or essentially comprising amino acid sequences selected from the group consisting of SEQ ID NOs. 9-17, 28-36, 47-55, 66-74, 85-93, and 104-112, or fragments, variants, or derivatives thereof.

2. The isolated influenza virus according to claim 1, comprising one or more of the PB2, PB1, PA, M, NP, and NS viral segments, independently comprising, comprising, or essentially comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-6, 20-25, 39-44, 58-63, 77-82, and 96-101, or fragments, variants, or derivatives thereof.

3. The isolated influenza virus according to claim 1, wherein the isolated influenza virus is capable of enhanced replication when grown in cells compared to a wild-type influenza virus isolate that does not contain one or more PA, PB1, PB2, NP, M, and NS virus segments.

4. The isolated influenza virus according to claim 1, wherein the isolated influenza virus allows for an enhanced yield of viral protein when grown in cells compared to a wild-type influenza virus isolate that does not contain one or more PA, PB1, PB2, NP, M, and NS viral segments.

5. The isolated influenza virus according to claim 4, wherein the viral protein is hemagglutinin (HA) protein.

6. The isolated influenza virus according to claim 3, wherein the cells are MDCK cells.

7. (a) Sequence IDs 9-17, or fragments, variants, or derivatives thereof (b) Sequence IDs 28-36, or fragments, variants, or derivatives thereof (c) Sequence IDs 47-55, or fragments, variants, or derivatives thereof (d) Sequence IDs 66-74, or fragments, variants, or derivatives thereof (e) Sequence IDs 85-93, or fragments, variants, or derivatives thereof, (f) An isolated influenza virus according to claim 1, comprising five or six of the PA, PB1, PB2, NP, M, and NS viral segments encoding one or more viral proteins independently comprising, consisting of, or essentially comprising amino acid sequences selected from sequence numbers 104-112 or fragments, variants, or derivatives thereof.

8. (a) Sequence IDs 1-6, or fragments, variants, or derivatives thereof, (b) Sequence IDs 20-25, or fragments, variants, or derivatives thereof (c) Sequence IDs 39-44, or fragments, variants, or derivatives thereof (d) Sequence IDs 58-63, or fragments, variants, or derivatives thereof (e) Sequence IDs 77-82, or fragments, variants, or derivatives thereof, (f) An isolated influenza virus according to claim 1, comprising five or six of the PA, PB1, PB2, NP, M, and NS viral segments, each independently comprising, consisting of, or essentially comprising nucleotide sequences selected from those shown in SEQ ID NOs: 96-101, or fragments, variants, or derivatives thereof.

9. The isolated influenza virus according to claim 1, further comprising a heterogeneous or chimeric HA virus segment and a heterogeneous or chimeric NA virus segment.

10. The isolated influenza virus according to claim 1, wherein the isolated influenza virus is of subtype N1, N2, N3, N7, or N9.

11. The isolated influenza virus according to claim 10, wherein the isolated influenza virus is of the N1 or N2 subtype.

12. The isolated influenza virus according to claim 1, wherein the isolated influenza virus is of the H1, H2, H3, H5, H7, or H9 subtype.

13. The isolated influenza virus according to claim 12, wherein the isolated influenza virus is of the H1 or H3 subtype.

14. The isolated influenza virus according to claim 1, wherein the isolated influenza virus is of the H1N1 subtype or the H3N2 subtype.

15. The isolated influenza virus according to claim 1, which is a recombinant influenza virus.

16. The isolated influenza virus according to claim 1, which is a reassembled influenza virus.

17. A method for preparing an influenza virus, the method comprising the step of contacting a cell with one or more gene constructs encoding one or more viral proteins, which independently comprise, consist of, or are essentially derived from, amino acid sequences selected from the group consisting of SEQ ID NOs: 9-17, 28-36, 47-55, 66-74, 85-93, and 104-112, or fragments, variants, or derivatives thereof.

18. The method according to claim 17, wherein the one or more gene constructs comprises, comprises, or encodes one or more PB2, PB1, PA, M, NP, and NS viral segments, independently comprising, comprising, or essentially comprising one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 1-6, 20-25, 39-44, 58-63, 77-82, and 96-101, or fragments, variants, or derivatives thereof.

19. The method according to claim 17, further comprising the step of isolating or collecting the influenza virus from the cells.

