Production of an influenza virus vaccine that does not use eggs

Abstract

Providing a method for producing an influenza virus vaccine that does not use eggs. [Solution] Currently, the steps taken before releasing influenza strains to vaccine manufacturers include the step of passage the influenza virus through eggs. The present invention aims to provide a useful procedure in the manufacture of influenza vaccines such that the use of eggs is reduced, and preferably completely avoided. For example, instead of using chicken eggs for influenza vaccine isolation, MDCK cells (Madinderby canine kidney cells) may be used, and the MDCK cells may grow in suspension, in serum-free medium, in protein-free medium, be non-tumoric, grow in the absence of stratified medium, etc.

Description

[Technical Field]

[0001] All references cited herein are incorporated herein by law in their entirety.

[0002] This invention belongs to the technical field of the manufacture of vaccines for protection against influenza viruses. [Background technology]

[0003] Current methods for preparing seasonal vaccines against human influenza virus infection include the following steps [1, 2 (Non-Patent Literature 1, Non-Patent Literature 2)]: (a) isolation of circulating virus strains; (b) analysis of the antigenicity and genes of the isolated viruses; (c) selection of virus strains to be used in the next season; (d) preparation of high-growth seed strains by the use of recombination or reverse genetics; (e) release of seed strains to vaccine manufacturers; (f) evaluation by manufacturers of the suitability of the strains for industrial production; and (g) production of viruses by growing seed strains and manufacturing vaccines from those viruses.

[0004] Steps (a) through (e) of this method are carried out by the FDA and government-approved International Influenza Center, typically under the auspices of the World Health Organization; steps (f) and (g) are carried out by the manufacturer themselves.

[0005] In step (d), the virus originally adapted to infect humans is converted into a virus that grows at a high titer under industrial growth conditions. For influenza A virus, this step typically involves generating a 6:2 reassorted strain, which contains the HA and NA encoding genomic segments from the strain selected in (c) and the remaining six genomic segments from a strain that grows efficiently in chicken eggs, and this strain is usually A / PR / 8 / 34. After this reassortment procedure, repeated passage of this strain in developing eggs allows for adaptation to eggs and increased growth. For influenza B virus, ancestral strains with good growth characteristics are usually obtained by repeated passage directly in developing eggs without attempting to generate a reassortment.

[0006] Therefore, the steps taken before release to vaccine manufacturers include the step of passage the influenza virus through eggs. Even if the manufacturer grows the virus on a cell culture medium instead of eggs in step (g), the virus will be passaged through eggs at some point between isolation in step (a) and receipt by the manufacturer in step (e).

[0007] For example, step (a) involves exposing a culture medium to a patient sample to infect the medium with any viruses present in the sample. The culture medium can then amplify the amount of virus present, and the amplified virus is available for further study. This step may be performed in eggs or mammalian cells. Cells known to be used for primary isolation include MRC-5 cells[3], Vero cells[4, 5], MDCK cells[6], HepG2 cells[7], LLC-MK2 cells[8], etc. However, eggs continue to be commonly used to isolate reference strains for the manufacture of influenza vaccines. The use of eggs is so important to current procedures that in the 2003-04 season, the FDA rejected the use of the most suitable H3N2 strain (A / Fujian / 411 / 2002) on the grounds that this strain was not originally isolated in eggs[2(Non-Patent Literature 2), 9] and that antigenically similar egg-isolated strains were unavailable.

[0008] It has long been proposed to eliminate the use of eggs from various stages of influenza virus production.

[0009] Reference 10 (Patent Document 1) suggests that if the isolate or reassembled virus has not been passaged in bird eggs, the vaccine should be grown in cell cultures using either (i) a highly viable strain of a passaged clinical isolate, or (ii) a reassembled virus derived from at least one naturally occurring mammalian influenza virus strain. Thus, the method described in Reference 10 (Patent Document 1) begins with a seed virus that has already been selected or manipulated for growth in a selected cell culture.

[0010] Reference 11 (Patent Document 2) compares viruses passaged through eggs with viruses passaged through MDCK cells, but specifically selects the former for vaccine production.

[0011] Reference 12 (Non-Patent Literature 3) suggests that seed viruses for pandemic influenza vaccines can be prepared by directly growing pandemic strains on mammalian cell cultures without using fertilized chicken eggs, but states that egg passage is mandatory for inter-pandemic production. The reason egg passage is mandatory is that it is thought to act as a "filter" against adventitious agents: regulatory bodies accept that a series of passages in avian lines between the original clinical isolation from humans and the final vaccine for human administration prevents mammalian adventitious agents from replicating alongside the influenza virus. [Prior art documents] [Patent Documents]

[0012] [Patent Document 1] U.S. Patent No. 6,344,354 [Patent Document 2] U.S. Patent No. 5,162,112 [Non-patent literature]

[0013] [Non-Patent Document 1] Gerdil Vaccine (2003) 21:1776-9 [Non-Patent Document 2] Palese Emerging Infectious Diseases(2006)12:61-65 [Non-Patent Document 3] Medema et al. Virus Res.(2004)103(1-2):9-15 [Overview of the Initiative] [Problems that the invention aims to solve]

[0014] The present invention aims to provide a useful further procedure in the manufacture of influenza vaccines such that the use of eggs is reduced, and preferably completely avoided. In one particular aspect, the present invention aims to provide a useful further improved procedure in the isolation of influenza viruses. [Means for solving the problem]

[0015] Disclosure of the invention While it has been conventionally proposed to eliminate the use of eggs from various stages of influenza virus production, the present invention differs from these proposals in several respects. This invention provides, for example, the following items: (Item 1) A method for preparing influenza seed viruses for vaccine production, (i) The step of infecting a cell line with influenza virus obtained directly from a patient or from a primary isolate; (ii) Passaging the virus from the infected cell line obtained in step (i) at least once; (iii) the step of culturing the infected cell line from step (ii) to generate an influenza virus for use as a seed virus A method comprising, wherein the influenza virus used in step (i) is either an influenza B virus or an H1 or H3 strain of influenza A virus. (Item 2) A method for preparing influenza seed viruses for vaccine production, (i) The step of infecting a cell line with influenza virus obtained directly from a patient or from a primary isolate; (ii) preparing cDNA of at least one viral RNA segment of the influenza virus produced by the infected cell line obtained in step (i), and using the cDNA in a reverse genetics procedure to prepare a new influenza virus having at least one viral RNA segment similar to the influenza virus of step (i); (iii) The step of infecting a cell line with the new influenza virus and then culturing the cell line to produce the new influenza virus for use as a seed virus; Methods that include... (Item 3) The method described in Item 1, wherein the passage in step (ii) is carried out using the same type of cells as those used in step (i). (Item 4) The method according to any one of Items 1 to 3, wherein step (i), (ii), or (iii) does not involve the growth or passage of influenza virus in an egg. (Item 5) The method according to any one of Items 1 to 4, wherein the cell line is a mammalian cell line other than human. (Item 6) The method according to any one of items 1 to 5, wherein the cell line is an MDCK cell line. (Item 7) The seed virus is sequenced according to the method described in any one of Items 1 to 6. (Item 8) The seed virus is used to induce an antiserum, as described in any one of Items 1 to 7. (Item 9) The method described in any one of Items 1 to 8, wherein the seed virus is used to prepare a viable seed lot. (Item 10) The seed virus is used for vaccine production, as described in any one of Items 1 to 9. (Item 11) The method according to any one of Items 1 to 10, wherein the genome of the seed virus does not have a PR / 8 / 34 segment. (Item 12) The method according to any one of Items 1 to 11, wherein the seed virus has a hemagglutinin that preferentially binds to oligosaccharides having a Sia(α2,6)Gal terminal disaccharide rather than to oligosaccharides having a Sia(α2,3)Gal terminal disaccharide. (Item 13) A method for preparing influenza viruses for vaccine production, (i) the step of obtaining an influenza virus circulating in a population, or an influenza virus having a hemagglutinin that antigenically represents an influenza virus circulating in a population; (ii) The step of infecting a cell line with the influenza virus obtained in step (i); (iii) A step of passing the virus from the infected cell line obtained in step (ii) at least once to produce a seed strain; (iv) The step of culturing the seed strain from step (iii) in order to generate an influenza virus Methods that include... (Item 14) A method for preparing influenza viruses for vaccine production, (i) the step of obtaining an influenza virus circulating in a population, or an influenza virus having a hemagglutinin that antigenically represents an influenza virus circulating in a population; (ii) The step of infecting a cell line with the influenza virus obtained in step (i); (iii) preparing cDNA of at least one viral RNA segment of the influenza virus produced by the infected cell line obtained in step (i), and using the cDNA in a reverse genetics procedure to prepare an influenza seed virus having at least one viral RNA segment similar to the influenza virus of step (i); (iv) Infecting a cell line with the influenza seed virus, and then culturing the passaged cell line from step (iii) to produce the influenza virus. Methods that include... (Item 15) A method for preparing an influenza virus vaccine, comprising steps (i) to (iv) of Item 13 or 14, and then (v) A step of processing the virus obtained in step (iv) to produce a vaccine and Methods that include... (Item 16) The method according to Item 15, wherein step (v) is a step of inactivating the virus. (Item 17) The method described in Item 16, wherein the vaccine is a whole virion vaccine. (Item 18) The method described in Item 16, wherein the vaccine is a hydrolyzable virion vaccine. (Item 19) The method according to Item 16, wherein the vaccine is a surface antigen vaccine. (Item 20) The method described in Item 16, wherein the vaccine is a virosomal vaccine. (Item 21) The method according to any one of Items 15 to 20, wherein the vaccine contains less than 10 ng of residual host cell DNA per dose. (Item 22) A method for producing a polyvalent influenza vaccine, The step of performing the method described in any one of items 15 to 21 for multiple individual influenza virus strains, The steps include: mixing the individual vaccines to produce the polyvalent influenza vaccine; Methods that include... (Item 23) The method according to Item 22, wherein the polyvalent influenza vaccine comprises two influenza A virus strains and one influenza B virus strain. (Item 24) The method described in any one of items 15 to 22, wherein the vaccine is substantially mercury-free. (Item 25) The vaccine is an adjuvant, as described in any one of items 15-24. (Item 26) A method for preparing antiserum from animals, (i) The step of administering purified influenza virus hemagglutinin to the animal; then (ii) The step of recovering serum containing an antibody that recognizes the hemagglutinin from the animal. A method comprising, wherein the hemagglutinin used in step (i) is derived from a virus grown in a cell line. (Item 27) A method for preparing antiserum from animals, (i) Steps for growing influenza virus in a cell line; (ii) A step of purifying hemagglutinin antigens from the virus grown in step (i); (iii) the step of administering the purified hemagglutinin from step (ii) to the animal; then (iv) The step of recovering serum containing an antibody that recognizes the hemagglutinin from the animal. Methods that include... (Item 28) The method described in Item 26 or 27, wherein the animal is a sheep. (Item 29) The method according to any one of items 26-28, further comprising the step of mixing the antiserum with a gel suitable for the SRID assay. (Item 30) Antiserum obtained by the method described in any one of items 26-28. (Item 31) A method for preparing antigen reference material, (i) Steps for growing influenza virus in a cell line; (ii) A step of purifying the virus grown in step (i); (iii) a step of inactivating the virus, wherein the influenza virus used in step (i) has never been grown in an egg; (iv) A method comprising the step of freeze-drying the inactivated virus. (Item 32) A method for isolating influenza virus from a patient sample, The step includes incubating the patient's sample with MDCK cells, The MDCK cells are grown in suspension culture. (Item 33) A method for isolating influenza virus from a patient sample, The step includes incubating the patient's sample with MDCK cells, The MDCK cells are grown in serum-free culture medium. (Item 34) A method for isolating influenza virus from a patient sample, The step includes incubating the patient's sample with MDCK cells, The MDCK cells are grown in a protein-free culture medium. (Item 35) A method for isolating influenza virus from a patient sample, A method comprising the step of incubating a sample from the patient with non-tumor-forming MDCK cells. (Item 36) A method for isolating influenza virus from a patient sample, The step includes incubating the patient's sample with MDCK cells, The MDCK cells are not provided in a stratified culture medium in this method. (Item 37) A method for isolating influenza virus from a patient sample, The step includes incubating the patient's sample with MDCK cells, The MDCK cells are grown in serum-free suspension culture. (Item 38) A method for isolating influenza virus from a patient sample, The step includes incubating the patient's sample with MDCK cells, The MDCK cells are grown in a protein-free suspension culture. (Item 39) A method for isolating influenza virus from a patient sample, The step includes incubating a sample from the patient with non-tumor-forming MDCK cells, The MDCK cells are grown in suspension culture. (Item 40) A method for isolating influenza virus from a patient sample, The step includes incubating a sample from the patient with non-tumor-forming MDCK cells, The MDCK cells are grown in serum-free suspension culture. (Item 41) A method for isolating influenza virus from a patient sample, The step includes incubating a sample from the patient with non-tumor-forming MDCK cells, The MDCK cells are grown in a protein-free suspension culture. (Item 42) Influenza virus isolated by any one of the methods described in items 32-41. (Item 43) A method for preparing reassembled influenza virus, (i) Infecting a cell line with both a first strain of influenza virus having a first set of genomic segments and a second strain of influenza virus having a second set of genomic segments, wherein the first strain has an HA segment encoding a desired hemagglutinin; (ii) The step of culturing the infected cells from step (i) to produce an influenza virus having at least one segment from the first set of genomic segments and at least one segment from the second set of genomic segments, The method wherein at least one segment from the first set of genomic segments comprises the HA segment from the first strain. (Item 44) A method for preparing influenza virus antigen for use in a vaccine, (i) the step of receiving an influenza virus that has never been grown in an egg culture medium; (ii) the step of infecting a cell line with the influenza virus; (iii) The step of culturing the infected cells from step (ii) in order to generate an influenza virus Methods that include... (Item 45) A method for preparing influenza virus antigen for use in a vaccine, (i) The step of receiving the influenza virus isolated in MDCK33016 cells; (ii) the step of infecting a cell line with the influenza virus; (iii) The step of culturing the infected cells from step (ii) in order to generate an influenza virus Methods that include... (Item 46) A method for preparing influenza virus antigen for use in a vaccine, (i) The step of receiving an influenza virus that has never been grown in a culture medium in which it grows in serum-containing medium; (ii) the step of infecting a cell line with the influenza virus; (iii) The step of culturing the infected cells from step (ii) in order to generate an influenza virus Methods that include... (Item 47) A method for preparing influenza virus antigen for use in a vaccine, (i) the step of receiving an influenza virus generated using reverse genetics technology; (ii) the step of infecting a cell line with the influenza virus; (iii) The step of culturing the infected cells from step (ii) in order to generate an influenza virus Methods that include... (Item 48) A method for preparing influenza virus antigen for use in a vaccine, (i) The step of receiving an influenza A virus having fewer than six viral segments from the PR / 8 / 34 influenza virus; (ii) the step of infecting a cell line with the influenza virus; (iii) The step of culturing the infected cells from step (ii) in order to generate an influenza virus Methods that include... (Item 49) A method for preparing influenza virus antigen for use in a vaccine, (i) The step of receiving an influenza A virus having fewer than six viral segments from an AA / 6 / 60 influenza virus; (ii) the step of infecting a cell line with the influenza virus; (iii) The step of culturing the infected cells from step (ii) in order to generate an influenza virus Methods that include... (Item 50) A method for preparing influenza virus antigen for use in a vaccine, (i) A step of receiving an influenza virus having a hemagglutinin that preferentially binds to oligosaccharides having a Sia(α2,6)Gal terminal disaccharide rather than to oligosaccharides having a Sia(α2,3)Gal terminal disaccharide; (ii) the step of infecting a cell line with the influenza virus; (iii) The step of culturing the infected cells from step (ii) in order to generate an influenza virus Methods that include... (Item 51) A method for preparing influenza virus antigen for use in a vaccine, (i) A step of receiving an influenza virus having hemagglutinins and / or neuraminidase glycoforms not found in chicken eggs; (ii) the step of infecting a cell line with the influenza virus; (iii) The step of culturing the infected cells from step (ii) in order to generate an influenza virus Methods that include...

[0016] Preparation of seed viruses A first aspect of the present invention provides a method for preparing an influenza seed virus for vaccine production, the method comprising: (i) infecting a cell line with an influenza virus obtained directly from a patient or from a primary isolate; (ii) passage the virus from the infected cell line obtained in step (i) at least once; and (iii) culturing the infected cells from step (ii) to produce an influenza virus. The influenza virus purified from the culture in step (iii) can be used as a seed virus.

