Methods for producing adenovirus

Abstract

Methods for producing adenovirus suitable for use in vaccines and for increasing the yield of adenovirus during production include adding adenovirus to a cell population in culture, culturing the cell population under conditions permissive for infection of the cell population by the adenovirus to provide a cell population comprising adenovirus-infected cells, culturing the cell population comprising adenovirus-infected cells under conditions permissive for replication of the adenovirus, and recovering the adenovirus from the culture.

Description

[Technical Field] 【0001】 The present invention relates to a method for producing adenoviruses. More specifically, the present invention relates to a method for producing adenoviruses suitable for use in vaccines, and to a method for increasing the yield of adenoviruses during production. [Background technology] 【0002】 Adenoviruses are double-stranded DNA viruses with a genome of approximately 26–46 kb. Adenoviruses are species-specific, with various serotypes isolated from a wide range of mammalian species. Human adenoviruses are ubiquitous, and most people are infected with one or more serotypes, resulting in lifelong immunity. 【0003】 Modified adenoviruses can be used as vectors for delivering DNA encoding foreign antigens. Such adenovirus vectors are often replication-deficient adenovirus vectors in which the essential E1A and E1B genes are deleted and replaced by an expression cassette having a highly active promoter, such as a cytomegalovirus pre-early promoter that drives the expression of heterologous genes. 【0004】 Replication-deficient adenovirus vectors are widely used in vaccines because they induce strong humoral and T-cell responses to heterologous genes encoded by the vector. Clinical trials investigating replication-deficient Ad5-based vaccines for use in the treatment of tuberculosis show very promising results (Non-Patent Literature 1). Nevertheless, current methods for producing such adenoviruses are inefficient and lack scalability. [Prior art documents] [Non-patent literature] 【0005】 [Non-Patent Document 1] Smail et al.2013;Sci.Transl.Med.Oct 2;5(205):205ra134 [Overview of the project] [Problems that the invention aims to solve] 【0006】 Therefore, improved methods for adenovirus production are needed. [Means for solving the problem] 【0007】 The present invention relates, at least in part, to the development of an improved adenovirus production method that is highly scalable and provides increased adenovirus vector titers compared to alternative production methods. Thus, the method of the present invention may be particularly useful when large quantities of adenovirus vectors are required, such as for providing adenovirus-based vaccines for epidemic and pandemic diseases. 【0008】 Therefore, in one embodiment, a method for producing adenovirus for use in a vaccine: (a) Adding adenovirus to a cell population in culture with a MOI that is insufficient to infect all cells in the cell population; (b) To provide a cell population containing adenovirus-infected cells by culturing a cell population under conditions that allow infection of the cell population with adenovirus; (c) Culturing a cell population containing adenovirus-infected cells under conditions that allow for adenovirus replication; and (d) A method is provided which includes recovering adenovirus from a culture. 【0009】 In another embodiment, a method is provided for producing an adenovirus for use in a vaccine, comprising culturing a cell population comprising a first fraction of adenovirus-infected cells under conditions that allow infection of a second fraction of the cell population with adenovirus, wherein the second fraction of the cell population is infected with adenovirus released by the first fraction of adenovirus-infected cells. 【0010】 In yet another embodiment, a method for producing adenovirus for use in a vaccine: (a) Adding adenovirus to a population of cells in culture; (b) To provide a cell population containing adenovirus-infected cells by culturing a cell population under conditions that allow infection of the cell population; (c) Culturing a cell population containing adenovirus-infected cells under conditions that allow for adenovirus replication; and (d) A method is provided which includes recovering the adenovirus from the culture approximately 96 to 144 hours after adding the adenovirus to the cell population. 【0011】 In another embodiment, a method for producing adenovirus for use in a vaccine: (a) at least 0.5 × 10 6 To provide a cell population during culture by seeding cells into a cell culture vessel at an initial cell density of cells / mL; (b) Adding adenovirus to a cultured cell population; (c) To provide a cell population containing adenovirus-infected cells by culturing a cell population under conditions that allow infection of the cell population; (d) Culturing a cell population containing adenovirus-infected cells under conditions that allow adenovirus replication; and (e) A method is provided which includes recovering adenovirus from a culture. 【0012】 In another embodiment, a method for producing adenovirus for use in a vaccine: (a) at least about 1 × 10 6 Adding adenovirus to a cultured cell population having a viable cell density of cells / mL; (b) To provide a cell population containing adenovirus-infected cells by culturing a cell population under conditions that allow infection of the cell population with adenovirus; (c) Culturing a cell population containing adenovirus-infected cells under conditions that allow for adenovirus replication; and (d) A method is provided which includes recovering adenovirus from a culture. 【0013】 In another embodiment, a method for producing adenovirus for use in a vaccine: (a) At an MOI insufficient to infect all cells in the cell population, at least about 1 × 10⁻⁶ 6 Adding adenovirus to a cultured cell population having a viable cell density of cells / mL; (b) To provide a cell population containing adenovirus-infected cells by culturing a cell population under conditions that allow infection of the cell population; (c) Culturing a cell population containing adenovirus-infected cells under conditions that allow for adenovirus replication; and (d) Approximately 96 to 144 hours after the adenovirus was added to the cell population, the adenovirus is recovered from the culture. A method is provided which includes switching the temperature to which a cell population is exposed from a first temperature to a second temperature, wherein the first and second temperatures allow the cell population to be infected with an adenovirus. 