20. A method for preparing an influenza virus, comprising the step of contacting a cell with an isolated influenza virus comprising one or more PA, PB1, PB2, NP, M, and NS viral segments encoding one or more viral proteins independently comprising, consisting of, or essentially comprising amino acid sequences selected from the group consisting of SEQ ID NOs: 9-17, 28-36, 47-55, 66-74, 85-93, and 104-112, or fragments, variants, or derivatives thereof.

21. The method according to claim 20, wherein the isolated influenza virus is as described in claim 1.

22. The method according to claim 20, further comprising the step of isolating or collecting the influenza virus from the cells.

23. An isolated influenza virus prepared by the method of claim 17.

24. An isolated influenza virus prepared by the method of claim 20.

25. Isolated cells infected with the isolated influenza virus described in claim 1.

26. The isolated cell according to claim 25, wherein the cell is an MDCK cell.

27. A gene construct encoding one or more viral proteins that independently comprises, consist of, or are essentially derived from amino acid sequences selected from the group consisting of SEQ ID NOs. 9-17, 28-36, 47-55, 66-74, 85-93, and 104-112, or fragments, variants, or derivatives thereof.

28. Multiple gene constructs encoding one or more viral proteins, each independently comprising, consisting of, or essentially comprising amino acid sequences selected from the group consisting of SEQ ID NOs. 9-17, 28-36, 47-55, 66-74, 85-93, and 104-112, or fragments, variants, or derivatives thereof.

29. The gene construct according to claim 27 or the plurality of gene constructs according to claim 26, wherein the gene construct or plurality of gene constructs comprises, comprises, or encodes one or more of the PB2, PB1, PA, M, NP, and NS virus segments, independently comprising, comprising, or essentially comprising, one or more of the nucleotide sequences selected from the group consisting of SEQ ID NOs: 1-6, 20-25, 39-44, 58-63, 77-82, and 96-101, or nucleotide sequences complementary thereto, or fragments, variants, or derivatives thereof.

30. The plurality of gene constructs according to claim 28, wherein the gene construct or the plurality of gene constructs comprises or encodes one or more PB2, PB1, PA, M, NP, and NS virus segments, independently comprising, comprising, or essentially comprising one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 1-6, 20-25, 39-44, 58-63, 77-82, and 96-101, or nucleotide sequences complementary thereto, or fragments, variants, or derivatives thereof.

31. A method for preparing an immunogenic composition, (a) the step of providing an isolated influenza virus and / or a viral protein derived therefrom as described in claim 1, (b) A method comprising the steps of combining the isolated influenza virus and / or the viral protein with an adjuvant, and / or treating the isolated influenza virus with an agent that inactivates the virus.

32. The method according to claim 31, wherein the adjuvant comprises an immunostimulatory DNA sequence, a bacterial component, an aluminum salt (alum), or a squalene oil-in-water emulsion system.

33. An immunogenic composition produced according to the method of claim 31.

34. An immunogenic composition comprising an isolated influenza virus and / or viral protein derived therefrom as described in claim 1, and a pharmaceutically acceptable carrier, diluent, or excipient.

35. A method for inducing an immune response in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of an isolated influenza virus according to claim 1, a viral protein derived therefrom, or an immunogenic composition containing the isolated influenza virus, thereby inducing the immune response in the subject.

36. A method for preventing and / or treating an influenza-related disease, disorder, or condition in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of an isolated influenza virus and / or viral protein derived therefrom according to claim 1, or an immunogenic composition comprising the isolated influenza virus, thereby preventing and / or treating the influenza-related disease, disorder, or condition.

37. Use of an isolated influenza virus, a viral protein derived therefrom, or an immunogenic composition containing the isolated influenza virus, as described in claim 1, in the manufacture of a pharmaceutical product for inducing an immune response in a subject.

38. Use of an isolated influenza virus, a viral protein derived therefrom, or an immunogenic composition containing the isolated influenza virus, in the manufacture of a pharmaceutical product for preventing and / or treating an influenza-related disease, disorder, or condition in a subject.

39. An immunogenic composition comprising the isolated influenza virus according to claim 1, a viral protein derived therefrom, or the isolated influenza virus, for use in therapy.

40. An immunogenic composition comprising the isolated influenza virus, a viral protein derived therefrom, or the isolated influenza virus, as described in claim 1, for use in a method for inducing an immune response in a subject.

41. An immunogenic composition comprising the isolated influenza virus, a viral protein derived therefrom, or the isolated influenza virus, as described in claim 1, for use in a method of preventing and / or treating influenza-related diseases, disorders, or conditions in a subject.

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