[0017] In contrast to reference 12, the influenza virus used in step (i) is either influenza B virus or a non-pandemic influenza A virus, i.e., currently H1N1 or H3N2 strains of influenza A.

[0018] Steps (i), (ii), and (iii) do not involve viral growth or passage in the egg. Preferably, at least two of these steps, and ideally all three, are carried out within the same cell type, for example, all within MDCK cells.

[0019] The passage in step (ii) typically includes the following steps: replicating the influenza virus in a cell culture; recovering the replicated virus from, for example, the culture supernatant; and transferring the recovered replicated virus to an uninfected cell culture. This method can be repeated. After at least one passage, in step (iii), the virus is replicated and recovered, but this virus is used as a seed virus rather than being transferred to an uninfected culture for further passage.

[0020] The cell line used in the first phase is preferably not a human cell line. By avoiding the use of human cells, accidental "filtration" of substances can be maintained without using eggs. Due to its close relationship with humans, the cell line is also preferably not a primate cell line, for example, not Vero cells (derived from monkey kidneys). Reference 10 suggests using Vero cells in vaccine production, but since these cells tolerate many human viruses, any other human viruses present in the influenza virus sample used in step (i) can grow alongside the influenza virus, resulting in contamination of the final seed virus.

[0021] A preferred cell line for use in conjunction with the first aspect of the present invention is a canine cell line, such as the MDCK cell line (Madin Darby canine kidney), which is incompatible with the specific teachings of Reference 10. Further details of MDCK cells are given below. It has now been found that MDCK cells exhibit a "filtration" effect against incidental substances equivalent to that obtained from bird eggs. Therefore, based on this finding, MDCK cells can be used instead of eggs without increasing the risk of regulation, and although Reference 13 reports that egg passage in influenza virus is advantageous for growth in MDCK culture, growth in eggs is avoided.

[0022] Reference 14 discloses that influenza strains isolated from human patients without subculturing in eggs or cell cultures can be efficiently grown in MDCK cell cultures, including serum-free cultures. Therefore, infection in step (i) may use influenza viruses from clinical samples obtained directly from patients (e.g., from pharyngeal swabs), or it may use viruses that have already been primary isolated. In some situations, primary isolation prior to step (i) may be performed in eggs, but the primary isolates that are preferably used in conjunction with the present invention are those obtained without using eggs, for example, in mammalian cells. Cells known to be used for primary isolation include, but are not limited to, MRC-5 cells[3], Vero cells[4,5], MDCK cells[6], HepG2 cells[7], LLC-MK2 cells[8], etc.

[0023] When the present invention uses a primary isolate in step (i), it is preferable that the primary isolation is performed in the same cell type as in steps (i) to (iii). It is already known that MDCK is suitable for the primary isolation, passage, and growth of influenza virus, but improvements in MDCK isolation are described below.

[0024] To maximize knowledge about the history of influenza virus isolates, it is preferable to use viruses obtained directly from clinical isolates rather than primary isolates. Current influenza surveillance systems involve primary isolation in hospitals, and the target strains are sent to national and international influenza centers. In addition to using patient samples for primary isolation, a portion of the sample is typically set aside (e.g., by freezing) for storage so that it can be returned to the original material for re-isolation. When such stored samples are available, they can be used in place of the primary isolate in step (i).

[0025] When the history of the isolate is unclear, particularly when starting from a virus not directly obtained from a clinical sample, reverse genetics can be used between steps (i) and (iii) to generate a new viral strain having at least one viral genome segment from the parent virus. By using reverse genetics between steps (i) and (iii), the viral product used in step (i) is separated from the viral product used in step (iii), thus acting as a filter for any accidental material that may have been introduced before step (i). In addition to being used as a “filter” in this embodiment, reverse genetics techniques may be used for other reasons, such as generating reassembled structures, manipulating coding sequences, or substituting specific segments. Further details are shown below in relation to a second aspect of the present invention.

[0026] Using reverse genetics in conjunction with influenza virus cell culture A second aspect of the present invention provides a method for preparing an influenza seed virus for vaccine production, the method comprising: (i) infecting a cell line with an influenza virus obtained directly from a patient or from a primary isolate; (ii) preparing cDNA of at least one viral RNA segment of the influenza virus produced by the infected cell line obtained in step (i), and using the cDNA in a reverse genetics procedure to prepare a novel influenza virus having at least one viral RNA segment similar to the influenza virus of step (i); and (iii) infecting a cell line with the novel influenza virus and then culturing the cell line to produce the novel influenza virus.

[0027] The virus used in step (i) may be any subtype of influenza A virus or influenza B virus. Preferred subtypes of influenza A virus are H1, H3, and H5.

[0028] Steps (i), (ii), and (iii) do not involve viral growth or passage in the egg. Preferably, all of these steps are carried out within the same cell type, for example, all within MDCK cells.

[0029] Further features of this second aspect of the present invention are the same as those described above for the first aspect, namely, the preference for the use of MDCK cells.

[0030] The reverse genetics technique is described in more detail below. The genomic segment transferred in step (ii) includes an HA segment and may also include an NA segment and / or one or more further segments.

[0031] Seed virus The first and second aspects of the present invention provide seed viruses. These seed viruses can be used in a variety of ways.

[0032] Characterizing seed viruses allows us to, for example, determine the sequences of their nucleic acids and / or proteins, check their antigenic relationships to other strains (e.g., circulating strains), and check their immunogenicity. Typically, this involves determining the sequence of the viral HA gene to reveal the HA amino acid sequence.

[0033] Seed viruses may be used to induce antisera.

[0034] Seed viruses may be distributed to vaccine manufacturers.

[0035] Seed viruses may be stored for future use.

[0036] Seed viruses may be used to prepare working seed lots. This system allows for the safe storage of the original seed virus while using a valid seed lot for daily use. The valid seed may be frozen until needed. Preparation of a valid seed lot may preferably include a step of growing the virus in a cell culture of the same cell type used to prepare the seed virus. Growth in eggs is not used in the preparation of a valid seed lot.

[0037] Seed viruses may be used to infect cell lines for growth to provide the virus for vaccine production, or for use in the preparation of diagnostic tests.

[0038] The seed virus of the present invention shares many characteristics with existing egg-derived seed viruses, but may differ in various aspects.

[0039] For example, as described in more detail below, the preferred influenza A seed virus of the present invention comprises fewer than six (i.e., 0, 1, 2, 3, 4, or 5) viral segments from the PR / 8 / 34 influenza virus.

[0040] Egg-free vaccine manufacturing A third aspect of the present invention provides a method for preparing an influenza virus for vaccine production, the method comprising: (i) obtaining an influenza virus circulating in a population or an influenza virus having a hemagglutinin antigenically representative of an influenza virus circulating in a population; (ii) infecting a cell line with the influenza virus obtained in step (i); (iii) passage the virus obtained from the infected cell line in step (ii) at least once to produce a seed strain; and (iv) culturing the seed strain from step (iii) to produce an influenza virus.

[0041] In the same manner as the first and second aspects are distinct from each other (see above), a fourth aspect of the present invention provides a method for preparing an influenza virus for vaccine production, the method comprising: (i) obtaining an influenza virus circulating in a population or an influenza virus having a hemagglutinin antigenically representative of an influenza virus circulating in a population; (ii) infecting a cell line with the influenza virus obtained in step (i); (iii) preparing cDNA of at least one viral RNA segment of the influenza virus produced by the infected cell line obtained in step (i), and using the cDNA in a reverse genetics procedure to prepare an influenza seed virus having at least one viral RNA segment similar to the influenza virus of step (i); and (iv) infecting a cell line with the influenza seed virus and then culturing the cell line passaged from step (iii) to produce an influenza virus.

[0042] The present invention also provides a method for preparing an influenza virus vaccine, the method comprising steps (i) to (iv) of a third or fourth phase, and a subsequent step (v) of processing the virus obtained in step (iv) to produce a vaccine. Details of the technique used in step (v) are shown below.

[0043] Steps (i), (ii), (iii), (iv), or (v) do not involve the growth or passage of the virus in the egg.

[0044] The virus used in step (i) may be any subtype of influenza A virus or influenza B virus. Preferred subtypes of influenza A virus are H1, H3, and H5, such as H1N1 or H3N2.

[0045] The virus used in step (i) has not been passaged in the egg since its primary isolation, and preferably has not been passaged in the egg at any stage between the patient who originally provided the virus and the start of step (i).

[0046] The term "antigenically expressive" is used in the field of influenza vaccine technology to refer to a viral strain that may not actually be present in widespread population circulation, but is convenient for manufacturing purposes and elicits an immune response that can protect against circulating strains. Serum elicited by an "antigenically expressive" strain can inhibit circulating strains, for example, in hemagglutination inhibition assays.

[0047] Step (i) may include a step of selecting a strain for further use, starting from several different strains. For example, this step may include a step of selecting a strain for further use, starting from several different H1N1 strains of influenza A virus. This step may include a step of selecting a strain for further use, starting from several different H3N2 strains of influenza A virus. This step may include a step of selecting a strain for further use, starting from several different strains of influenza B virus. This selection is based on immunological and serological criteria routinely used when selecting strains for an influenza virus contaminant, such as selecting a strain that antigenically represents the most common and / or most pathogenic strains in circulation.

[0048] Step (iii) may be followed by a step to confirm that the seed virus antigenically represents the strain obtained in step (i). This verification step may be performed before step (iv) begins or concurrently with step (iv).

[0049] The present invention also provides a method for preparing an influenza virus vaccine, the method comprising (a) obtaining a virus prepared by a method comprising either steps (i) to (iii) of the first phase or steps (i) to (iv) of the second phase; and (b) processing the virus to produce a vaccine.

[0050] Therefore, as a whole, the third and fourth aspects of the present invention enable the production of an influenza vaccine from a patient sample (or primary isolate), and the influenza virus used to prepare the vaccine is not passaged through eggs at any stage.

[0051] Virus regrouping A fifth aspect of the present invention provides a method for preparing a reassembled influenza virus, the method comprising the step of (i) infecting a cell line with both a first strain of influenza virus having a first set of genomic segments and a second strain of influenza virus having a second set of genomic segments, wherein the first strain has an HA segment encoding a desired hemagglutinin, and the method further comprises (ii) culturing the infected cells from step (i) to produce an influenza virus having at least one segment from the first set of genomic segments and at least one segment from the second set of genomic segments, wherein the at least one segment from the first set of genomic segments contains an HA segment from the first strain. Thus, the method can generate a new reassembled strain having a different set of viral genomic segments from either the first or second strain without using eggs, by transferring at least an HA segment from the first strain to the second strain.

[0052] The influenza virus purified from the culture in step (ii) may be used as described elsewhere in this specification. The reassembled strain may exhibit improved growth characteristics compared to the first strain.

[0053] This reassembled material may include an NA segment from a first strain encoding a desired neuraminidase.

[0054] This reassembly typically contains segments from the first and second stocks in ratios of 1:7, 2:6, 3:5, 4:4, 5:3, 6:2, or 7:1. Typically, the majority of the stock comes from the second stock.

[0055] This method may be used to generate a reassembly of influenza A virus and influenza B virus. In some embodiments, the second strain for influenza A virus is PR / 8 / 34, but other strains may also be used that share only one, two, three, four, or five of the segments NP, M, NS, PA, PB1, or PB2 with PR / 8 / 34.

[0056] The present invention also provides influenza viruses that can be obtained by a fifth-phase reassembly method. The present invention also provides the use of such viruses in vaccine production.

[0057] Neither step (i) nor (ii) involves the growth, reassembly, or passage of the virus in the egg. As described elsewhere in this specification, the reassembly method can be conveniently carried out in the same cell type (e.g., MDCK cells) used for isolation and passage.

[0058] The first strain may conveniently be an influenza virus obtained directly from a patient or an influenza virus obtained from a primary isolate.

[0059] Virus isolation As mentioned above, eggs are strongly used for the isolation of influenza viruses. A sixth aspect of the present invention is the use of MDCK cells instead. In some embodiments, MDCK cells are grown in a suspension. In other embodiments, MDCK cells are grown in serum-free or protein-free medium. In other embodiments, MDCK cells are non-tumorogenic. In other embodiments, MDCK cells are not grown in the presence of stratified medium.

[0060] Therefore, the present invention provides a method for isolating influenza virus from a patient sample, the method comprising the step of incubating the patient sample with MDCK cells, where the MDCK cells are grown in suspension culture.

[0061] In addition, the present invention provides a method for isolating influenza virus from a patient sample, the method comprising the step of incubating the patient sample with MDCK cells, where the MDCK cells are grown in serum-free medium.

[0062] In addition, the present invention provides a method for isolating influenza virus from a patient sample, the method comprising the step of incubating the patient sample with MDCK cells, where the MDCK cells are grown in a protein-free medium.

[0063] In addition, the present invention provides a method for isolating influenza virus from a patient sample, the method comprising the step of incubating the patient sample with non-tumor-forming MDCK cells.

[0064] In addition, the present invention provides a method for isolating influenza virus from a patient sample, the method comprising the step of incubating the patient sample with MDCK cells, wherein the MDCK cells are not provided in stratified medium.

[0065] In addition, the present invention provides a method for isolating influenza virus from a patient sample, the method comprising the step of incubating the patient sample with MDCK cells, where the MDCK cells are grown in serum-free suspension culture.

[0066] In addition, the present invention provides a method for isolating influenza virus from a patient sample, the method comprising the step of incubating the patient sample with MDCK cells, in which the MDCK cells are grown in a protein-free suspension culture.

[0067] In addition, the present invention provides a method for isolating influenza virus from a patient sample, the method comprising the step of incubating the patient sample with non-tumor-forming MDCK cells, where the MDCK cells are grown in suspension culture.

[0068] In addition, the present invention provides a method for isolating influenza virus from a patient sample, the method comprising the step of incubating the patient sample with non-tumor-forming MDCK cells, where the MDCK cells are grown in serum-free suspension culture.

[0069] In addition, the present invention provides a method for isolating influenza virus from a patient sample, the method comprising the step of incubating the patient sample with non-tumor-forming MDCK cells, where the MDCK cells are grown in a protein-free suspension culture.

[0070] The present invention also provides influenza viruses isolated by one of these methods. The present invention also provides the use of such viruses in vaccine production.

[0071] Incubation of patient samples with MDCK cells typically results in infection of MDCK cells with influenza viruses, such as human influenza viruses, particularly human influenza A viruses. The viruses can replicate within the cells, and the replicated viruses can then be recovered. Optionally, these viruses can be used in downstream method steps, such as detection, characterization, analysis, seed virus preparation, and manipulation.

[0072] After isolation in MDCK cells, the virus may be passaged and / or grown in MDCK cells. Alternatively, the virus may be passaged and / or grown in non-MDCK cells, eggs, or another culture medium.

[0073] A sixth aspect of the present invention involves the use of the MDCK cell line. While the original MDCK cell line is available from ATCC, the sixth aspect of the present invention utilizes derivatives of this cell line. Isolation of these derivatives has been shown to be superior to isolation of the original MDCK cell line, as described below. Preferred MDCK cells and their characteristics are discussed in more detail below.

[0074] For example, some embodiments of the sixth phase use MDCK cell lines that can spontaneously replicate in suspension culture. Reference 30 discloses an MDCK cell line adapted for growth in suspension culture, and this cell line ("MDCK33016") is particularly useful for the methods of the sixth phase. MDCK33016 can grow in serum-free cultures and can grow without the need for stratified medium. Another MDCK cell line that can grow in suspension cultures, including serum-free cultures, is the "B-702" cell line [36; see below].

[0075] Non-tumor-forming MDCK cell lines for use in conjunction with the sixth phase include the cell lines disclosed in Reference 37, such as "MDCK-S," "MDCK-SF101," "MDCK-SF102," and "MDCK-SF103" (see below).

[0076] In some embodiments of the sixth aspect, MDCK cells are grown in serum-free and / or protein-free media.

[0077] Unlike some specific prior art methods, a sixth aspect of the present invention is that it avoids the need to use stratified media when isolating influenza viruses.

[0078] As mentioned above, it is preferable that the virus does not grow in the egg before being exposed to MDCK cells. In addition, as mentioned above, it is preferable that the virus that has grown in MDCK cells does not subsequently grow in the egg.

[0079] Regarding patient samples used in the sixth phase, clinical samples used for influenza virus isolation can take various forms, but typically include, but are not limited to, respiratory secretions such as: direct aspirates; gargles; nasal irrigation solutions; nasal swabs; throat swabs; pharyngeal swabs, etc. These samples are generally taken from patients suspected of having influenza virus infection, including those who may carry new strains of the influenza virus.