【0014】 In another embodiment, a method for producing adenovirus for use in a vaccine: (a) At an MOI insufficient to infect all cells in the cell population, at least about 1 × 10⁻⁶ 6 Adding adenovirus to a cultured cell population having a viable cell density of cells / mL; (b) Culturing the cell population under conditions that allow infection of a first fraction of cells in the cell population with adenovirus; (c) Culturing a cell population containing a first fraction of adenovirus-infected cells under conditions that allow infection of a second fraction of cells in the cell population with adenovirus, wherein the second fraction of cells is infected with adenovirus released into the culture by the first fraction of adenovirus-infected cells; (d) Culturing a cell population containing adenovirus-infected cells under conditions that allow adenovirus replication; and (e) Approximately 96 to 144 hours after the adenovirus was added to the cell population, the adenovirus is recovered from the culture. A method is provided which includes switching the temperature to which a cell population is exposed from a first temperature to a second temperature, wherein the first and second temperatures allow the cell population to be infected with an adenovirus. 【0015】 In another embodiment, a method for preparing a vaccine containing an adenovirus: (a) Adding adenovirus to a cell population in culture with a MOI that is insufficient to infect all cells in the cell population; (b) To provide a cell population containing adenovirus-infected cells by culturing a cell population under conditions that allow infection of the cell population; (c) Culturing a cell population containing adenovirus-infected cells under conditions that allow for adenovirus replication; (d) Recovering adenovirus from the culture; (e) Purifying adenovirus; and (f) A method is provided which includes preparing a vaccine containing purified adenovirus. 【0016】 In another embodiment, a method for preparing a vaccine containing an adenovirus: (a) Culturing a cell population containing a first fraction of adenovirus-infected cells under conditions that allow infection of a second fraction of the cell population with adenovirus, wherein the second fraction of the cell population is infected with adenovirus released into the culture by the first fraction of adenovirus-infected cells; (b) Recovering adenovirus from the culture; (c) Purifying adenovirus; and (d) A method is provided which includes preparing a vaccine containing purified adenovirus. 【0017】 In another embodiment, a method for preparing a vaccine containing an adenovirus: (a) Adding adenovirus to a population of cells in culture; (b) To provide a cell population containing adenovirus-infected cells by culturing a cell population under conditions that allow infection of the cell population; (c) Culturing a cell population containing adenovirus-infected cells under conditions that allow for adenovirus replication; (d) Approximately 96 to 144 hours after adding the adenovirus to the cell population, the adenovirus is collected from the culture; (e) Purifying adenovirus; and (f) A method is provided which includes preparing a vaccine containing purified adenovirus. 【0018】 In another embodiment, a method for preparing a vaccine containing an adenovirus: (a) at least 0.5 × 10 6 To provide a cell population during culture by seeding cells into a cell culture vessel at an initial cell density of cells / mL; (b) Adding adenovirus to a cultured cell population; (c) To provide a cell population containing adenovirus-infected cells by culturing a cell population under conditions that allow infection of the cell population; (d) Culturing a cell population containing adenovirus-infected cells under conditions that allow adenovirus replication; and (e) Recovering adenovirus from the culture; (f) Purifying adenovirus; and (g) A method is provided which includes preparing a vaccine containing purified adenovirus. 【0019】 In another embodiment, a method for preparing a vaccine containing an adenovirus: (a) at least about 1 × 10 6 Adding adenovirus to a cultured cell population having a viable cell density of cells / mL; (b) To provide a cell population containing adenovirus-infected cells by culturing a cell population under conditions that allow infection of the cell population with adenovirus; (c) Culturing a cell population containing adenovirus-infected cells under conditions that allow for adenovirus replication; (d) Recovering adenovirus from the culture; (e) Purifying adenovirus; and (f) A method is provided which includes preparing a vaccine containing purified adenovirus. 【0020】 In another embodiment, a method for preparing a vaccine containing an adenovirus: (a) At an MOI insufficient to infect all cells in the cell population, at least about 1 × 10⁻⁶ 6 Adding adenovirus to a cultured cell population having a viable cell density of cells / mL; (b) To provide a cell population containing adenovirus-infected cells by culturing a cell population under conditions that allow infection of the cell population; (c) Culturing a cell population containing adenovirus-infected cells under conditions that allow for adenovirus replication; (d) Approximately 96 to 144 hours after adding the adenovirus to the cell population, the adenovirus is collected from the culture; (e) Purifying adenovirus; and (f) preparing a vaccine containing purified adenovirus, A method is provided which includes switching the temperature to which a cell population is exposed from a first temperature to a second temperature, wherein the first and second temperatures allow the cell population to be infected with an adenovirus. 