[0080] The influenza virus isolated by the method of the sixth phase may be used to prepare an influenza seed virus for vaccine production. Thus, the method of the sixth phase may include a further step of passage the virus from the infected MDCK cell line at least once. The method may then include a step of culturing the infected cells to produce the influenza virus. The influenza virus purified after this culturing step may be used as a seed virus as described elsewhere in this specification.

[0081] The virus isolated according to the sixth phase may be used as a source for reverse genetics techniques. That is, cDNA may be prepared from at least one viral RNA segment of the influenza virus isolated according to the sixth phase. This cDNA may then be used in a reverse genetics procedure to prepare a new influenza virus having at least one viral RNA segment similar to that of the isolated influenza virus. This new influenza virus may then be used to infect cell lines, for example, for further culture.

[0082] A sixth aspect of the present invention may be used to isolate any suitable influenza virus, including human influenza viruses. These influenza viruses may be influenza A viruses, influenza B viruses, or influenza C viruses. Influenza A viruses are typical, and useful influenza A virus subtypes are H1, H3, and H5.

[0083] Vaccines in the current interpandemic phase typically contain two influenza A strains (H1N1 and H3N2) and one influenza B strain. The sixth phase may be used to isolate such strains, or to isolate pandemic virus strains such as H2, H5, H7, or H9 subtype strains. Generally, the sixth phase may be used to isolate influenza A viruses having one of the HA subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16.

[0084] Influenza viruses isolated according to the sixth aspect of the present invention may contain hemagglutinins that preferentially bind to oligosaccharides having a Sia(α2,6)Gal terminal disaccharide rather than oligosaccharides having a Sia(α2,3)Gal terminal disaccharide. Advantageously, the isolation method of the present invention has been found to help stably preserve the HA sequence of the virus and its oligosaccharide preference.

[0085] Viruses isolated according to the sixth phase may be used in vaccine manufacturing and treatment methods. Thus, the present invention also provides the use of antigens prepared from viruses isolated according to the sixth phase in the manufacture of drugs for enhancing the immune response in patients.

[0086] Receptor binding Human influenza viruses bind to receptor oligosaccharides that have a Sia(α2,6)Gal terminal disaccharide (sialic acid linked to galactose via an α-2,6 bond), but eggs instead have receptor oligosaccharides with a Sia(α2,3)Gal terminal disaccharide. When human influenza viruses are grown in eggs, hemagglutinins are subjected to selective pressure that causes them to move away from Sia(α2,6)Gal binding and towards Sia(α2,3)Gal binding.

[0087] Similar to eggs, Vero cells primarily express the Sia(α2,3)Gal receptor

[15] . In contrast, MDCK cells and PER.C6 cells express both Sia(α2,3)Gal and Sia(α2,6)Gal. Reference 16 reports that overexpression of α-2,6-sialyltransferase by transfection of MDCK cells favors the selection of Sia(α2,6)Gal binding. However, it is possible to grow influenza viruses in MDCK cells without directing them towards Sia(α2,3)Gal binding even without such manipulation. Therefore, the present invention can use cells that express both Sia(α2,3)Gal and Sia(α2,6)Gal, but can generate influenza viruses that preferentially bind to oligosaccharides having a Sia(α2,6)Gal terminal disaccharide rather than oligosaccharides having a Sia(α2,3)Gal terminal disaccharide.

[0088] In preferred embodiments of the first and second aspects of the present invention, the influenza virus used for infection in step (i) preferentially binds to oligosaccharides having a Sia(α2,6)Gal terminal disaccharide rather than oligosaccharides having a Sia(α2,3)Gal terminal disaccharide. This binding preference is maintained between steps (ii) and (iii), so that the influenza virus generated in step (iii) preferentially binds to oligosaccharides having a Sia(α2,6)Gal terminal disaccharide rather than oligosaccharides having a Sia(α2,3)Gal terminal disaccharide.

[0089] In preferred embodiments of the third and fourth aspects of the present invention, the influenza virus used for infection in step (ii) preferentially binds to oligosaccharides having a Sia(α2,6)Gal terminal disaccharide rather than oligosaccharides having a Sia(α2,3)Gal terminal disaccharide. This binding preference is maintained between steps (iii) and (iv), so that the influenza virus generated in step (iv) preferentially binds to oligosaccharides having a Sia(α2,6)Gal terminal disaccharide rather than oligosaccharides having a Sia(α2,3)Gal terminal disaccharide.

[0090] Various assays may be used to determine whether a particular virus preferentially binds to oligosaccharides having a Sia(α2,6)Gal terminal disaccharide rather than oligosaccharides having a Sia(α2,3)Gal terminal disaccharide. For example, reference 17 describes a solid-phase enzyme binding assay for influenza virus receptor binding activity that provides a highly sensitive quantitative measurement of affinity constants. In the solid-phase assay used in reference 18, the binding of the virus to two different sialic acid glycoproteins was evaluated (ovomucoid, having a Sia(α2,3)Gal determinant; and porcine α2-macroglobulin, having a Sia(α2,6)Gal determinant), and the assay further described evaluated the binding of the virus to the following two receptor analogues: free sialic acid (Neu5Ac) and 3'-sialyl lactose (Neu5Acα2-3Galβ1-4Glc). Reference 19 reports an assay using a polysaccharide array that can clearly distinguish the receptor preference for α2,3 or α2,6 binding. Reference 20 reports an assay based on the agglutination of human erythrocytes enzymatically modified to contain either Sia(α2,6)Gal or Sia(α2,3)Gal. Depending on the type, the assay may be performed directly with the virus itself or indirectly with hemagglutinins purified from the virus.

[0091] Reference material Current methods for producing influenza viruses involve the preparation of reference reagents for each strain, namely (i) anti-HA serum and (ii) purified whole virions. These calibrated reagents are used in an SRID assay to determine the level of HA in the bulk antigen produced by the manufacturer, enabling the manufacturer to dilute the bulk to provide a vaccine with the desired amount of HA per dose.

[0092] The serum and antigens in the reference reagent are well-suited to the current method in which the reference strain is passaged through eggs and the resulting strain is optimized for growth in eggs. However, it has been found that serum may not be well-suited to antigens generated in cell culture, which is likely due to different selective pressures in different systems. Poor reactivity between reference serum and antigen means that HA levels are underestimated, which in turn leads to (i) a smaller dose from a given bulk and (ii) an excess of HA in the vaccine.

[0093] To overcome the problem of compatibility between antigens derived from cell cultures and serums produced for egg-derived materials, the present invention provides a virus-based reference material that is not adapted for egg-based growth.

[0094] Therefore, the present invention provides a method for preparing antiserum from animals, the method comprising the steps of (i) administering purified influenza virus hemagglutinin to an animal; and (ii) recovering serum from the animal containing antibodies that recognize the hemagglutinin, characterized in that the hemagglutinin used in step (i) is from a virus grown in a cell line.

[0095] The hemagglutinin used in step (i) is preferably derived from a virus that has never grown in an egg. For example, this hemagglutinin may preferentially bind to oligosaccharides having a Sia(α2,6)Gal terminal disaccharide rather than to oligosaccharides having a Sia(α2,3)Gal terminal disaccharide.

[0096] The preferred hemagglutinins used to produce antiserum are glycosylated with polysaccharides that can be obtained from growths in mammalian cell lines such as MDCK (e.g., the cell lines described herein).

[0097] Antisera may be produced against influenza A virus and influenza B virus.

[0098] The animal is preferably a mammal, such as a goat, or more preferably a sheep. The antiserum can be conveniently prepared in sheep by extracting HA from the purified virus by treatment with bromelain, followed by purification by sedimentation on a sucrose gradient. A dose of approximately 50 μg of hemagglutinin is administered intramuscularly to the sheep in combination with Freund's complete adjuvant (FCA). A 10 μg dose may be given after two weeks, followed by 2-4 further doses at one-week intervals. The serum may then be collected. Before use, the serum may be diluted (e.g., with PBS buffer containing sodium azide) and filled into containers. This serum may be exposed to an acidic pH (e.g., pH 5 for 2 hours) to meet foot-and-mouth disease regulations.

[0099] The present invention also provides a method for preparing antiserum from animals, the method comprising: (i) growing influenza virus in a cell line; (ii) purifying hemagglutinin antigen from the virus grown in step (i); (iii) administering the purified hemagglutinin from step (ii) to an animal; and (iv) recovering serum from the animal containing antibodies that recognize hemagglutinin.

[0100] The present invention also provides antiserums that can be obtained by these methods.

[0101] The present invention also provides a gel containing this antiserum. That is, the above-described method for preparing antiserum from an animal may include a further step of mixing the antiserum with a gel. This gel is suitable for performing SRID assays, and for example, this gel is an agarose gel.

[0102] In addition to providing antiserum, the present invention provides antigen reference material. That is, the present invention provides a method for preparing antigen reference material, the method comprising (i) growing influenza virus in a cell line; (ii) purifying the virus grown in step (i); and (iii) inactivating the virus, characterized in that the influenza virus used in step (i) has never been grown in an egg. The method may further include (iv) freeze-drying the inactivated virus.

[0103] The virus used in step (i) has never been grown in an egg. For example, the hemagglutinin of this virus may preferentially bind to oligosaccharides having a Sia(α2,6)Gal terminal disaccharide rather than oligosaccharides having a Sia(α2,3)Gal terminal disaccharide.

[0104] The reference material does not contain egg-derived materials (e.g., it does not contain ovalbumin, ovomucoid, or chicken DNA). The glycoproteins in this reference material are glycosylated with polysaccharides that can be obtained from growths in mammalian cell lines such as MDCK (e.g., the cell lines described herein). The reference material may be generated for influenza A virus and influenza B virus.

[0105] Since reference materials are usually used in pairs, the present invention also provides a kit comprising (i) an antiserum that can be obtained by these methods and (ii) an antigen reference material that can be obtained by these methods.

[0106] The present invention also provides a method for preparing a kit, the method comprising (i) the step of preparing an antiserum as described above; (ii) the step of preparing an antigen reference material as described above; and (iii) the step of combining the products of steps (i) and (ii) to form a kit.

[0107] This antigen and antiserum are suitable and intended for use in SRID assays, and the present invention provides a single radial immunodiffusion assay against influenza virus hemagglutinin, characterized by the use of antiserum and / or antigen reference material that can be obtained by these methods. The SRID assay comprises the steps of preparing a gel containing the antiserum, applying the antigen reference material (converted in aqueous medium if necessary) to the gel (typically in wells), and then diffusing the antigen radially into the gel. This antigen may be treated with a surfactant such as Zwittergent surfactant before use.

[0108] Viruses (including seed viruses) prepared or isolated by the technology of the present invention The preferred influenza A viruses of the present invention (including seed viruses, viruses isolated from patient samples using MDCK cells, reassembled viruses, etc.) contain fewer than six (i.e., 0, 1, 2, 3, 4, or 5) viral segments from the PR / 8 / 34 influenza virus. Preferably, this virus does not contain any PR / 8 / 34 segments. When any PR / 8 / 34 segments (one or more) are present, the segment does not contain the PR / 8 / 34 HA segment and usually does not contain the PR / 8 / 34 NA segment. Thus, in the preferred viruses, at least one of the segments NP, M, NS, PA, PB1, and / or PB2 does not originate from PR / 8 / 34. More preferably, at least one of the segments NP, M, PA, PB1, and / or PB2 does not originate from PR / 8 / 34. Thus, the present invention can improve existing vaccines by adding one or more additional epitope-containing antigens representing a circulating strain to the usual HA and NA antigens.

[0109] Similarly, a preferred influenza A virus contains fewer than six (i.e., 0, 1, 2, 3, 4, or 5) viral segments from the AA / 6 / 60 influenza virus (A / Ann Arbor / 6 / 60). Preferably, this virus does not contain any AA / 6 / 60 segments. When any AA / 6 / 60 segments (one or more) are present, they do not contain the AA / 6 / 60 HA segment and usually do not contain the AA / 6 / 60 NA segment. Thus, in a preferred virus, at least one of the segments NP, M, NS, PA, PB1, and / or PB2 does not originate from AA / 6 / 60. More preferably, at least one of the segments NP, M, PA, PB1, and / or PB2 does not originate from AA / 6 / 60.

[0110] A preferred influenza B virus contains fewer than six (i.e., 0, 1, 2, 3, 4, or 5) viral segments from the AA / 1 / 66 influenza virus (B / Ann Arbor / 1 / 66). Preferably, this virus does not contain any AA / 1 / 66 segments. When any AA / 1 / 66 segments (one or more) are present, they do not contain the AA / 1 / 66 HA segment and usually do not contain the AA / 1 / 66 NA segment. Thus, in a preferred virus, at least one of the segments NP, M, NS, PA, PB1, and / or PB2 does not originate from AA / 1 / 66. More preferably, at least one of the segments NP, M, PA, PB1, and / or PB2 does not originate from AA / 1 / 66.

[0111] The preferred influenza viruses of the present invention (including seed viruses, viruses isolated from patient samples using MDCK cells, and reassembled viruses) contain hemagglutinins that preferentially bind to oligosaccharides having a Sia(α2,6)Gal terminal disaccharide rather than oligosaccharides having a Sia(α2,3)Gal terminal disaccharide. This binding preference is discussed in more detail above.

[0112] The preferred influenza viruses of the present invention (including seed viruses, viruses isolated from patient samples using MDCK cells, reassembled viruses, etc.) contain glycoproteins (including hemagglutinins) having a glycosylation pattern different from that of egg-derived viruses. In other words, these glycoproteins contain glycoforms not found in viruses grown in chicken eggs, and may have sugar bonds other than those of birds, such as mammalian sugar bonds.

[0113] cell line The present invention involves the use of a cell line that supports the replication of the influenza virus and avoids the use of eggs. This cell line is typically derived from mammals. Suitable mammalian cells from which the cell line may be derived include, but are not limited to, cells from hamsters, cattle, primates (including humans and monkeys), and dogs, although the use of primate cells is not preferred. Various cell types may be used, such as kidney cells, fibroblasts, retinal cells, lung cells, etc. An example of a suitable hamster cell is a cell line having the name BHK21 or HKCC. Suitable monkey cells include, for example, African green monkey cells, kidney cells such as the Vero cell line [21-22, 23]. Suitable dog cells include, for example, kidney cells such as the CLDK and MDCK cell lines.

[0114] Suitable cell lines include, but are not limited to, MDCK;CHO;CLDK;HKCC;293T;BHK;Vero;MRC-5;PER.C6

[24] ;FRhL2;WI-38;and others. Suitable cell lines are widely available from, for example, the American Type Cell Culture (ATCC) collection

[25] , Coriell Cell Repositories

[26] , or the European Collection of Cell Cultures (ECACC). For example, ATCC supplies various different Vero cells under catalog numbers CCL-81, CCL-81.2, CRL-1586 and CRL-1587, and MDCK cells under catalog number CCL-34. PER.C6 is available from ECACC under deposit number 96022940. Any of these cell types can be used for growth, reassortment and / or passaging according to the present invention.

[0115] The most preferred cell lines are those with mammalian-type glycosylation. Less preferred alternatives to mammalian cell lines include avian cell lines, such as those derived from ducks (e.g., duck retina) or hens, e.g., chicken embryo fibroblasts (CEF) [e.g., references 27-28, 29], but using mammalian cells means that the vaccine can be free from avian DNA and egg proteins (such as ovalbumin and ovomucoid), thus reducing allergenicity.

[0116] The most preferred cell line for growing influenza viruses is the MDCK cell line derived from Madinderby canine kidneys [30-31, 32, 33]. The original MDCK cell line is available from ATCC as CCL-34, but derivatives of this cell line may be used. For example, reference 30 discloses an MDCK cell line adapted for growth in suspension culture ("MDCK33016" or "33016-PF", deposited as DSM ACC2219; see also references 34 and 35). Similarly, reference 36 discloses an MDCK-derived cell line for growth in suspension in serum-free culture ("B-702", deposited as FERM BP-7449). Reference 37 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" (ATCC PTA-6503). Reference 38 discloses susceptible MDCK cell lines, including "MDCK.5F1" cells (ATCC CRL-12042). Any of these MDCK cell lines can be used in conjunction with the present invention.

[0117] The virus may grow in cells in adherent culture or suspension. Microcarrier culture may be used. In some embodiments, cells may be adapted for growth in suspension.

[0118] Preferably, cell lines are grown in serum-free and / or protein-free media. In the context of the present invention, a medium that does not contain additives from human or animal serum is referred to as a serum-free medium. While cells growing in such cultures naturally contain their own proteins, a protein-free medium is understood to mean a medium in which cell growth occurs even by the exclusion (not adding) of proteins, growth factors, other protein additives, and non-serum proteins, but which may optionally contain proteins such as trypsin or other proteases that may be necessary for viral growth.

[0119] Cell lines that support influenza virus replication are preferably grown at temperatures lower than 37°C during viral replication

[39] (e.g., 30–36°C, or approximately 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C). For example, in the sixth phase, MDCK cells may be grown at these temperatures in particular during viral replication (before, during, or after the isolation step).

[0120] A method for growing influenza virus in cultured cells (for example, to grow influenza virus in MDCK cells cultured according to the sixth phase) typically includes the steps of: inoculating a cell culture with an inoculum of the strain to be grown; culturing the infected cells for a desired period for viral growth, such as determined by viral titer or antigen expression (e.g., 24 to 168 hours after inoculation); and collecting the grown virus. 50The cells are inoculated so that the ratio to cells (measured by ) is 1:500 to 1:1, preferably 1:100 to 1:5, more preferably 1:50 to 1:10. The virus is added to a cell suspension or applied to a cell monolayer, and the virus is absorbed by the cells at 25°C to 40°C, preferably 28°C to 37°C for at least 60 minutes, usually less than 300 minutes, preferably 90 to 240 minutes. The infected cell culture (e.g., monolayer) may be removed by freeze-thawing or by enzymatic action to increase the virus content of the collected culture supernatant. The collected liquid is then inactivated or cryopreserved. The cultured cells may be infected with a multiplicity of infection ("moi") of about 0.0001 to 10, preferably 0.002 to 5, more preferably 0.001 to 2. Even more preferably, the cells are infected with a moi of about 0.01. Infected cells may be collected 30 to 60 hours after infection. Preferably, cells are collected 34 to 48 hours after infection. More preferably, cells are collected 38 to 40 hours after infection. Typically, the virus is released by adding a protease (typically trypsin) during cell culture, which may be added at any suitable stage during culture, for example, before inoculation, at the same time as inoculation, or after inoculation

[39] .

[0121] In a preferred embodiment, particularly with respect to MDCK cells, the cell line is not passaged beyond 40 divisions from the master working cell bank.

[0122] The viral inoculum and viral cultures are preferably free from herpes simplex virus, respiratory syncytial virus, parainfluenza virus 3, SARS coronavirus, adenovirus, rhinovirus, reovirus, polyomavirus, birnavirus, circovirus, and / or parvovirus (i.e., tested for these viruses and giving a negative result for their contamination)

[40] . Similarly, preferred MDCK cell lines used in conjunction with the sixth phase are free from herpes simplex virus, respiratory syncytial virus, parainfluenza virus 3, SARS coronavirus, adenovirus, rhinovirus, reovirus, polyomavirus, birnavirus, circovirus, and / or parvovirus (i.e., tested for these viruses and giving a negative result for their contamination). The absence of herpes simplex virus is particularly preferred.

[0123] The MDCK cell line used in conjunction with the present invention preferably does not contain a marker for G418 resistance (see Reference 16). Therefore, this cell line may be sensitive to G418 treatment.

[0124] The cell lines used in conjunction with the present invention preferably do not contain exogenous plasmids, except for those that may be necessary for reverse genetics techniques (see Reference 16).

[0125] Reverse genetics technology As described above, the present invention can be used directly with clinical isolates or primary isolates. In addition, the present invention can be used with reassembled strains, including those generated using reverse genetics techniques [e.g., 41-42, 43, 44, 45]. Reverse genetics techniques can facilitate manipulation of coding or non-coding sequences within viral segments, or introduce mutations, by generating combinations of viral segments using in vitro manipulation of plasmids. This technique can be used for both influenza A and influenza B viruses.

[0126] Reverse genetics typically involves the expression of both types of DNA in a cell, resulting in a complete collection of infectious virions, by (a) the expression of DNA molecules encoding a desired viral RNA molecule from, for example, a polI promoter, a bacterial RNA polymerase promoter, or a bacteriophage polymerase promoter, and (b) the expression of DNA molecules encoding viral proteins from, for example, a polII promoter. This DNA preferably provides all viral RNA and proteins, but it is also possible to use helper viruses to provide some of the RNA and proteins. Plasmid-based methods using separate plasmids to generate each viral RNA are preferred [46-47, 48], and these methods also involve the use of plasmids to express all or some of the viral proteins (e.g., PB1, PB2, PA, and NP proteins only), with some methods using 12 plasmids.

[0127] To reduce the number of plasmids required, recent approaches

[49] combine multiple RNA polymerase I transcription cassettes (for viral RNA synthesis) on the same plasmid (e.g., sequences encoding all 1, 2, 3, 4, 5, 6, 7, or 8 influenza A vRNA segments) and combine regions encoding multiple proteins with RNA polymerase II promoters on a separate plasmid (e.g., sequences encoding all 1, 2, 3, 4, 5, 6, 7, or 8 influenza A mRNA transcripts). Preferred aspects of the method in reference 49 include: (a) regions encoding PB1, PB2, and PA mRNA on a single plasmid; and (b) segments encoding all 8 vRNAs on a single plasmid. The situation can also be facilitated by including the NA and HA segments on one plasmid and the other six segments on separate plasmids.

[0128] Because the polI promoter is species-specific, the canine polI promoter may be used when performing reverse genetics in MDCK cells

[50] . Instead of using the polI promoter to encode a viral RNA segment, a bacteriophage polymerase promoter can be used

[51] . For example, promoters for SP6, T3, or T7 polymerase can be conveniently used. Because the polI promoter is species-specific, bacteriophage polymerase promoters may be more convenient for many cell types (e.g., MDCK), but the cells need to be transfected with a plasmid encoding an exogenous polymerase enzyme.

[0129] In other technologies, dual polI and polII promoters can be used to simultaneously encode viral RNA and expressible mRNA from a single template [52, 53].

[0130] The strains used for egg development typically contain six RNA segments from the PR / 8 / 34 influenza A virus (the HA and N segments are from the vaccine strain, i.e., a 6:2 reassembly), but egg avoidance according to the present invention means that the PR / 8 / 34 segments can be eliminated. Thus, the influenza A virus may contain fewer than six (i.e., 0, 1, 2, 3, 4, or 5) viral segments from the PR / 8 / 34 influenza virus. A preferred virus is one in which at least one of the segments NP, M, NS, PA, PB1, and / or PB2 is not derived from PR / 8 / 34. The virus may also contain an NS segment derived from an avian influenza virus.

[0131] When the present invention employs reverse genetics, it becomes possible to transfer a viral RNA segment from a source influenza virus to the genome of a target influenza virus. Thus, these two viruses have at least one common viral RNA segment. The term “common” here means an identical copy of the entire segment, but may be broadly defined to mean a modified copy of the segment having modifications in the coding region and / or non-coding region. When the modification is made in the coding region, the modification does not substantially alter the immunogenicity and / or activity of the encoded protein. Thus, the HA segment may be manipulated around the HA1 / HA2 cleavage site without altering its ability to induce effective anti-HA antibodies when administered to a patient. Thus, reverse genetics may be used to modify the native HA of a virus isolated according to the sixth phase in order to remove a determinant that enhances viral pathogenicity in, for example, bird species (e.g., an overly basic region around the HA1 / HA2 cleavage site).

[0132] Vaccine preparation Currently, various forms of influenza virus vaccines are available (e.g., see Chapters 17 and 18 of Reference 54). Generally, vaccines are based on either live or inactivated viruses. Inactivated vaccines may be based on whole virions, "split" virions, or purified surface antigens. Influenza antigens may be supplied in the form of virosomals. The present invention can be used when manufacturing any of these types of vaccines.

[0133] The live virus includes MedImmune's FLUMIST® product (trivalent live virus). The vaccine is prepared by a method comprising the steps of growing the virus on a suitable culture medium and then purifying the virions from a liquid containing virions. For example, this liquid may be purified by centrifugation and stabilized with a buffer (e.g., containing sucrose, potassium phosphate, and monosodium glutamate).

[0134] When inactivated viruses are used, the vaccine may contain whole virions, degraded virions, or purified surface antigens (containing hemagglutinins, and usually neuraminidases). Chemical means for inactivating the virus include treatment with effective amounts of one or more of the following agents: surfactants, formaldehyde, β-propiolactone, methylene blue, psoralen, carboxyfullerene (C60), binary ethylamine, acetylethyleneimine, or combinations thereof. Non-chemical methods for virus inactivation, such as UV light or gamma irradiation, are known in the art.

[0135] Virions can be collected from a virus-containing liquid by various methods. For example, the purification method may include zone centrifugation using a linear sucrose gradient solution containing a surfactant to destroy the virions. The antigen may then be purified by membrane separation after any dilution.

[0136] Degraded virions are obtained by treating purified virions with surfactants (e.g., ethyl ether, polysorbate 80, deoxycholate, tri-N-butyl phosphate, Triton X-100, Triton N101, cetyltrimethylammonium bromide, Tergitol NP9, etc.) to produce subvirion preparations, including the "tween-ether" degradation method. Methods for degrading influenza viruses are well known in the art; see, for example, references 55-56, 57, 58, 59, 60. Viral degradation is typically carried out by destroying or fragmenting the entire virus, and the virus becomes infectious or non-infectious depending on the concentration of the degrading agent. Destruction results in complete or partial solubilization of viral proteins, altering the integrity of the virus. Preferred decomposition agents include nonionic and ionic (e.g., cationic) surfactants, such as alkyl glycosides, alkyl thioglycosides, acyl sugars, sulfobetaines, betaines, polyoxyethylene alkyl ethers, N,N-dialkyl-glucamides, Hecameg, alkylphenoxy-polyethoxyethanol, NP9, quaternary ammonium compounds, sarcosyl, CTAB (cetyl trimethyl ammonium bromides), tri-N-butyl phosphate, cetabrone, myristyltrimethylammonium salts, lipofectin, lipofectamine, and DOT-MA, as well as octyl- or nonylphenoxy polyoxyethanol (e.g., Triton surfactants, such as Triton X-100 or Triton N101), polyoxyethylene sorbitan esters (twin surfactants), polyoxyethylene ethers, and polyoxyethylene esters. One useful decomposition procedure involves the sequential effects of sodium deoxycholate and formaldehyde, where decomposition can occur during the initial virion purification (e.g., in a sucrose density gradient solution).Therefore, the decomposition method may include purifying the virion-containing material (to remove non-virion material), concentrating the collected virions (e.g., using an adsorption method such as CaHPO4 adsorption), separating all virions from the non-virion material, decomposing the virions by using a decomposing agent in a density gradient centrifugation step (e.g., using a sucrose gradient containing a decomposing agent such as sodium deoxycholate), and removing undesirable materials by subsequent filtration (e.g., ultrafiltration). The decomposed virions can be usefully resuspended in sodium phosphate-buffered isotonic saline. BEGRIVAC®, FLUARIX®, FLUZONE®, and FLUSHIELD® products are decomposed vaccines.

[0137] Purified surface antigen vaccines contain influenza surface antigen hemagglutinins and typically neuraminidases. Methods for preparing these proteins in a purified form are well known in the art. FLUVIRIN®, AGRIPPAL®, and INFLUVAC® products are subunit vaccines.

[0138] Another form of inactivated influenza antigen is virosomes

[61] (virus-like liposome particles that do not contain nucleic acids). Virosomes can be prepared by solubilizing the influenza virus with a surfactant, then removing the nucleocapsid and reconstructing the membrane containing the viral glycoprotein. An alternative method for preparing virosomes involves adding an excess amount of viral membrane glycoprotein to phospholipids to give liposomes having the viral protein within the membrane. The present invention may also be used to store bulk virosomes, as with INFLEXAL V® and INVAVAC® products.

[0139] Influenza viruses may be attenuated. Influenza viruses may be temperature-sensitive. Influenza viruses may be adapted to low temperatures. These three characteristics are particularly useful when using live viruses as antigens.

[0140] HA is the primary immunogen in current inactivated influenza vaccines, and vaccine doses are typically standardized by reference to HA levels measured by SRID. Existing vaccines typically contain about 15 μg of HA per strain, but lower doses may be used, for example, for children, during pandemics, or when adjuvants are used. Partial doses such as 1 / 2 (i.e., 7.5 μg of HA per strain), 1 / 4, and 1 / 8 have been used [81, 82], and higher doses (e.g., 3x or 9x doses [62, 63]) have also been used. Thus, vaccines may contain 0.1 μg to 150 μg of HA per influenza strain, preferably 0.1 μg to 50 μg, for example, 0.1-20 μg, 0.1-15 μg, 0.1-10 μg, 0.1-7.5 μg, 0.5-5 μg, etc. Specific doses include, for example, approximately 45, 30, 15, 10, 7.5, 5, 3.8, 1.9, and 1.5 per strain.

[0141] The dosage for live vaccines is determined not by the HA content, but by the 50% tissue culture infectious dose (TCID). 50 ) Measured by, 10 per plant 6 from 10 8 (preferably 10) 6.5 ~10 7.5 ) TCID 50 This is typical.

[0142] The strains used in this invention may have natural HA, as found in wild-type viruses, or modified HA. For example, it is known that HA can be modified to remove determinants that enhance viral pathogenicity in bird species (e.g., excessively basic regions around HA1 / HA2 cleavage sites).

[0143] The influenza virus strains used in vaccines change from season to season. In the current interpandemic period, vaccines typically contain two influenza A strains (H1N1 and H3N2) and one influenza B strain, and trivalent vaccines are typical. The present invention may also use pandemic virus strains (i.e., strains of influenza A virus that vaccine recipients and the general human population have not immunologically received), such as H2, H5, H7, or H9 subtype strains, and influenza vaccines against pandemic strains may be monovalent or based on a standard trivalent vaccine supplemented with pandemic strains. However, depending on the season and the nature of the antigens contained in the vaccine, the present invention may provide protection against one or more of the HA subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16. The present invention may provide protection against one or more of the NA subtypes N1, N2, N3, N4, N5, N6, N7, N8, or N9 of the influenza A virus.

[0144] The compositions of the present invention are not only suitable for immunization against pandemic strains, but are particularly useful for immunization against pandemic strains. The characteristics that give an influenza strain the potential to cause a pandemic are as follows: (a) it contains a new hemagglutinin compared to the hemagglutinins in currently circulating human strains, i.e., one that has not appeared in human populations for more than 10 years (e.g., H2), or one that has never been seen in human populations in the past (e.g., H5, H6, or H9, which are generally found only in bird populations), so that human populations have not been immunologically exposed to the hemagglutinin of that strain; (b) it is horizontally transmissible in human populations; and (c) it is pathogenic to humans. For immunization against pandemic influenza, viruses having the H5 hemagglutinin type, such as the H5N1 strain, are preferred. Other possible strains include H5N3, H9N2, H2N2, H7N1, and H7N7, as well as any other potentially pandemic strains that may emerge. Within the H5 subtype, the virus may be in HA clade 1, HA clade 1', HA clade 2, or HA clade 3

[64] , with clades 1 and 3 being particularly related.

[0145] Other strains having antigens that can be usefully incorporated into the composition are strains resistant to antiviral therapy (e.g., resistant to oseltamivir

[65] and / or zanamivir), including resistant pandemic strains

[66] .

[0146] Therefore, the composition of the present invention may contain antigens from one or more (e.g., 1, 2, 3, 4 or more) influenza virus strains, including influenza A virus and / or influenza B virus. When the vaccine contains two or more influenza strains, the different strains are typically grown separately and mixed after the viruses have been collected and the antigens have been prepared. Therefore, the method of the present invention may include the step of mixing antigens from two or more influenza strains. A trivalent vaccine containing antigens from two influenza A virus strains and one influenza B virus strain is preferred.

[0147] In some embodiments of the present invention, the composition may contain antigens from a single influenza A strain. In some embodiments, the composition may contain antigens from two influenza A strains, provided that these two strains are not H1N1 and H3N2. In some embodiments, the composition may contain antigens from two or more influenza A strains.