【0021】 In another embodiment, a method for preparing a vaccine containing an adenovirus: (a) At an MOI insufficient to infect all cells in the cell population, at least about 1 × 10⁻⁶ 6 Adding adenovirus to a cultured cell population having a viable cell density of cells / mL; (b) Culturing the cell population under conditions that allow infection of a first fraction of cells in the cell population with adenovirus; (c) Culturing a cell population containing a first fraction of adenovirus-infected cells under conditions that allow infection of a second fraction of cells in the cell population with adenovirus, wherein the second fraction of cells is infected with adenovirus released into the culture by the first fraction of adenovirus-infected cells; (d) Culturing a cell population containing adenovirus-infected cells under conditions that allow adenovirus replication; (e) Approximately 96 to 144 hours after adding the adenovirus to the cell population, the adenovirus is collected from the culture; (f) Purifying adenovirus; and (g) preparing a vaccine containing purified adenovirus, A method is provided which includes switching the temperature to which a cell population is exposed from a first temperature to a second temperature, wherein the first and second temperatures allow the cell population to be infected with an adenovirus. 【0022】 In another embodiment, a method is provided for increasing the yield of adenovirus during adenovirus production, comprising culturing a population of cells in culture at a first temperature in the presence of adenovirus, and switching the temperature to which the cell population is exposed to a second temperature, wherein the first and second temperatures allow for infection of the cell population with adenovirus. 【0023】 In another embodiment, a method for producing adenovirus, as shown in Figure 2, is provided. 【0024】 In another embodiment, an adenovirus for use in a vaccine is provided, which can be obtained by the method of the present invention or is obtained by the method of the present invention. 【0025】 In another embodiment, a vaccine comprising an adenovirus that can be obtained by or obtained by the method of the present invention is provided. 【0026】 In any embodiment described herein, the method may include switching the temperature to which the cell population is exposed from a first temperature to a second temperature, the first and second temperatures being suitable for infection of the cell population with adenovirus. 【0027】 Aspects and embodiments of the present invention are described in the appended claims. These and other aspects and embodiments of the present invention are also described herein. [Brief explanation of the drawing] 【0028】 [Figure 1] Figure 1 shows an exemplary high MOI process. According to this method, cells are grown until they reach a cell density of approximately 3–5 × 10⁶ cells / mL, at which point they are diluted 1:1 and infected with adenovirus at an MOI of 10. Infected cells are cultured for a further 42 ± 2 hours before being harvested for adenovirus purification. [Figure 2] Figure 2 shows an exemplary low-MOI process according to the present invention. Cells are seeded and infected with adenovirus at a low MOI of up to 1 approximately 24 hours after seeding. Infected cells are cultured for approximately 6 days and then harvested for adenovirus purification. [Figure 3] Figure 3 shows a comparison of viable cell density (VCD), viability, and adenovirus titer during high-MOI and low-MOI processes. Figure 3A VCD; Figure 3B Viability; Figure 3C qPCR titer; Figure 3D A260:A280 ratio. [Figure 4] Figure 4 provides a comparison of high-MOI and low-MOI processes. Figure 4A: Viral genome concentration; infectivity titer; and viral particle titer. Figure 4B: Viral genome:infectivity unit ratio and A260:A280 ratio. [Figure 5] Figure 5 shows the effect of MOI on VCD, survival rate, and adenovirus titer. Figure 5A VCD; Figure 5B Survival rate; Figure 5C Adenovirus qPCR titer. MOI [Figure 6] Figure 6 shows different initial cell seeding densities and adenovirus titers at the time of infection. Figure 6A: Day 0 of infection; Figure 6B: Day 1 of infection. [Figure 7] Figure 7 shows the effect of dilution on viral titer in low-MOI processes with infection at different time points. Figure 7A: Day 0 of infection; Figure 7B: Day 1 of infection. [Figure 8] Figure 8 shows the effects of various cell culture additives on infectivity titers. [Figure 9] Figure 9 shows the effect of temperature shift on VCD, survival rate, and adenovirus titer. Figure 9A VCD; Figure 9B Survival rate; Figure 9C Adenovirus qPCR titer. [Figure 10] Figure 10 shows the scalability of an exemplary low-MOI process. Figure 10A VCD; Figure 10B Survival rate; Figure 10C Adenovirus qPCR titer. [Modes for carrying out the invention] 【0029】 A brief explanation of sequence listings 【0030】 [Table 1] 【0031】 [Table 2] 【0032】 Adenovirus production Adenoviruses are non-enveloped viruses with a linear double-stranded DNA (dsDNA) genome 26–46 kb in length. Defective adenovirus vectors have been used as vaccine vectors to deliver infectious pathogen antigens in several clinical trials. However, current methods for producing adenoviruses for vaccine use are not scalable and are limited by the final adenovirus titer. We have, surprisingly, discovered a novel adenovirus production method that is highly scalable, provides increased adenovirus vector titers compared to alternative methods, and represents a suitable option for producing adenovirus vectors for inclusion in adenovirus-based vaccines for epidemic and pandemic diseases. 【0033】 It will be understood that the method of the present invention may be for the production of adenovirus for use in vaccines, for example, for use in COVID-19 vaccines. 【0034】 The inventors have surprisingly demonstrated that adenoviruses added to a cell population at a low MOI can provide high viral titers (see Examples 1-2). Furthermore, products obtained from the low-MOI process had comparable quality to those derived from exemplary high-MOI processes (see Example 1). As is readily apparent, the use of a low-MOI process significantly reduces viral seed requirements compared to a high-MOI process. As used herein, "MOI" refers to the ratio of the number of infectious viral particles to the number of target cells in a cell population. 