[0148] The present invention provides a method for preparing an influenza virus antigen for use in a vaccine, the method comprising (i) receiving an influenza virus; (ii) infecting a cell line with this influenza virus; and (iii) culturing the infected cells from step (ii) to produce an influenza virus. The virus obtained in step (iii) can be used to prepare a vaccine by methods including, for example, inactivation and compounding. The influenza virus received in step (i) has one or more of the following characteristics: (a) has never been grown in an egg culture medium; (b) has been isolated in MDCK cells, e.g., MDCK33016 cells and / or MDCK cells grown in serum-free medium; (c) has never been grown in a culture medium that contains serum; (d) has been produced using reverse genetics techniques; (e) is an influenza A virus having fewer than six viral segments from a PR / 8 / 34 influenza virus, and / or an influenza A virus having fewer than six viral segments from an AA / 6 / 60 influenza virus, or an influenza B virus having fewer than six viral segments from an AA / 1 / 66 influenza virus; (f) contains hemagglutinin that preferentially binds to oligosaccharides having a Sia(α2,6)Gal terminal disaccharide rather than oligosaccharides having a Sia(α2,3)Gal terminal disaccharide; and / or (g) has glycoproteins (including hemagglutinin) with a glycosylation pattern different from that of an egg-derived virus. Therefore, the influenza virus received in step (i) may be obtained as described elsewhere in this specification.

[0149] host cell DNA When viruses are grown in cell lines, it is standard practice to minimize the amount of residual cell line DNA in the final vaccine in order to minimize any tumorigenic activity of the DNA.

[0150] Therefore, the vaccine composition prepared according to the present invention preferably contains less than 10 ng (preferably less than 1 ng, more preferably less than 100 pg) of residual host cell DNA per dose, although trace amounts of host cell DNA may be present.

[0151] Vaccines containing <10 ng (e.g., <1 ng, <100 pg) of host cell DNA per 15 μg of hemagglutinin are preferred, such as vaccines containing <10 ng (e.g., <1 ng, <100 pg) of host cell DNA per 0.25 ml volume. More preferably, vaccines containing <10 ng (e.g., <1 ng, <100 pg) of host cell DNA per 50 μg of hemagglutinin are preferred, such as vaccines containing <10 ng (e.g., <1 ng, <100 pg) of host cell DNA per 0.5 ml volume.

[0152] The average length of all residual host cell DNA is preferably less than 500 bp, for example less than 400 bp, less than 300 bp, less than 200 bp, less than 100 bp, etc.

[0153] Contaminating DNA can be removed during vaccine preparation using standard purification procedures, such as chromatography. Removal of residual host cell DNA can be improved by nuclease treatment, such as the use of DNases. Convenient methods for reducing host cell DNA contamination are disclosed in references 67 and 68, which involve a two-step process: first using a DNase (e.g., benzonase), which may also be used during viral growth; and then using a cationic surfactant (e.g., CTAB), which may be used during virion disruption. Host cell DNA may also be removed by treatment with an alkylating agent such as β-propiolactone, which may also be used to inactivate virions

[69] .

[0154] The measurement of residual host cell DNA is now a routine regulatory requirement for biologics and is within the ordinary capabilities of those skilled in the art. The assays used to measure DNA are typically validated assays [70, 71]. The performance characteristics of validated assays can be mathematically and quantitatively described, and potential sources of error are identified. The assay is generally tested for characteristics such as accuracy, precision, and specificity. Once an assay is calibrated and tested (e.g., against a known standard amount of host cell DNA), quantitative DNA measurements can be performed routinely. Three main techniques can be used for DNA quantification: hybridization methods, e.g., Southern blotting or slot blotting

[72] ; immunoassays, e.g., the Threshold™ system

[73] ; and quantitative PCR

[74] . While all these methods are well known to those skilled in the art, the precise characteristics of each method may depend on the host cells in question, e.g., the selection of probes for hybridization, the selection of primers and / or probes for amplification, etc. The Threshold® system from Molecular Devices is a quantitative assay for picogram-level total DNA and is used to monitor the level of contaminating DNA in biopharmaceuticals

[73] . A typical assay involves the sequence-nonspecific formation of a reaction complex between a biotinylated ssDNA-binding protein, a urease-bound anti-ssDNA antibody, and DNA. All assay components are included in complete whole DNA assay kits available from manufacturers. Various commercial manufacturers, such as AppTec® Laboratory Services, BioReliance®, and Althea Technologies, offer quantitative PCR assays for detecting residual host cell DNA. A comparison of the chemiluminescence hybridization assay for measuring host cell DNA contamination in human viral vaccines with the Whole DNA Threshold® system can be found in reference 75.

[0155] Pharmaceutical composition Vaccine compositions manufactured in accordance with the present invention are pharmaceutically acceptable. These compositions typically contain, in addition to the influenza antigen, other components, such as, for example, one or more pharmaceutical carriers and / or excipients. Adjuvants may also be included, as described below. A detailed discussion of these components can be found in Reference 76.

[0156] Vaccine compositions are generally in aqueous form.

[0157] The vaccine composition may contain preservatives such as thiomersal or 2-phenoxyethanol. However, preferably, the vaccine should be substantially free of mercury materials (i.e., less than 5 μg / ml), and should not contain thiomersal, for example [59, 77]. A mercury-free vaccine is more preferable. Alpha-tocopherol succinate may be included as a substitute for mercury compounds

[59] . A vaccine that does not contain preservatives is particularly preferred.

[0158] To control the tonicity, it is preferable to include physiological salts such as sodium salts. Sodium chloride (NaCl) is preferred, and may be present in concentrations of 1 mg / ml to 20 mg / ml. Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, anhydrous disodium phosphate, magnesium chloride, and calcium chloride.

[0159] The osmolality of the vaccine composition is generally between 200 mOsm / kg and 400 mOsm / kg, preferably 240 to 360 mOsm / kg, and more preferably 290 to 310 mOsm / kg. It has been previously reported that the osmolality does not affect the pain caused by vaccination

[78] , but it is still preferable to keep the osmolality within this range.

[0160] The vaccine composition may contain one or more buffers. Typical buffers include: phosphate buffer; Tris buffer; borate buffer; succinate buffer; histidine buffer (especially with aluminum hydroxide adjuvant); or citrate buffer. The buffers are typically present in concentrations ranging from 5 to 20 mM.

[0161] The pH of vaccine compositions is generally between 5.0 and 8.1, more typically between 6.0 and 8.0, for example, between 6.5 and 7.5, or between 7.0 and 7.8. Therefore, the method of the present invention may include a step of adjusting the pH of the bulk vaccine before packaging.

[0162] The vaccine composition is preferably sterile. The vaccine composition is preferably non-pyrogenic, for example, containing <1 EU (endotoxin unit, a standard unit) per dose, preferably <0.1 EU per dose. The vaccine composition is preferably gluten-free.

[0163] The vaccine composition of the present invention may contain surfactants, such as polyoxyethylene sorbitan ester surfactants (known as "Twin"), octoxynol (e.g., octoxynol-9 (Triton X-100) or t-octylphenoxypolyethoxyethanol), cetyltrimethylammonium bromide ("CTAB"), or sodium deoxycholate, particularly for degradable or surface antigen vaccines. Surfactants may be present in trace amounts. Thus, the vaccine may contain less than 1 mg / ml each of octoxynol-10 and polysorbate 80. Other trace residual components may be antibiotics (e.g., neomycin, kanamycin, polymyxin B).

[0164] The vaccine composition may contain materials for a single immunization or materials for multiple immunizations (i.e., a "multidose" kit). Preservatives are preferably included in the multidose configuration. As an alternative to (or addition to) including preservatives in the multidose composition, the composition may be contained in a container having a sterile adapter for material removal.

[0165] Influenza vaccines are typically administered in a dose volume of approximately 0.5 ml, but children may be given half the dose (i.e., approximately 0.25 ml).

[0166] The compositions and kits are preferably stored at 2°C to 8°C. The compositions and kits should not be frozen. Ideally, the compositions and kits should be kept away from direct light.

[0167] Adjuvant The compositions of the present invention may advantageously include an adjuvant, which may function to enhance the immune response (humoral and / or cellular) induced in patients receiving the composition. The use of adjuvants in conjunction with influenza vaccines has been previously described. Aluminum hydroxide was used in references 79 and 80, and a mixture of aluminum hydroxide and aluminum phosphate was used in reference 81. Reference 82 also describes the use of aluminum salt adjuvants. FLUAD® products from Chiron Vaccines contain oil-in-water emulsions.

[0168] Adjuvants that may be used in conjunction with the present invention include, but are not limited to, the following: • An inorganic-containing composition comprising calcium salts and aluminum salts (or mixtures thereof). The calcium salt includes calcium phosphate (e.g., “CAP” particles disclosed in Reference 83). The aluminum salt includes aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc., and these salts can take any suitable form (e.g., gel, crystal, amorphous, etc.). Adsorption to these salts is preferred. The inorganic-containing composition may be formulated as particles of metal salts

[84] . Aluminum salt adjuvants are described in more detail below.

[0169] • Cytokine inducers (see below for more details).

[0170] • Saponins [Reference 112, Chapter 22]. Saponins are a heterogeneous group of sterol glycosides and triterpenoid glycosides found in the bark, leaves, trunks, roots, and even flowers of a wide range of plant species. Saponins from the bark of the tree Quillaia saponaria Molina have been widely studied as adjuvants. Saponins can also be commercially obtained from Smilax ornata (sarsaparilla), Gypsophila paniculata (brides' veil), and Saponaria officianalis (baby's breath). Saponin adjuvant formulations include purified formulations, e.g., QS21, and lipid formulations, e.g., ISCOM. QS21 is commercially available as Stimulon®. Saponin compositions have been purified using HPLC and RP-HPLC. Specific purified fractions have been identified using these techniques, and these fractions include QS7, QS17, QS18, QS21, QH-A, QH-B, and QH-C. Preferably, the saponin is QS21. A method for producing QS21 is disclosed in reference 85. The saponin formulation may also contain sterols such as cholesterol

[86] . Combinations of saponins and cholesterol can be used to form unique particles called immunostimulating complexes (ISCOMs) [Chapter 23 of reference 112]. Typically, ISCOMs also contain phospholipids, such as phosphatidylethanolamine or phosphatidylcholine. Any known saponin may be used as an ISCOM. Preferably, the ISCOM contains one or more of QuilA, QHA, and QHC. ISCOMs are further described in references 86-87-88. Optionally, the ISCOM may not contain additional surfactants

[89] . An overview of the development of adjuvants primarily composed of saponins can be found in references 90 and 91.

[0171] • Fatty adjuvants (see more detailed description below).

[0172] • Bacterial ADP-ribosylated toxins (e.g., E. coli heat-labile enterotoxin "LT", cholera toxin "CT", or pertussis toxin "PT") and their detoxified derivatives, such as mutant toxins known as LT-K63 and LT-R72

[92] . The use of detoxified ADP-ribosylated toxins as mucosal adjuvants is described in reference 93, and their use as parenteral adjuvants is described in reference 94.

[0173] • Bioadhesives and mucoadhesives, such as esterified hyaluronic acid microspheres

[95] , or chitosan and its derivatives

[96] .

[0174] Fine particles (i.e., particles with a diameter of about 100 nm to about 150 μm, more preferably about 200 nm to about 30 μm, or about 500 nm to about 10 μm, or about 500 nm to about 10 μm) formed from biodegradable and non-toxic materials (e.g., poly(α-hydroxy acid), polyhydroxybutyric acid, polyorthoester, polyanhydride, polycaprolactone, etc.), where poly(lactide-co-glycolide) is preferred, and optionally treated to have a negatively charged surface (e.g., by SDS) or a positively charged surface (e.g., by a cationic surfactant such as CTAB).

[0175] • Liposomes (Chapters 13 and 14 of Reference 112). Examples of liposome formulations suitable for use as adjuvants are described in References 97-98 and 99.

[0176] • Polyoxyethylene ethers and polyoxyethylene esters

[0100] . These formulations further include combinations of polyoxyethylene sorbitan ester surfactants with octoxynol

[0101] and combinations of polyoxyethylene alkyl ether or ester surfactants with at least one additional nonionic surfactant such as octoxynol

[0102] . Preferred polyoxyethylene ethers are selected from the following group: polyoxyethylene-9-lauryl ether (laureth-9), polyoxyethylene-9-steolyl ether, polyoxyethylene-8-steolyl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl ether.

[0177] Muramyl peptides, such as N-acetylmuramyl-L-threonyl-D-isoglutamine ("thr-MDP"), N-acetyl-normuramyl-L-alanyl-D-isoglutamine ("nor-MDP"), and N-acetylglucosaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide. Examples include propylamide ("DTP-DPP" or "Theramide®"), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine ("MTP-PE"), etc.

[0178] A combination of an outer membrane protein proteasome preparation prepared from a first Gram-negative bacterium and a liposaccharide preparation derived from a second Gram-negative bacterium, wherein the outer membrane protein proteasome and the liposaccharide preparation form a stable non-covalent adjuvant complex. Such a complex includes "IVX-908," which is a complex containing the outer membrane and lipopolysaccharide of Neisseria meningitidis. These have been used as adjuvants for influenza vaccines

[0103] .

[0179] Methyl inosine 5'-monophosphate ("MIMP")

[0104] .

[0180] • Polyhydroxylated pyrrolizidine compounds

[0105] , such as those having the following formula:

[0181] [ka] Here, R is selected from the group comprising hydrogen, linear or branched chains, unsubstituted or substituted, saturated or unsaturated acyl, alkyl (e.g., cycloalkyl), alkenyl, alkynyl and aryl groups, or pharmaceutically acceptable salts or derivatives thereof. Examples include, but are not limited to, casuarine, casuarine-6-α-D-glucopyranose, 3-epi-casuarine, 7-epi-casuarine, 3,7-diep-casuarine, etc.

[0182] Gamma inulin

[0106] or its derivatives, such as algammulin.

[0183] CD1d ligands, such as α-galactosylceramide.

[0184] • Polyoxidonium polymer [107, 108] or other N-polyethylene oxide-piperazine derivatives.

[0185] These and other adjuvant active substances are discussed in more detail in references 112 and 113.

[0186] The composition may contain two or more of the above adjuvants. For example, the composition may advantageously contain both an oil-in-water emulsion and a cytokine inducer, because this combination improves the cytokine response induced by the influenza vaccine, such as the interferon-gamma response, and this improvement is considerably greater than that seen when either the emulsion or the drug is used alone.

[0187] The antigen and adjuvant in the composition are typically found in a mixture.

[0188] Oil-in-water emulsion adjuvant Oil-in-water emulsions have been found to be particularly suitable for use as adjuvants in influenza virus vaccines. Various such emulsions are known, typically comprising at least one oil and at least one surfactant, the oils and surfactants being biodegradable (metabolic) and biocompatible. The oil droplets in the emulsions are generally less than 5 μm in diameter, and may be less than a micron in diameter; such small sizes are obtained by microfluidizers to provide stable emulsions. Droplets smaller than 220 nm are preferred because they can be filter-sterilized.

[0189] The present invention may be used in conjunction with oils, such as oils from animal (fish, etc.) or plant sources. Sources of plant oils include nuts, seeds, and grains. Peanut oil, soybean oil, coconut oil, and olive oil are examples of the most commonly available nut oils. Jojoba oil, for example, obtained from jojoba beans, may be used. Seed oils include safflower oil, cottonseed oil, sunflower oil, sesame oil, and the like. Among the grain group, 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. The 6-10 carbon fatty acid esters of glycerol and 1,2-propanediol do not occur spontaneously in seed oils, but may be prepared by hydrolysis, separation, and esterification of suitable materials starting from nuts and seed oils. Fats and oils from mammalian milk are metabolizable and may therefore be used in the implementation of the present invention. Procedures for separation, purification, saponification, and other means necessary to obtain pure oils from animal sources are well known in the art. Most fish contain readily recoverable metabolic oils. For example, cod liver oil, shark liver oil, and whale oil, such as whale wax, are some examples of fish oils that may be used herein. Some branched-chain oils are biochemically synthesized from 5-carbon isoprene structural units and are generally called terpenoids. Shark liver oil contains squalene, a branched-chain unsaturated terpenoid known as 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene, which is particularly preferred herein. Squalane, a saturated analogue of squalene, is also a preferred oil. Fish oils containing squalene and squalane are readily available from commercial sources and may be obtained by methods known in the art. Other preferred oils are tocopherols (see below). Mixtures of oils may also be used.

[0190] Surfactants can be classified by their "HLB" (hydrophile / lipophile balance). The HLB of preferred surfactants of the present invention is at least 10, preferably at least 15, and more preferably at least 16. The present invention can be used with surfactants including, but not limited to, the following: polyoxyethylene sorbitan ester surfactants (commonly called twin), particularly polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO), sold under the trade name DOWFAX®, e.g., linear EO / PO block copolymers; octoxynol, where the number of repeating ethoxy(oxy-1,2-ethanediyl) groups can vary, with octoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol) being of particular interest; and (octylphenoxy)polyethoxyethanol (IGEPAL). CA-630 / NP-40); phospholipids, such as phosphatidylcholine (lecithin); nonylphenol ethoxylates, such as the Tergitol® NP series; polyoxyethylene fatty ethers derived from lauryl alcohol, cetyl alcohol, stearyl alcohol, and oleyl alcohol (known as Brij surfactants), such as triethylene glycol monolauryl ether (Brij 30); and sorbitan esters (commonly known as SPAN), such as sorbitan trioleate (SPAN 85) and sorbitan monolaurate. Nonionic surfactants are preferred. Preferred surfactants for inclusion in the emulsion are Twin 80 (polyoxyethylene sorbitan monooleate), SPAN 85 (sorbitan trioleate), lecithin, and Triton X-100.

[0191] A mixture of surfactants, such as a Twin 80 / Span 85 mixture, can be used. A combination of a polyoxyethylene sorbitan ester, such as polyoxyethylene sorbitan monooleate (Twin 80), and an octoxynol, such as t-octylphenoxypolyethoxyethanol (Triton X-100), is also suitable. Another useful combination includes laureth-9 and a polyoxyethylene sorbitan ester and / or an octoxynol.

[0192] The preferred amounts (by weight) of surfactants are as follows: polyoxyethylene sorbitan ester (such as Twin 80) in amounts of 0.01% to 1%, particularly about 0.1%; octyl- or nonylphenoxy polyoxyethanol (such as Triton X-100 or other Triton series surfactants) in amounts of 0.001% to 0.1%, particularly 0.005% to 0.02%; and polyoxyethylene ether (such as Laureth 9) in amounts of 0.1% to 20%, preferably 0.1% to 10%, particularly 0.1% to 1% or about 0.5%.

[0193] Specific oil-in-water emulsion adjuvants useful with the present invention include, but are not limited to, the following: • Submicron emulsions of squalene, twin 80, and span 85. The volumetric composition of the emulsion may be approximately 5% squalene, approximately 0.5% polysorbate 80, and approximately 0.5% span 85. By weight, these ratios are 4.3% squalene, 0.5% polysorbate 80, and 0.48% span 85. This adjuvant is known as "MF59" [109-110, 111] and is described in more detail in Chapter 10 of Reference 112 and Chapter 12 of Reference 113. The MF59 emulsion advantageously contains citrate ions, for example, 10 mM sodium citrate buffer.

[0194] • Emulsions of squalene, tocopherol, and Twin 80. These emulsions may also contain phosphate-buffered saline. These emulsions may also contain Span 85 (e.g., 1%) and / or lecithin. These emulsions may have 2% to 10% squalene, 2% to 10% tocopherol, and 0.3% to 3% Twin 80, and the weight ratio of squalene:tocopherol is preferably ≤1 to provide a more stable emulsion. Squalene and Twin 80 may be present in a volume ratio of about 5:2. One such emulsion can be prepared by dissolving Twin 80 in PBS to give a 2% solution, then mixing 90 ml of this solution with a mixture of (5 g DL-α-tocopherol and 5 ml squalene), and then microfluidizing the mixture. The resulting emulsion may have oil droplets smaller than a micron, for example, the average diameter of the droplets may be 100 nm to 250 nm, preferably about 180 nm.

[0195] • Emulsion of squalene, tocopherol, and a Triton surfactant (e.g., Triton X-100). This emulsion may also contain 3d-MPL (see below). This emulsion may also contain phosphate buffer.

[0196] • An emulsion comprising polysorbate (e.g., polysorbate 80), Triton surfactant (e.g., Triton X-100), and tocopherol (e.g., α-tocopherol succinate). This emulsion may contain these three components in a mass ratio of approximately 75:11:10 (e.g., 750 μg / ml polysorbate 80, 110 μg / ml Triton X-100, and 100 μg / ml α-tocopherol succinate), and these concentrations should include some contribution of these components from the antigen. This emulsion may also contain squalene. This emulsion may also contain 3d-MPL (see below). The aqueous phase may contain phosphate buffer.

[0197] · An emulsion of squalane, polysorbate 80, and poloxamer 401 (“Pluronic (trademark) L121”). This emulsion can be formulated in phosphate-buffered saline at pH 7.4. This emulsion is a useful delivery vehicle for muramyl dipeptide and has been used with threonyl-MDP in the “SAF-1” adjuvant

[0114] (0.05 - 1% Thr-MDP, 5% squalane, 2.5% Pluronic L121, and 0.2% polysorbate 80). It can also be used without Thr-MDP, like the “AF” adjuvant

[0115] (5% squalane, 1.25% Pluronic L121, and 0.2% polysorbate 80). Microfluidization is preferred. <00…​​​​​​​​​​· An emulsion having 0.5 to 50% oil, 0.1 to 10% phospholipid, and 0.05 to 5% nonionic surfactant. As described in Reference 118, preferred phospholipid components are phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, sphingomyelin, and cardiolipin. Droplet sizes below micron are advantageous.

[0201] · A water-in-oil emulsion below micron of non-metabolic oil (such as light oil) and at least one surfactant (such as lecithin, Tween 80 or Span 80). Additives such as Quil A saponin, cholesterol, saponin lipophilic conjugate (such as GPI-0100 described in Reference 119, which is produced by adding aliphatic amine to desacylsaponin via the carboxyl group of glucuronic acid), dimethyldioctadecylammonium bromide, and / or N,N-dioctadecyl-N,N-bis(2-hydroxyethyl)propanediamine may be included.

[0202] · An emulsion containing mineral oil, nonionic lipophilic ethoxylated fatty alcohol, and nonionic hydrophilic surfactant (e.g., ethoxylated fatty alcohol and / or polyoxyethylene-polyoxypropylene block copolymer)

[0120] .

[0203] · An emulsion containing mineral oil, nonionic hydrophilic ethoxylated fatty alcohol, and nonionic lipophilic surfactant (e.g., ethoxylated fatty alcohol and / or polyoxyethylene-polyoxypropylene block copolymer)

[0120] .

[0204] · An emulsion in which saponin (e.g., Quil A or QS21) and sterol (e.g., cholesterol) are bound as helical micelles

[0121] .

[0205] The emulsion may be immediately mixed with the antigen at the time of delivery. That is, the adjuvant and antigen may be stored separately in the packaged or distributed vaccine so that the final formulation is ready at the time of use. Generally, the antigen is in aqueous form, and the vaccine is ultimately prepared by mixing the two liquids. The volume ratio of the two liquids for mixing can vary (e.g., 5:1 to 1:5), but is generally about 1:1.

[0206] After the antigen and adjuvant are mixed, the hemagglutinin antigen generally remains in the aqueous solution, but may be positioned around the oil / water interface. Generally, even if hemagglutinin enters the oil phase of the emulsion, it is only in very small amounts.

[0207] When the composition contains tocopherol, any of α, β, γ, δ, ε, or ζ tocopherol may be used, but α-tocopherol is preferred. Tocopherol can take several forms, such as different salts and / or isomers. Salts include organic salts, such as succinates, acetates, and nicotinates. Both D-α-tocopherol and DL-α-tocopherol can be used. Tocopherol is advantageously included in vaccines for use in elderly patients (e.g., 60 years of age or older) because vitamin E has been reported to have a positive effect on the immune response in such patient groups

[0122] . Tocopherol also has antioxidant properties and may help stabilize the emulsion

[0123] . The preferred α-tocopherol is DL-α-tocopherol, and the preferred salt of this tocopherol is succinate. Succinate has been found to cooperate with TNF-related ligands in vivo. Furthermore, alpha-tocopherol succinate is known to be suitable for influenza vaccines and is a useful preservative as a substitute for mercury compounds.

[59]

[0208] Cytokine inducers Cytokine inducers to be included in the composition of the present invention can induce the immune system to release cytokines, including interferon and interleukin, when administered to a patient. Cytokine responses are known to be associated with the early decisive stages of the host's defense against influenza infection

[0124] . Preferred agents can induce the release of one or more of the following: interferon-γ; interleukin-1; interleukin-2; interleukin-12; TNF-α; TNF-β; and GM-CSF. Preferred agents induce the release of cytokines associated with the Th1 immune response, such as interferon-γ, TNF-α, interleukin-2, etc. Stimulation of both interferon-γ and interleukin-2 is preferred.

[0209] Therefore, as a result of receiving the composition of the present invention, patient T cells release desired cytokines in an antigen-specific manner when stimulated by the influenza antigen. For example, T cells purified from the blood of those patients release γ-interferon when exposed to influenza virus hemagglutinin in vitro. Methods for measuring such responses in peripheral blood mononuclear cells (PBMCs) are known in the art and include ELISA, ELISPOT, flow cytometry, and real-time PCR. For example, in a study reported in Reference 125, antigen-specific T cell-mediated immune responses to tetanus toxoid, particularly the γ-interferon response, were monitored, and ELISPOT was found to be the most sensitive method for distinguishing antigen-specific TT-induced responses from innate responses, while intracytoplasmic cytokine detection by flow cytometry was found to be the most efficient method for detecting the restorative effect.

[0210] Suitable cytokine inducers include, but are not limited to, the following: • Immunostimulant oligonucleotides, such as those containing a CpG motif (a dinucleotide sequence containing unmethylated cytosine linked to guanosine by a phosphate bond), or oligonucleotides containing double-stranded RNA, or palindromic sequences, or poly(dG) sequences.

[0211] 3-O-deacylated monophosphoryl lipid A (also known as "3dMPL" or "MPL (trademark)") [126-127, 128, 129].

[0212] • Imidazocinoline compounds, e.g., imiquimod ("R-837") [130, 131], reximod ("R-848")

[132] , and their analogues; as well as their salts (e.g., hydrochloride). Further details on immunostimulant imidazoquinolines can be found in references 133 to 134, 135, 136, 137.

[0213] • Thiosemiccarbazone compounds, such as those disclosed in Reference 138. Methods for compounding, manufacturing, and screening active compounds are also described in Reference 138. Thiosemiccarbazones are particularly effective for stimulating human peripheral blood mononuclear cells for cytokine production, such as TNF-α.

[0214] • Tryptanthrin compounds, such as those disclosed in Reference 139. Methods for compounding, manufacturing, and screening active compounds are also described in Reference 139. Thiosemicarbazone is particularly effective for stimulating human peripheral blood mononuclear cells for cytokine production, such as TNF-α.

[0215] • Nucleoside analogs, e.g.: (a) Isatorabine (ANA-245; 7-thia-8-oxoguanosine):

[0216] [ka] and its prodrugs; (b) ANA975; (c) ANA-025-1; (d) ANA380; (e) the compounds disclosed in References 140 to 141, 142; (f) compounds having the following formula:

[0217]

Chemical formula

[0220] [ka] This bond

[0221] [ka] Achieved in the combination shown by, R 10 and R 11 Each of these is independently H, Halo, C 1-6 Alkoxy, substituted C 1-6 Alkoxy, -NR a R b , or -OH; R a and R b Each of these is independently H, C 1-6 Alkyl, substituted C 1-6 Alkyl, -C(O)R d , C 6-10 It is an allele; Each R c These are independently H, phosphate, diphosphate, triphosphate, and C 1-6 Alkyl or substituted C 1-6 It is alkyl; Each R d These are H, Halo, and C independently. 1-6Alkyl, substituted C 1-6 Alkyl, C 1-6 Alkoxy, substituted C 1-6 Alkoxy, -NH2, -NH(C 1-6 Alkyl), -NH (substituted C) 1-6 Alkyl), -N(C 1-6 Alkyl)2,-N(substituted C 1-6 Alkyl)2, C 6-10 It is an aryl or heterocyclyl; Each R e H and C are independent of each other. 1-6 Alkyl, substituted C 1-6 Alkyl, C 6-10 Aryl, substituted C 6-10 It is an aryl, heterocyclyl, or substituted heterocyclyl; Each R f H and C are independent of each other. 1-6 Alkyl, substituted C 1-6 Alkyl, -C(O)R d , phosphoric acid, diphosphate, or triphosphate; Each n is independently 0, 1, 2, or 3; Each p is independently 0, 1, or 2; or (g) A pharmaceutically acceptable salt of any of (a) to (f), a tautomer of any of (a) to (f), or a pharmaceutically acceptable salt of the tautomer thereof.

[0222] • Loxoribine (7-allyl-8-oxoguanosine)

[0143] .

[0223] Compounds disclosed in Reference 144, including the following: acylpiperazine compounds, indoledione compounds, tetrahydraisoquinoline (THIQ) compounds, benzocyclodione compounds, aminoazavinyl compounds, aminobenzimidazole quinolinone (ABIQ) compounds [145, 146], hydrapthalamide compounds, benzophenone compounds, isoxazole compounds, sterol compounds, quinazilinone compounds, pyrrole compounds

[0147] , anthraquinone compounds, quinoxaline compounds, triazine compounds, pyrazalopyrimidine compounds, and benzazole compounds

[0148] .

[0224] • Compounds disclosed in Reference 149.

[0225] • Derivatives of aminoalkylglucosaminid phosphates, such as RC-529 [15 0, 151].

[0226] • Phosphazenes, such as poly[di(carboxylatophenoxy)phosphazene] ("PCPP"), as described in references 152 and 153.

[0227] • Small molecule immunopotentiators (SMIPs), such as the following: N2-methyl-1-(2-methylpropyl)-1H-imidazo[4,5-c]quinoline-2,4-diamine N2,N2-dimethyl-1-(2-methylpropyl)-1H-imidazo[4,5-c]quinoline-2,4-diamine N2-Ethyl-N2-methyl-1-(2-methylpropyl)-1H-imidazo[4,5-c]quinoline-2,4-diamine N2-methyl-1-(2-methylpropyl)-N2-propyl-1H-imidazo[4,5-c]quinoline-2,4-diamine 1-(2-methylpropyl)-N2-propyl-1H-imidazo[4,5-c]quinoline-2,4-diamine N2-Butyl-1-(2-methylpropyl)-1H-imidazo[4,5-c]quinoline-2,4-diamine N2-Butyl-N2-methyl-1-(2-methylpropyl)-1H-imidazo[4,5-c]quinoline-2,4-diamine N2-methyl-1-(2-methylpropyl)-N2-pentyl-1H-imidazo[4,5-c]quinoline-2,4-diamine N2-methyl-1-(2-methylpropyl)-N2-prop-2-enyl-1H-imidazo[4,5-c]quinoline-2,4-diamine 1-(2-methylpropyl)-2-[(phenylmethyl)thio]-1H-imidazo[4,5-c]quinoline-4-amine 1-(2-methylpropyl)-2-(propylthio)-1H-imidazo[4,5-c]quinoline-4-amine 2-[[4-amino-1-(2-methylpropyl)-1H-imidazo[4,5-c]quinoline-2-yl](methyl)amino]ethanol 2-[[4-amino-1-(2-methylpropyl)-1H-imidazo[4,5-c]quinoline-2-yl](methyl)amino]ethyl acetate 4-amino-1-(2-methylpropyl)-1,3-dihydro-2H-imidazo[4,5-c]quinoline-2-one N2-Butyl-1-(2-methylpropyl)-N4,N4-bis(phenylmethyl)-1H-imidazo[4,5-c]quinoline-2,4-diamine N2-Butyl-N2-methyl-1-(2-methylpropyl)-N4,N4-bis(phenylmethyl)-1H-imidazo[4,5-c]quinoline-2,4-diamine N2-methyl-1-(2-methylpropyl)-N4,N4-bis(phenylmethyl)-1H-imidazo[4,5-c]quinoline-2,4-diamine N2,N2-dimethyl-1-(2-methylpropyl)-N4,N4-bis(phenylmethyl)-1H-imidazo[4,5-c]quinoline-2,4-diamine 1-{4-amino-2-[methyl(propyl)amino]-1H-imidazo[4,5-c]quinoline-1-yl}-2-methylpropane-2-ol 1-[4-amino-2-(propylamino)-1H-imidazo[4,5-c]quinoline-1-yl]-2-methylpropane-2-ol N4,N4-Dibenzyl-1-(2-methoxy-2-methylpropyl)-N2-propyl-1H-imidazo[4,5-c]quinoline-2,4-diamine.