【0035】 Thus, in some embodiments, the method of the present invention includes adding an adenovirus to a cell population in culture. In a preferred embodiment, the method of the present invention includes adding an adenovirus to a cell population in culture at a multiplicity of infection (MOI) that is insufficient to infect all of the cells in the cell population. In some embodiments, the MOI is from about 0.003 to about 1, preferably from about 0.03 to about 0.3, and most preferably about 0.1. For example, in some embodiments, the MOI is 0.025, 0.030, 0.052, 0.075, 0.090, 0.100, 0.120, 0.180 or 0.270. 【0036】 In some embodiments, prior to adding an adenovirus to a cell population in culture, the method of the present invention includes seeding cells in a cell culture vessel to provide a cell population in culture. As used herein, "cell culture vessel" refers to a vessel suitable for culturing cells. In some embodiments, a cell culture medium is used for cell seeding. As used herein, "cell culture medium" means a liquid solution containing cell culture nutrients and salts used in the initial cell seeding step that is designed to support the growth and viability of cells in culture. In some embodiments, the cell culture medium used for cell seeding is BalanCD™ HEK Medium. In some embodiments, the cell culture medium used for cell seeding is not 293 SFM II. 【0037】 The adenovirus can be added to the cells at various times after seeding the cells in the cell culture vessel. Thus, in some embodiments, the method includes adding the adenovirus to the cell population in culture about 0 to 48 hours after seeding the cells in the cell culture vessel, preferably about 24 hours after seeding the cells in the cell culture vessel. For example, the adenovirus can be added to the cell population in culture within 6 hours, 12 hours, 18 hours, 24 hours, 32 hours, or 48 hours after seeding the cells in the cell culture vessel. 【0038】 Conventional approaches to adenovirus production are 5×10 at the time of infection 5The method relies on using a cell density in the range of cells / mL, because higher cell densities eliminate the production of infectious particles (as outlined in Kamen & Henry 2004 J. Gene Med. 2004 Feb; 6 Suppl1: S184~92). Here, the inventors have shown that increasing the seeded cell density surprisingly increases the viral titer (see Example 3). In a preferred embodiment, the method uses at least about 0.5 × 10⁻¹⁶ cells. 6 Cells / mL, preferably at least about 0.8 × 10⁶ 6 cells / mL, most preferably at least about 1.2 × 10⁶ 6 The method involves seeding cells into a cell culture vessel at an initial cell density of cells / mL. Increasing the initial cell seeding density can increase the viable cell density of the cell population at the time of infection. In some embodiments, the method involves seeding cells at least about 0.5 × 10⁻¹⁶. 6 cells / mL, preferably at least about 0.75 × 10⁶ 6 cells / mL, at least about 1 × 10⁶ 6 Cells / mL, at least approximately 1.5 × 10⁶ 6 cells / mL, at least about 2 × 10⁶ 6 cells / mL, or at least 2.5 × 10⁶ 6 cells / mL, most preferably at least about 1 × 10⁶ 6 The method involves adding adenovirus to a cell population in culture having a viable cell density of cells / mL. In some embodiments, the method involves adding adenovirus to a cell population having a viable cell density of approximately 0.5 × 10⁻⁶. 6 cells / mL ~ approx. 1×10 7 Cells / mL, preferably about 0.5 × 10⁶ 6 cells / mL ~ approx. 5×10 6 Cells / mL, most preferably about 0.5 × 10⁶ 6 cells / mL ~ approx. 2.5×10 6 This involves adding an adenovirus having a viable cell density of cells / mL to a cell population in culture. 【0039】 After adenovirus is added to a cell population, the adenovirus particles attach to target cells before endocytosis, thereby infecting the target cells. Therefore, in some embodiments, the method of the present invention involves culturing a cell population under conditions that allow infection of the cell population with adenovirus in order to provide a cell population containing adenovirus-infected cells. As used herein, “conditions that allow infection” means any suitable method of culturing cells that allows the entry of adenovirus DNA into the cells. Such conditions depend on the cell population being cultured and the adenovirus used to infect the cells. Techniques for determining the entry of adenovirus DNA into cells are well known in the art and include qPCR. 【0040】 When using a low MOI to infect a cell population, there may not be enough viral particles to infect all cells in the cell population. Therefore, in some embodiments, the method of the present invention includes culturing the cell population under conditions that allow infection of a first fraction of cells in the cell population with adenovirus. In some embodiments, the method further includes culturing the cell population containing the first fraction of infected cells under conditions that allow infection of a second fraction of cells in the cell population with adenovirus, where the second fraction of cells is infected with adenovirus released into the culture by the first fraction of infected cells. Therefore, in a preferred embodiment, the method is characterized by a first infection and a second infection, the first infection providing a first fraction of adenovirus-infected cells induced by adding adenovirus to the cell population, and the second infection providing a second fraction of adenovirus-infected cells induced by adenovirus released into the culture by the first fraction of adenovirus-infected cells. In some embodiments, the conditions for allowing infection of the first and second fractions of the cell population are the same. In some embodiments, the conditions for allowing infection of the first and second fractions of the cell population vary. In some embodiments, the method comprises culturing a cell population containing the first and second fractions of infected cells under conditions that allow infection of a third fraction of cells in the cell population with adenovirus, the third fraction of cells being infected with adenovirus released by the first and / or second fractions of adenovirus-infected cells. 【0041】 In a preferred embodiment, the method of the present invention comprises culturing a cell population containing a first fraction of adenovirus-infected cells under conditions that allow for adenovirus infection of a second fraction of cells in the cell population, the second fraction of cells being infected with adenovirus released into the culture by the first fraction of infected cells. In some embodiments, the method comprises culturing the cell population containing a first fraction of adenovirus-infected cells under conditions that allow for adenovirus infection of the second fraction of cells in the cell population, before culturing the cell population containing a first fraction of adenovirus-infected cells under conditions that allow for adenovirus infection of the second fraction of cells in the cell population. In some embodiments, the method comprises adding adenovirus to the cell population in culture before culturing the cell population under conditions that allow for adenovirus infection of the first fraction of cells in the cell population. 【0042】 In some embodiments of the method of the present invention, the conditions for allowing infection of a cell population with adenovirus include adding a cell culture additive to the cell population. As shown in Example 5, such additives can increase viral titer. Therefore, in some embodiments, the method of the present invention includes adding a cell culture additive to a cell population. In some embodiments, the conditions for allowing infection of a cell population with adenovirus include culturing the cell population in the presence of a cell culture additive as defined herein. In some embodiments, the method includes adding a cell culture additive to a cell population while culturing the cell population under conditions that allow infection of the cell population. As used herein, “cell culture additive” means a cell culture additive that is not present during the initial cell seeding step. 【0043】 In some embodiments, the cell culture additive contains DMSO. In some embodiments, the cell culture additive contains sodium butyrate. In some embodiments, the cell culture additive contains CaCl2. In preferred embodiments, the cell culture additive contains DMSO, sodium butyrate, and / or CaCl2. In particularly preferred embodiments, the cell culture additive contains DMSO, sodium butyrate, and CaCl2. In some embodiments, after adding the cell culture additive to the cell population, the cell population is exposed to about 0.1% to about 4% DMSO, preferably about 0.5% to about 2% DMSO, most preferably about 0.5% or about 1% DMSO. In some embodiments, after adding the cell culture additive to the cell population, the cell population is exposed to about 0.2 mM to about 10 mM sodium butyrate, preferably about 0.5 mM to about 2.5 mM sodium butyrate, most preferably about 1 mM sodium butyrate. In some embodiments, after adding a cell culture additive to the cell population, the cell population is exposed to about 0.5 mM to about 10 mM CaCl2, preferably about 1 mM to about 5 mM CaCl2, most preferably about 2 mM CaCl2. 【0044】 Cell culture additives can be added to the cell population at various points in time. For example, in some embodiments, the method of the present invention includes adding the cell culture additive to the cell population about 0 to 148 hours after the adenovirus has been added to the cell population, preferably about 48 to 120 hours after the adenovirus has been added to the cell population, and most preferably about 72 to 120 hours after the adenovirus has been added to the cell population. In some embodiments, the method includes adding the cell culture additive to the cell population at least every 12 to 96 hours, preferably at least every 24 to 72 hours, and most preferably every 48 hours. 【0045】 In some embodiments of the method of the present invention, the conditions for allowing infection of a cell population with adenovirus include adding a feed to the cell population. Therefore, in some embodiments, the method of the present invention includes adding a feed to a cell population. In some embodiments, the conditions for allowing infection of a cell population with adenovirus include culturing the cell population in the presence of a feed as defined herein. In some embodiments, the method includes adding a feed to a cell population while culturing the cell population under conditions that allow infection of the cell population. As used herein, “feed” means cell culture nutrients (e.g., amino acids and / or glucose) that are not present during the initial cell seeding step. Therefore, in some embodiments, the feed includes amino acids, vitamins and / or glucose. In preferred embodiments, the feed includes amino acids, vitamins and glucose. In some embodiments, the feed is BalanCD® HEK 293 feed. 【0046】 The feed can be added to the cell population at various points in time. For example, in some embodiments, the method includes adding the feed to the cell population about 0 to 120 hours after the adenovirus has been added to the cell population, preferably about 24 to 96 hours after the adenovirus has been added to the cell population, and most preferably about 24 to 48 hours after the adenovirus has been added to the cell population. In some embodiments, the method includes adding the feed to the cell population at least every 12 to 96 hours, preferably at least every 24 to 72 hours, and most preferably every 48 hours. In some embodiments, the method includes adding the feed to the cell population at a final concentration of up to about 10% v / v, preferably up to about 7.5% v / v, and most preferably about 5% v / v. In a preferred embodiment, the method includes adding the feed to the cell population about 24 to 48 hours after the adenovirus has been added to the cell population at a final concentration of about 5% v / v. 