[0228] The cytokine inducers used in the present invention may be modulators and / or agonists of Toll-like receptors (TLRs). For example, these cytokine inducers may be agonists of one or more human TLR1, TLR2, TLR3, TLR4, TLR7, TLR8, and / or TLR9 proteins. Preferred agents are agonists of TLR7 (e.g., imidazoquinoline) and / or TLR9 (e.g., CpG oligonucleotides). These agents are useful for activating intrinsic immune pathways.

[0229] Cytokine inducers may be added to the composition at various stages during its preparation. For example, the cytokine inducer may be present in the antigen composition, and this mixture may then be added to an oil-in-water emulsion. Alternatively, the cytokine inducer may be present in the oil-in-water emulsion, in which case the drug may be added to the emulsion components before or after emulsification. Similarly, the drug may be coacervated into emulsion droplets. The location and distribution of the cytokine inducer in the final composition depends on the hydrophilic / lipophilic properties of the cytokine inducer; for example, the drug may be located in the aqueous phase, the oil phase, and / or at the oil-water interface.

[0230] Cytokine inducers may be conjugated to other drugs, such as antigens (e.g., CRM197). A general overview of conjugation techniques for small molecules is given in reference 154. Alternatively, adjuvants may be non-covalently associated with additional drugs, for example, by hydrophobic or ionic interactions.

[0231] Two preferred cytokine inducers are (a) immunostimulatory oligonucleotides and (b) 3dMPL. Immunostimulatory oligonucleotides may contain nucleotide modifications / analogs such as phosphorothioate modifications and may be double-stranded or single-stranded (except RNA). References 155, 156 and 157 disclose possible analog substitutions, such as substitution of guanosine with 2'-deoxy-7-deazaguanosine. References 158-159, 160, 161, 162 and 163 further discuss the adjuvant effects of CpG oligonucleotides. The CpG sequence may be directed to TLR9, for example, the motif GTCGTT or TTCGTT

[0164] . The CpG sequence may be specific to induce a Th1 immune response, such as CpG-A ODN (oligodeoxynucleotide), or more specific to induce a B cell response, such as CpG-B ODN. CpG-A ODNs and CpG-B ODNs are discussed in references 165-166 and 167. Preferably, the CpG is a CpG-A ODN. Preferably, the CpG oligonucleotide is configured so that its 5' end is accessible for receptor recognition. Optionally, the 3' ends of two CpG oligonucleotide sequences may be joined to form an "immunomer." See, for example, references 164, 168-169, and 170. A useful CpG adjuvant is CpG7909, also known as ProMune® (Coley Pharmaceutical Group, Inc.).

[0232] TpG sequences may be used as an alternative to, or in addition to, the use of CpG sequences

[0171] . These oligonucleotides do not have to contain unmethylated CpG motifs.

[0233] Immunostimulating oligonucleotides may be pyrimidine-rich. For example, an immunostimulating oligonucleotide may contain two or more consecutive thymidine nucleotides (e.g., TTTT, as disclosed in Reference 171) and / or have a nucleotide composition having >25% thymidine (e.g., >35%, >40%, >50%, >60%, >80%, etc.). For example, an immunostimulating oligonucleotide may contain two or more consecutive cytosine nucleotides (e.g., CCCC, as disclosed in Reference 171) and / or have a nucleotide composition having >25% cytosine (e.g., >35%, >40%, >50%, >60%, >80%, etc.). These oligonucleotides do not need to contain unmethylated CpG motifs.

[0234] Immunostimulating oligonucleotides typically contain at least 20 nucleotides. They may also contain fewer than 100 nucleotides.

[0235] 3dMPL (also known as 3-de-O-acylated monophosphoryl lipid A or 3-O-deacyl-4'-monophosphoryl lipid A) is an adjuvant in which the reducing terminal glucosamine at position 3 in monophosphoryl lipid A is deacylated. 3dMPL is prepared from a heptose-deficient mutant of Salmonella minnesota and is chemically similar to lipid A but lacks the acid-unstable phosphoryl group and the base-unstable acyl group. 3dMPL activates monocyte / macrophage cells and stimulates the release of several cytokines, including IL-1, IL-12, TNF-α, and GM-CSF (see also reference 172). The preparation of 3dMPL was originally described in reference 173.

[0236] 3dMPL may take the form of a mixture of multiple related molecules with different acylation (e.g., having 3, 4, 5, or 6 acyl chains, which may be of different lengths). Two glucosamine (also known as 2-deoxy-2-amino-glucose) monosaccharides have their carbons at the 2-position (i.e., positions 2 and 2’) N-acylated, and there is also O-acylation at the 3’ position. The group attached to carbon 2 has the formula -NH-CO-CH2-CR 1 R 1’ and. The group attached to carbon 2’ has the formula -NH-CO-CH2-CR 2 R 2’ and. The group attached to carbon 3’ has the formula -O-CO-CH2-CR 3 R 3’ and. Representative structures are as follows:

[0237]

Chemical Structure

[0238] The group R 1’ 、R 2’ and R 3’ each may independently be (a) -H; (b) -OH; or (c) -O-CO-R 4 where R 4 is either -H or -(CH2) m -CH3, where the value of m is preferably from 8 to 16, more preferably 10, 12, or 14. At the 2-position, m is preferably 14. At the 2’-position, m is preferably 10. At the 3’-position, m is preferably 12. Thus, the groups R 1’ 、R 2’ and R 3’ are preferably -O-acyl groups from dodecanoic acid, tetradecanoic acid, or hexadecanoic acid.

[0239] R 1’ 、R 2’ and R 3’ are all -H, 3dMPL has only three acyl chains (one at each of positions 2, 2' and 3'). R 1’ 、R 2’ and R 3’ When only two of them are -H, 3dMPL can have four acyl chains. R 1’ 、R 2’ and R 3’ When only one of them is -H, 3dMPL can have five acyl chains. R 1’ 、R 2’ and R 3’ When none of them is -H, 3dMPL can have six acyl chains. The 3dMPL adjuvant used according to the present invention may be a mixture of these forms having three to six acyl chains, but it is preferred to include 3dMPL having six acyl chains in the mixture, and in particular it is preferred to ensure that the hexaacyl chain form occupies at least 10% by weight of the total 3dMPL, for example ≥20%, ≥30%, ≥40%, ≥50%, or more. 3dMPL having six acyl chains has been found to be the most adjuvant-active form.

[0240] Therefore, the most preferred form of 3dMPL for inclusion in the composition of the present invention has the following formula (IV).

[0241] When 3dMPL in the form of a mixture is used, reference to the amount or concentration of 3dMPL in the composition of the present invention refers to the combined 3dMPL species in the mixture.

[0242] Under aqueous conditions, 3dMPL can form micelle aggregates or particles of different sizes, such as diameters <150 nm or >500 nm. Either or both of these can be used in conjunction with the present invention, and better particles can be selected by routine assays. For use according to the present invention, smaller particles (e.g., small enough to give a clear aqueous suspension of 3dMPL) are preferred because they have superior activity

[0174] . Preferred particle average diameters are less than 220 nm, more preferably less than 200 nm, or less than 150 nm, or less than 120 nm, and may have an average diameter of less than 100 nm. However, in most cases, the average diameter will not be smaller than 50 nm. These particles are small enough to be suitable for filter sterilization. Particle diameter can be estimated by routine techniques of dynamic light scattering that reveal the average particle diameter. When a particle is said to have a diameter of x nm, generally a distribution of particles exists with respect to this average value, but at least 50% of the particles (e.g., ≥60%, ≥70%, ≥80%, ≥90%, or more) have a diameter within the range of x ± 25%.

[0243] 3dMPL may be advantageously used in combination with an oil-in-water emulsion. Substantially all 3dMPL may be located in the aqueous phase of the emulsion.

[0244] 3dMPL may be used alone or in combination with one or more further compounds. For example, it is known to be used in combination with QS21 saponin

[0175] (included in oil-in-water emulsion

[0176] ), immunostimulatory oligonucleotides, both QS21 and immunostimulatory oligonucleotides, aluminum phosphate

[0177] , aluminum hydroxide

[0178] , or both aluminum phosphate and aluminum hydroxide.

[0245] [ka] Fat adjuvant Fatty adjuvants that may be used in conjunction with the present invention include the oil-in-water emulsions described above, and further include, for example, the following: • Compounds of formulas I, II, or III, or salts thereof:

[0246] [ka] As defined in Reference 179, for example, "ER803058", "ER803732", "ER804053", "ER804058", "ER804059", "ER804442", "ER804680", "ER804764", "ER803022", or "ER804057", etc.

[0247] [ka] • Derivatives of lipid A from Escherichia coli, such as OM-174 (described in references 180 and 181).

[0248] • Preparations of cationic lipids and (usually neutral) copolymers, such as aminopropyl-dimethyl-myristoleyloxy-propanaminonium bromide-diphytanoylphosphatidyl-ethanolamine ("Vaxfectin" or "Vaxfectin") or aminopropyl-dimethyl-bis-dodecyloxy-propanaminonium bromide-dioleoylphosphatidyl-ethanolamine ("GAP-DLRIE:DOPE"). Preparations containing (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(syn-9-tetradeceneyloxy)-1-propanaminonium salt are preferred

[0182] .

[0249] • 3-O-deacylated monophosphoryl lipid A (see above).

[0250] • Compounds containing lipids bound to a phosphate-containing acyclic backbone, such as the TLR4 antagonist E5564 [183, 184]:

[0251] [ka] Aluminum salt adjuvant Adjuvants known as aluminum hydroxide and aluminum phosphate may be used. These names are conventional but are used merely for convenience and do not accurately describe any actual chemical compounds (see, e.g., Chapter 9 of Reference 112). The present invention can use any "hydroxide" or "phosphate" adjuvant commonly used as an adjuvant.

[0252] Adjuvants known as "aluminum hydroxide" are typically aluminum oxyhydroxide salts, which are usually at least partially crystalline. Aluminum oxyhydroxide can be represented by the formula AlO(OH) and is known by infrared (IR) spectroscopy, particularly at 1070 cm⁻¹. -1 Adhesion zone in and 3090~3100cm -1 The presence of a strong shoulder in aluminum hydroxide adjuvant distinguishes it from other aluminum compounds, such as aluminum hydroxide Al(OH)3 [Reference 112, Chapter 9]. The crystallinity of aluminum hydroxide adjuvants is reflected in the width of the diffraction band at half height (WHH), with less crystalline particles showing greater line broadening due to smaller crystal size. As WHH increases, the surface area increases, and it has been shown that adjuvants with higher WHH values ​​have a greater capacity for antigen adsorption. A fibrous morphology (e.g., as seen in transmission electron microscopy) is typical for aluminum hydroxide adjuvants. The pI of aluminum hydroxide adjuvants is typically about 11, meaning that the adjuvant itself has a positive surface charge at physiological pH. For aluminum hydroxide adjuvants, 1 mg of Al at pH 7.4 +++ A protein adsorption capacity of 1.8–2.6 mg per unit has been reported.

[0253] Adjuvants known as "aluminum phosphate" are typically aluminum phosphate hydroxide, often containing small amounts of sulfate as well (i.e., aluminum phosphate hydroxide sulfate). This adjuvant may also be obtained by precipitation, and the reaction conditions and concentrations during precipitation affect the degree of phosphoric acid substitution of hydroxyl groups in the salt. Generally, the PO4 / Al molar ratio of phosphate hydroxides is 0.3 to 1.2. Phosphate hydroxides can be distinguished from strict AlPO4 by the presence of hydroxyl groups. For example, 3164 cm⁻¹. -1 The IR spectral band in this region (e.g., when heated to 200°C) indicates the presence of structural hydroxyl groups [Reference 112, Chapter 9].

[0254] PO4 / Al aluminum phosphate adjuvant 3+ The molar ratio is generally 0.3 to 1.2, preferably 0.8 to 1.2, and more preferably 0.95 ± 0.1. Aluminum phosphate is generally amorphous, especially with respect to phosphate hydroxides. A typical adjuvant is 0.6 mg of Al 3+ It is amorphous aluminum hydroxide phosphate with a PO4 / Al molar ratio of 0.84 to 0.92 contained in / ml. Aluminum phosphate is generally particulate (e.g., plate-like morphology seen in transmission electron microscopy). Typical particle diameters range from 0.5 to 20 μm (e.g., approximately 5 to 10 μm) after adsorption of any antigen. For aluminum phosphate adjuvants, 1 mg of Al at pH 7.4 +++ A protein adsorption capacity of 0.7 to 1.5 mg per unit has been reported.

[0255] The point of zero charge (PZC) of aluminum phosphate is inversely proportional to the degree of substitution of phosphate with respect to the hydroxyl groups, and this degree of substitution can vary depending on the reaction conditions and concentrations of the reactants used for the preparation of the salt by precipitation. The PZC can also be altered by changing the concentration of free phosphate ions in the solution (more phosphate = more acidic PZC) or by adding a buffer such as histidine buffer (making the PZC more basic). Aluminum phosphate used according to the present invention generally has a PZC of 4.0 to 7.0, more preferably 5.0 to 6.5, for example, about 5.7.

[0256] The aluminum salt suspension used to prepare the compositions of the present invention may contain a buffer (e.g., phosphate or histidine or Tris buffer), but this is not always necessary. The suspension is preferably sterile and free of pyrogens. The suspension may also contain free aqueous phosphate ions, which may be present at concentrations of, for example, 1.0 mM to 20 mM, preferably 5 mM to 15 mM, and more preferably about 10 mM. The suspension may also contain sodium chloride.

[0257] The present invention can use a mixture of both aluminum hydroxide and aluminum phosphate

[81] . In this case, aluminum phosphate may be present in greater quantities than aluminum hydroxide, for example, at least 2:1, for example, ≥5:1, ≥6:1, ≥7:1, ≥8:1, ≥9:1 by weight.

[0258] Al in a composition for administration to patients +++ The concentration is preferably less than 10 mg / ml, for example, ≤5 mg / ml, ≤4 mg / ml, ≤3 mg / ml, ≤2 mg / ml, ≤1 mg / ml, etc. The preferred range is 0.3 mg / ml to 1 mg / ml. A maximum of 0.85 mg / dose is preferred.

[0259] In addition to containing one or more aluminum salt adjuvants, the adjuvant component may contain one or more further adjuvants or immunostimulants. Such additional components include, but are not limited to, 3-O-deacylated monophosphoryl lipid A adjuvants ("3d-MPL"); and / or oil-in-water emulsions.

[0260] Packaging of vaccine composition Suitable containers for the composition (or kit components) of the present invention include liquid medicine bottles, syringes (e.g., disposable syringes), nasal sprays, and the like. These containers should be sterilized.

[0261] When the composition / component is placed in a liquid medicine bottle, the bottle is preferably made of glass or plastic material. Preferably, the liquid medicine bottle is sterilized before the composition is added. To avoid problems associated with patients with latex hypersensitivity, the liquid medicine bottle is preferably sealed with a latex-free stopper, and it is preferable that all packaging materials are latex-free. The liquid medicine bottle may contain a single dose of vaccine or two or more doses, for example, 10 doses ("multi-dose" liquid medicine bottle). Preferably, the liquid medicine bottle is made of colorless glass.

[0262] The liquid medicine bottle may have a cap (e.g., a Luer lock) that is fitted so that a pre-filled syringe can be inserted into the cap, the contents of the syringe can be released into the liquid medicine bottle (e.g., to reconstitute the lyophilized material inside), and the contents of the liquid medicine bottle can be returned into the syringe. After being removed from the liquid medicine bottle, the syringe can be fitted with a needle and the composition can be administered to the patient. It is preferable that the cap is located inside a seal or cover so that the seal or cover must be removed before the cap can be accessed. Particularly for multi-dose liquid medicine bottles, the liquid medicine bottle may have a cap that allows for the aseptic removal of its contents.

[0263] When the ingredients are packaged in a syringe, the syringe may have a needle attached. When the needle is not attached, a separate needle may be supplied with the syringe for assembly and use. Such needles may be coated. Safety needles are preferred. Typical needles are 1-inch 23 gauge, 1-inch 25 gauge, and 5 / 8-inch 25 gauge. Syringes may be provided with peel-off labels, which may be printed with the lot number of the contents, the flu season, and the expiration date to facilitate record-keeping. The plunger in the syringe preferably has a stopper to prevent the plunger from accidentally coming off during aspiration. The syringe may have a latex rubber cap and / or plunger. Disposable syringes contain a single dose of vaccine. Syringes generally have a tip cap to seal the tip before needle attachment, and this tip cap is preferably made of butyl rubber. When the syringe and needle are packaged separately, the needle is preferably fitted into a butyl rubber shield. Preferred syringes are those sold under the trade name "Tip-Lok" (trademark).