【0047】 In some embodiments, the conditions that allow infection of a cell population with adenovirus are those that maintain a cell viability of more than 80%, preferably more than 85%, and most preferably more than 90%. In some embodiments of the method of the present invention, the cell population is cultured under conditions that maintain a cell viability of more than 80%, preferably more than 85%, and most preferably more than 90%. Cell viability can be determined by a number of techniques known in the art. For example, dye exclusion techniques use indicator dyes to identify cell membrane damage. Cells that absorb the dye are stained and considered unviable. Dyes such as trypan blue, erythrosine, and nigrosine are commonly used. Cell viability can be calculated using automated machines such as the Vi-CELL® XR Cell Viability Analyzer. 【0048】 In some embodiments, the conditions that allow infection of a cell population with adenovirus include agitating the cell population. Therefore, in some embodiments of the method of the present invention, the method includes agitating the cell population. For example, in some embodiments, the cell population is subjected to about 1 to about 100 W / m 3 For example, approximately 5 to 90 W / m 3 Preferably about 15 to about 70 W / m 3 The cells are cultured in a cell culture vessel (e.g., a bioreactor) set to have a stirring speed that provides the power input. 【0049】 The inventors have surprisingly found that exposing a cell population to both the first and second temperatures can result in a higher yield of adenovirus production when the first temperature is higher than the second temperature (see Example 6). As used herein, “first temperature” refers to the temperature at which the cell population is cultured before the adenovirus is added to the cell population, e.g., about 31–40°C, preferably about 35–38°C, most preferably about 37°C, and “second temperature” is a different (e.g., lower) temperature than the first temperature, e.g., about 27–40°C, preferably about 31–35°C, most preferably about 33°C. In some embodiments, the second temperature is about 1–10°C lower than the first temperature, preferably about 3–7°C lower than the first temperature, most preferably about 4°C lower than the first temperature. 【0050】 Accordingly, in some embodiments of the method of the present invention, the method includes culturing a cell population at a first temperature. In some embodiments, conditions that allow infection of the cell population include culturing the cell population at a first temperature. For example, conditions that allow infection of the cell population may include culturing the cell population at a first temperature of about 31 to 40°C, preferably about 35 to 38°C, and most preferably about 37°C. In some embodiments, the method includes culturing the cell population at a second temperature. In some embodiments, conditions that allow infection of the cell population include culturing the cell population at a second temperature, i.e., a temperature different from (e.g., lower than) the first temperature. For example, conditions that allow infection of the cell population may include culturing the cell population at a second temperature of about 31 to 40°C, preferably about 31 to 35°C, and most preferably about 33°C. In a preferred embodiment, conditions that allow infection of the cell population with adenovirus include culturing the cell population at a first temperature, followed by culturing the cell population at a second temperature. 【0051】 In some embodiments, the conditions for allowing infection of a first fraction of cells in a cell population include culturing the cell population at a first temperature. For example, in some embodiments, the conditions for allowing infection of a first fraction of cells include culturing the cell population at a first temperature of about 31-40°C, preferably about 35-38°C, and most preferably about 37°C. In some embodiments, the conditions for allowing infection of a second fraction of cells in a cell population include culturing the cell population at a first temperature. In some embodiments, the conditions for allowing infection of a second fraction of cells in a cell population include culturing the cell population at a second temperature, i.e., a temperature different from (e.g., lower than) the first temperature. For example, in some embodiments, the conditions for allowing infection of a second fraction of cells include culturing the cell population at a second temperature of about 27-40°C, preferably about 31-35°C, and most preferably about 33°C. In a preferred embodiment, the conditions for allowing infection of a first fraction of cells include culturing the cell population at a first temperature, and the conditions for allowing infection of a second fraction of cells include culturing the cell population containing the first fraction of infected cells at a second temperature. 【0052】 Following cell infection, the adenovirus is transported to the cell nucleus. The viral DNA is then released, entering the cell nucleus and becoming capable of replication. In some embodiments, the method of the present invention involves culturing a cell population containing adenovirus-infected cells under conditions that allow for adenovirus replication. As used herein, “conditions that allow for adenovirus replication” means any suitable conditions that enable the proliferation of adenovirus within cells. Such conditions depend on the cell population being cultured and the adenovirus used to infect the cells. In preferred embodiments, the pH of the culture is maintained at about 6.5–7.5, more preferably about 6.9–7.3. Preferably, pH and / or other conditions are maintained to optimize glucose metabolism by the cells. The pH of the cell culture can be controlled by any suitable method, preferably in a manner that does not substantially inhibit adenovirus production. Several suitable techniques for modifying pH are known in the art, including the addition of buffers (e.g., bicarbonates or Tris buffers). Proper mixing of the culture is another condition that may be important for cell proliferation and adenovirus production. Other factors that may be considered include temperature, stirring speed, oxygen concentration, CO2 perfusion rate, cell concentration, cell sedimentation rate and flow rate in the culture, and levels of certain nutrients and / or intermediates (e.g., glutamine) that affect cell proliferation and metabolic rate. Techniques for determining intracellular adenovirus proliferation are well known in the art and include qPCR for determining gene copy number, plaque assay for determining infectious viral titer, and HPLC for determining viral particle count. 【0053】 In some embodiments, conditions that allow adenovirus replication include adding a cell culture additive as defined herein to a cell population. In some embodiments, conditions that allow adenovirus replication include culturing a cell population in the presence of a cell culture additive. In some embodiments, the method includes adding a cell culture additive to a cell population while culturing a cell population containing adenovirus-infected cells under conditions that allow adenovirus replication. 【0054】 In some embodiments, conditions that allow adenovirus replication include adding a feed as defined herein to a cell population. In preferred embodiments, conditions that allow adenovirus replication include culturing a cell population in the presence of a feed. In some embodiments, the method includes adding a feed to a cell population while culturing a cell population containing adenovirus-infected cells under conditions that allow adenovirus replication. 【0055】 In some embodiments, the conditions for allowing adenovirus replication are those that maintain a cell viability of more than 80%, preferably more than 85%, and most preferably more than 90%. In preferred embodiments, the conditions for allowing infection of a cell population by adenovirus and for allowing adenovirus replication are those that maintain a cell viability of more than 80%, preferably more than 85%, and most preferably more than 90%. 【0056】 In some embodiments, the conditions that allow adenovirus replication include agitating the cell population. For example, in some embodiments, the cell population is agitated at about 1 to about 100 W / m². 3 For example, approximately 5 to 90 W / m 3 Preferably about 15 to about 70 W / m 3 The cells are cultured in a container (e.g., a bioreactor) set to have a stirring speed that provides the necessary power input. 【0057】 In some embodiments, conditions that allow adenovirus replication include culturing the cell population at a first temperature as defined herein. In preferred embodiments, conditions that allow adenovirus replication include culturing the cell population at a second temperature as defined herein. In preferred embodiments, conditions that allow infection of the cell population with adenovirus include culturing the cell population at a first temperature as defined herein, and conditions that allow adenovirus replication include culturing the cell population at a second temperature as defined herein. 【0058】 Accordingly, in some embodiments, the method of the present invention includes switching the temperature to which a cell population is exposed from a first temperature to a second temperature. In a preferred embodiment, the first temperature allows infection of the cell population by adenovirus. In some embodiments, the second temperature allows infection of the cell population by adenovirus. In a preferred embodiment, the first and second temperatures allow infection of the cell population by adenovirus. In some embodiments, the first temperature allows replication of adenovirus. In a preferred embodiment, the second temperature allows replication of adenovirus. In a preferred embodiment, the first and second temperatures allow replication of adenovirus. In a preferred embodiment, the first temperature allows infection of the cell population by adenovirus, and the second temperature allows replication of adenovirus. In a preferred embodiment, the first and second temperatures allow infection of the cell population by adenovirus, and the first and second temperatures allow replication of adenovirus. 【0059】 In some embodiments, the method of the present invention includes switching the temperature to which the cell population is exposed from a first temperature to a second temperature about 3 to 96 hours after the adenovirus is added to the cell population, preferably about 48 to 96 hours after the adenovirus is added to the cell population, and most preferably about 72 hours after the adenovirus is added to the cell population. In some embodiments, the cell population is cultured at the first temperature for at least about 24 hours, preferably at least about 72 hours, and most preferably at least about 96 hours. In some embodiments, the cell population is cultured at the second temperature for at least about 24 hours, preferably at least about 36 hours, and most preferably at least about 48 hours. In some embodiments, after switching the temperature from the first temperature to the second temperature, the cell population is cultured at the second temperature until the adenovirus is recovered from the culture. 【0060】 In some embodiments of the method of the present invention, exposure of a cell population to a second temperature increases the stability of adenovirus in the culture. For example, exposure of a cell population to a second temperature can increase the stability of adenovirus in the culture compared to simply exposing the cell population to a first temperature. Adenovirus stability can be easily determined by any suitable method, for example, by measuring viral genome titer (e.g., by qPCR) or infectivity titer (e.g., by plaque assay) over time. If the virus is stable, the viral titer is not expected to decrease. Conversely, if the virus is unstable, the viral titer is expected to decrease. In some embodiments, exposure of a cell population to a second temperature decreases the adenovirus particle:infectious particle ratio in the culture. For example, exposure of a cell population to a second temperature may decrease the adenovirus particle:infectious particle ratio in the culture compared to simply exposing the cell population to a first temperature. The adenovirus particle:infectious particle ratio can be determined by any suitable method, for example, by separately measuring the virus particle titer and infectivity titer and then taking the ratio of the two values. In a preferred embodiment, exposure of the cell population to a second temperature increases the stability of the adenovirus in the culture and decreases the adenovirus particle:infectious particle ratio in the culture. 