[0264] For example, to facilitate delivery to children, containers may be marked to indicate half the dose volume. For instance, a syringe containing a 0.5 ml dose may have a mark indicating a 0.25 ml volume.

[0265] When glass containers (e.g., syringes or liquid medicine bottles) are used, it is preferable to use containers made of borosilicate glass rather than soda-lime glass.

[0266] The kit or composition may be packaged (e.g., in the same box) with a leaflet containing vaccine details, such as instructions for administration and details of the antigens in the vaccine. The instructions may also include warnings, such as keeping a solution of adrenaline readily available in case of an anaphylactic reaction after vaccination.

[0267] Treatment methods and vaccine administration This invention provides a vaccine manufactured in accordance with the present invention.

[0268] Vaccine compositions manufactured according to the present invention are suitable for administration to human patients, and the present invention provides a method for enhancing the immune response in a patient, the method comprising the step of administering the composition of the present invention to the patient.

[0269] The present invention also provides compositions for use as pharmaceuticals.

[0270] The present invention also provides the use of influenza virus antigens prepared according to the present invention in the manufacture of drugs for enhancing the immune response in patients.

[0271] The immune responses enhanced by these methods and their use generally include antibody responses, preferably protective antibody responses. Methods for evaluating antibody responses, neutralizing capacity, and protection after influenza virus vaccination are well known in the art. Studies in humans have shown that antibody titers against human influenza virus hemagglutinins correlate with protection (approximately 30-40 serum sample hemagglutination inhibitory titers provide approximately 50% protection from homologous virus infection)

[0185] . Antibody responses are typically measured by hemagglutination inhibition, microneutralization, single radial immunodiffusion (SRID), and / or single radial hemolysis (SRH). These assay techniques are well known in the art.

[0272] The compositions of the present invention can be administered in a variety of ways. The most preferred route of immunization is intramuscular injection (e.g., into the arm or leg), but other available routes include subcutaneous injection, intranasal injection [186, 187-188], oral injection

[0189] , intradermal injection [190, 191], transcutaneous injection, transdermal injection

[0192] , and the like.

[0273] Vaccines prepared according to the present invention may be used to treat both children and adults. Currently, influenza vaccines are recommended for use in pediatric and adult immunization starting from 6 months of age. Therefore, patients may be under 1 year old, 1-5 years old, 5-15 years old, 15-55 years old, or at least 55 years old. Patients who are preferably eligible for this vaccine include the elderly (e.g., ≥50 years, ≥60 years, and preferably ≥65 years), young children (e.g., ≤5 years), hospitalized patients, healthcare workers, military and armed personnel, pregnant women, patients with chronic diseases, immunocompromised patients, patients who have taken antiviral compounds (e.g., oseltamivir or zanamivir compounds; see below) within 7 days prior to vaccination, people with egg allergies, and international travelers. However, this vaccine is not only suitable for these groups and may be used more generally for the population. For pandemic strains, administration to all age groups is preferable.

[0274] Preferred compositions of the present invention satisfy one, two, or three of the CPMP criteria for efficacy. In adults (18–60 years), the criteria are: (1) ≥70% serological protection; (2) ≥40% serological conversion; and / or (3) ≥2.5-fold GMT increase. In older adults (>60 years), the criteria are: (1) ≥60% serological protection; (2) ≥30% serological conversion; and / or (3) ≥2-fold GMT increase. These criteria are based on open-label studies involving at least 50 patients.

[0275] Treatment may be a single-dose or multi-dose plan. Multi-dose plans may be used in primary immunization plans and / or booster immunization plans. In multi-dose plans, different doses may be administered via the same or different routes, for example, parenteral primary immunization and mucosal booster immunization, or mucosal primary immunization and parenteral booster immunization. Administration of two or more doses (typically two doses) is particularly useful in immunologically inexperienced patients, for example, those who have never received an influenza vaccine before, or for vaccination against new HA subtypes (such as during a pandemic). Multi-dose plans are typically administered at least one week apart (e.g., about two weeks, three weeks, four weeks, six weeks, eight weeks, ten weeks, twelve weeks, sixteen weeks, etc.).

[0276] The vaccines produced by the present invention may be administered to a patient substantially simultaneously with other vaccines (e.g., during the same medical examination or visit to a healthcare professional or immunization center), such as measles vaccine, mumps vaccine, rubella vaccine, MMR vaccine, varicella vaccine, MMRV vaccine, diphtheria vaccine, tetanus vaccine, pertussis vaccine, DTP vaccine, combined H. influenzae type b vaccine, inactivated poliovirus vaccine, hepatitis B virus vaccine, meningococcal combined vaccine (e.g., quadrivalent AC-W135-Y vaccine), respiratory syncytial virus vaccine, pneumococcal combined vaccine, etc. Substantially co-administration with pneumococcal vaccine and / or meningococcal vaccine is particularly useful in elderly patients.

[0277] Similarly, the vaccines of the present invention may be administered to the patient substantially simultaneously with antiviral compounds, particularly antiviral compounds active against influenza viruses (e.g., oseltamivir and / or zanamivir) (e.g., during the same medical consultation or visit to a healthcare professional). These antiviral substances include neuraminidase inhibitors, such as (3R,4R,5S)-4-acetylamino-5-amino-3(1-ethylpropoxy)-1-cyclohexene-1-carboxylic acid, or 5-(acetylamino)-4-[(aminoiminomethyl)-amino]-2,6-anhydrous-3,4,5-trideoxy-D-glycero-D-galactonone-2-enonic acid, and also include their esters (e.g., ethyl esters) and salts (e.g., phosphates). A preferred antiviral substance is (3R,4R,5S)-4-acetylamino-5-amino-3(1-ethylpropoxy)-1-cyclohexene-1-carboxylic acid, ethyl ester, phosphate (1:1), which is also known as oseltamivir phosphate (TAMIFLU®).

[0278] General The term "comprising" encompasses both "including" and "consisting." For example, a composition "comprising" X may consist only of X, or it may include something additional, such as X + Y.

[0279] The term "substantially" does not exclude "completely"; for example, a composition that "substantially does not contain" Y does not have to contain Y completely. The term "substantially" may be removed from the definition of this invention when necessary.

[0280] The term "approximately" in relation to a numerical value x means, for example, x ± 10%.

[0281] Unless otherwise specified, any method involving the step of mixing two or more components does not require any particular order of mixing. Therefore, multiple components can be mixed in any order. When there are three components, two components can be combined with each other, and then that combination can be combined with a third component, and so on.

[0282] When animal (especially bovine) material is used in cell culture, it should be obtained from a source that is free from transmissible spongiform encaphalopathies (TSEs), and particularly bovine spongiform encephalopathy (BSE). Overall, it is preferable to culture cells in a culture environment that is completely free of animal-derived material.

[0283] When a compound is administered to the body as part of a composition, that compound may be substituted by a suitable alternative prodrug. [Brief explanation of the drawing]

[0284] [Figure 1] This figure shows a method for isolating influenza virus from clinical samples. [Figure 2] This figure compares the HA titers of nine viral samples isolated from MDCK-33016 cells. For each sample, the bars on the left represent passage 2, and the bars on the right represent passage 5. [Figure 3] This figure compares the HA titers of influenza virus in 10 samples grown in three different MDCK cell types. For each sample, the three rods are as follows: left, 33016 in suspension; middle, adherent 33016; and right, CCL-34 MDCK cells. [Figure 4] This figure shows the binding of the three viruses to SNA or MAA lectin. Figure 4A shows the binding of the original isolate, Figure 4B shows the binding after growth in MDCK33016 cells, and Figure 4C shows the binding after growth in eggs. [Figure 5] This figure shows the binding of viruses to 3-SL or 6-SLN. The figure contains six columns: the leftmost three show binding to 3-SL at different concentrations (1 μM, 0.5 μM, 0.25 μM), and the rightmost three show binding to 6-SLN at different concentrations (0.25 μM, 0.125 μM, 0.0625 μM). Each column in each of the six groups represents a different virus. In Figure 5, the three columns from left to right represent: (i) cell-isolated viruses; (ii) egg-isolated viruses; and (iii) avian viruses. [Figure 6] This figure shows the binding of the virus to 3-SL or 6-SLN. The figure contains six columns: the three leftmost columns show binding to 3-SL at different concentrations (1 μM, 0.5 μM, 0.25 μM), and the three rightmost columns show binding to 6-SLN at different concentrations (0.25 μM, 0.125 μM, 0.0625 μM). Each column in each of the six groups represents a different virus. In Figure 6, the four columns from left to right show: (i) virus after 2 passages in an egg; (ii) virus after 2 passages in an MDCK; (iii) virus after 5 passages in an egg; (iv) virus after 5 passages in an MDCK. [Modes for carrying out the invention]

[0285] Isolation of virus from patient samples During the 2006-2007 Northern Hemisphere influenza season, clinical samples (nasal or throat swabs) containing influenza A and / or B virus subtypes were obtained from children and adults. The infectivity and reliability of the MDCK33016 cell line (DSM ACC2219), grown in serum-free suspension culture, were compared with the established MDCK CCL34 cell line (ATCC) and chicken eggs by hemagglutinin (HA) titer determination, polymerase chain reaction (PCR), and viral titration.

[0286] Diagnostic polymerase chain reaction (PCR) identified 248 influenza-positive samples. The reliability and infectivity of influenza virus replication and isolation in the MDCK33016 cell line and chicken eggs were evaluated by the following methods: (i) hemagglutinin (HA) titer; (ii) real-time polymerase chain reaction (PCR) for viral load measurement; and (iii) viral titration. Cellular replication accuracy was assessed by sequencing of the HA gene in the original clinical samples and in isolates from second passages in MDCK cells and chicken eggs. Viral titers obtained from isolates grown in suspended MDCK33016 cells were compared to those from MDCK33016 cells attached to plates.

[0287] The results showed that the isolation ability of the MDCK33016 suspension cell line was superior to that of the established MDCK CCL34 cell line and considerably higher than that of chicken eggs. After passage of viral samples in MDCK33016 cells, no amino acid substitutions were identified in any isolate. In contrast, almost all egg-passaged viruses contained one or more amino acid substitutions, mainly in the HA1 gene. Mutations in the antibody-binding site of the HA gene observed after passage in eggs may alter the antigenicity of the influenza virus.

[0288] Fifty-five percent of clinical samples obtained from patients with acute respiratory illness were identified as influenza-positive and possessed the following viral types: 79% A / H3N2; 12.5% ​​A / H1N1; 1.6% B; 0.4% H3 / B; and 6.5% untyped. Virus isolation from clinical samples was successful using MDCK33016 cells (Figure 1). In contrast, viruses injected into eggs from clinical samples could not be successfully isolated. Similar negative results were obtained with newly prepared chicken embryo fibroblasts (CEF). Isolation and establishment of influenza virus in eggs was only possible using the supernatant of MDCK33016 cultures with positive HA titers.

[0289] The first sample from each cell was further inoculated into eggs for reference. The number of successful virus isolations using each approach is shown in the box in Figure 1, along with the number of different virus types injected. All three virus subtypes isolated from MDCK33016 cells showed reasonable HA (>32) and viral titers (>1'10) after the second passage in eggs. 6 ) was acquired, and both titers increased with further passage (Figure 2).

[0290] MDCK33016 cells grown in suspension were superior to adherent cell lines (CCL-34) in isolating influenza viruses from clinical swabs for all three subtypes. As shown by the recovery rates (Table 1), the suspension cell lines showed higher sensitivity to positive influenza swab material. HA sequences between different passages of MDCK33016 cells and eggs were compared to the original isolates (Table 2). No mutations were found in influenza A strains isolated in MDCK33016 cells after 5 passages, whereas influenza A strains isolated in eggs showed mutations in the antibody-binding site of the HA protein after 2 passages. No mutations were found in influenza B strains isolated in MDCK33016 cells or eggs.

[0291] After replication of isolates in suspended MDCK33016 cells, a viral yield at least 1 log level higher was observed compared to adherent MDCK33016 cells (Figure 3).

[0292] Therefore, the MDCK33016 suspension cell line is an ideal system for the isolation and replication of wild-type influenza strains, as it offers greater isolation capacity than chicken eggs.

[0293] Furthermore, due to their high replication accuracy, using cell-based isolates in the production of human influenza vaccines may result in more reliable vaccines. Improving the compatibility between circulating wild-type strains and the strains included in the vaccine should provide greater protection against influenza.

[0294] In conclusion: (a) Compared to eggs, MDCK33016 cells successfully isolated all virus strains; (b) Virus strains isolated from MDCK33016 cells could be successfully propagated in eggs; (c) MDCK33016 cells grown in suspension had a superior recovery rate for all three influenza virus subtypes compared to adherent cells; and (d) no HA gene substitutions were present in any of the isolates grown in MDCK33016 cells compared to the original material, but were present in eggs after the second passage. Therefore, the MDCK33016 suspension cell line is a highly reliable culture medium for the isolation and propagation of human influenza virus subtypes, as it preserves the distinctive characteristics of wild-type viruses and is reliable for passage of wild-type influenza viruses from clinical isolates.

[0295] Receptor binding The receptor preferences for the original isolated virus, oviparous virus, and MDCK-developing virus were investigated. Lectins with 2,3-sialyl linkages (MAA) or 2,6-sialyl linkages (SNA), or sialyl glycopolymers of 2,3-sialyl lactose (3-SL, an analogue of the oviparous receptor) and 2,6-sialyl-N-acetyllactosamine (6-SLN, an analogue of the human receptor) were used in the study.

[0193]

[0296] Figure 4 shows the results of a representative study. Labeled peaks indicate binding to SNA or MAA lectin. The original virus (Figure 4A) and the virus grown with MDCK33016 (Figure 4B) have distinct peaks for SNA and MAA, but in the oviparous virus (Figure 4C), the SNA and MAA peaks are substantially overlapping.

[0297] Further experiments using 3-SL and 6-SLN investigated binding specificity. One example of the results is shown in Figure 5. Binding on the left side of the graph indicates preference for the avian receptor, while binding on the right side indicates preference for the human receptor. As can be seen in Figure 5, cell-isolated viruses strongly prefer human receptors.

[0298] Figure 6 shows data using Stuttgart isolates (A / H1N1) after 2 or 5 passages in MDCK33016 grown in eggs or suspensions. MDCK-passed viruses show strong preference for 6-SLN.

[0299] In conclusion, all MDCK-grown clinical human A and B viruses, with the exception of a few isolates that did not bind to either in this assay, bind to 6-SLN rather than 3-SL. Unlike the original clinical isolates, oocyte-compatible viruses either bind to 3-SL or not to either 3-SL or 6-SLN.

[0300] Changes in egg development Various strains of influenza A and B viruses were isolated in MDCK cells and then passaged up to five times in one of the following culture media: oocytes; MDCK cell CCL-34; MDCK cell 33016; Vero cells; or HEK293-T cells. After each passage, the viral HA gene was sequenced and the HA titer was measured.

[0301] The HA sequences for some strains (e.g., A / H1N1 / Bayern / 7 / 95) were stable between passages with eggs and MDCK33016, while others were not. For example, the HA sequence of A / H1N1 / Nordrhein Westfalen / 1 / 05 acquired the D203N mutation at antibody-binding site D after two passages with eggs, and additionally acquired R329K after two more passages. In contrast, the sequence remained unchanged in viruses passaged in parallel with MDCK33016.

[0302] This A / H1N1 / NRW / 1 / 05 strain did not grow when cultured in Vero cells. The other four culture media supported the growth of this strain, but the HA titer varied. For example, titers of 32-256 were observed in eggs, but 293-T cells gave lower titers (16-32), while MDCK33016 gave higher titers (32-512).

[0303] The present invention is described by illustration only, and it is understood that modifications may be made within the scope and spirit of the invention.

[0304] Table 1: Recovery rate of influenza-positive samples after first passage in MDCK33016 and ATCC(CCL-34) cell lines

[0305] [Table 1] * One was a double infection (H3 / B) that could be isolated in both MDCK cell lines.

[0306] Table 2: Comparison of hemagglutinin sequences after 2 or 5 passages in MDCK33016-PF cells or eggs compared to the original isolate.

[0307] [Table 2] 0 = No mutations detected * Only the HA1 sequence is available for the original isolate. ** Comparison with P2(MDCK33016) isolate.

[0308] References (their contents are incorporated herein by reference)

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Claims

[Claim 1] The influenza vaccine or method described in the specification.

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