【0061】 Conveniently, the number of viable cells in a cell population may increase after the addition of adenovirus to the cell population (see Examples 1, 2, and 7). In some embodiments of the method of the present invention, a first temperature allows for the growth of the cell population. In some embodiments, a second temperature allows for the growth of the cell population. In some embodiments, the first and second temperatures allow for the growth of the cell population. Thus, in some embodiments, the method of the present invention involves culturing the cell population under conditions that allow for the growth of the cell population. As used herein, “allowing for the growth of the cell population” means any suitable method of culturing a cell population that enables cell growth. The method of culturing such cells depends on the selected cell type. Suitable culturing methods are well known in the art and typically involve maintaining pH and temperature within a range suitable for cell growth. Preferred temperatures for culturing are about 27–40°C, more preferably 31–37°C, and optimally about 37°C. Preferably, the pH of the culture is maintained at about 6–8, more preferably about 6.7–7.8, and optimally about 6.9–7.5. Cell density can increase throughout the growth cycle of a cell population. The concentration of cells in a culture can be monitored throughout the process using numerous techniques known in the art. Techniques focusing on the total number of cells in a culture include determining the weight of the culture, assessing the turbidity of the culture, determining the metabolic activity in the culture, electronic cell counting, microscopic cell counting of culture samples, plate counting using serial dilutions, membrane filter counting, and radioisotope assays. In this invention, any acceptable technique for evaluating cell density is preferred. For example, the cell density of a culture can be determined by spectrophotometry or by using a counting chamber such as a hemocytometer. Cell density can be calculated using automated machines such as the Vi-CELL® XR Cell Viability Analyzer. 【0062】 In some embodiments, the conditions that allow infection of a cell population are the conditions that allow cell proliferation. In some embodiments, the conditions that allow infection of a first fraction of a cell population are the conditions that allow cell proliferation. In some embodiments, the conditions that allow infection of a second fraction of a cell population are the conditions that allow cell proliferation. In some embodiments, the conditions that allow infection of a third fraction of a cell population are the conditions that allow cell proliferation. In some embodiments, the conditions that allow replication of adenovirus are the conditions that allow cell proliferation. 【0063】 Following the initial cell infection, the adenovirus may undergo several subsequent infections of cell populations and / or replication within the cells. Therefore, in some embodiments of the method of the present invention, the peak viral titer is achieved approximately 2–8 days after the adenovirus is added to the cell population, preferably approximately 3–7 days after the adenovirus is added, and most preferably approximately 4–6 days after the adenovirus is added. 【0064】 After replication, the adenovirus can be conveniently isolated from the culture (for example, for purification purposes so that the adenovirus can be added to a vaccine). Therefore, in some embodiments, the method of the present invention includes recovering the adenovirus from the culture. In some embodiments, the step of recovering the adenovirus from the culture includes recovering the adenovirus from the cell culture medium in which the cell population was cultured. In preferred embodiments, the step of recovering the adenovirus from the culture includes lysing the cells of the cell population. In some embodiments, the step of recovering the adenovirus from the culture includes lysing the cells of the cell population and recovering the adenovirus from the cell lysates of the cell population. 【0065】 In a preferred embodiment, the cells of a cell population are lysed using a cell lysant (e.g., a surfactant). The use of surfactants for cell lysis has the advantage of being easy to implement and easily scalable. Surfactants that can be used for cell lysis are known in the art. Surfactants used for cell lysis in the method of the present invention include, but are not limited to, cationic, anionic, zwitterionic, and nonionic surfactants. In a preferred embodiment, the surfactant is a nonionic surfactant. Examples of suitable nonionic surfactants include polysorbate (e.g., polysorbate-20 or polysorbate-80) and Triton (e.g., Triton-X). In one embodiment, the nonionic surfactant is polysorbate-20. The optimal concentration of the nonionic surfactant used to lyse the host cell population may vary, for example, within the range of about 0.005 to 0.025 kg surfactant / kg cell culture vessel, about 0.01 to 0.02 kg surfactant / kg cell culture vessel, or about 0.011 to 0.016 kg surfactant / kg cell culture vessel. As used herein, “kg cell culture vessel” means the total mass of the cell population and cell culture medium in the cell culture vessel. In a preferred embodiment, the concentration of the nonionic surfactant (e.g., polysorbate-20) used to lyse the host cell population is about 0.013 kg surfactant / kg cell culture vessel. The host cells may be incubated with the nonionic surfactant (e.g., polysorbate-20) for a time sufficient to lyse all or substantially all of the cells in the host cell population. In an embodiment, the host cells are incubated with the nonionic surfactant (e.g., polysorbate-20) for at least about 15 minutes prior to the nuclease treatment step. In an embodiment, the host cells are incubated with the nonionic surfactant (e.g., polysorbate-20) for up to 30 minutes prior to the nuclease treatment step. In an embodiment, the host cells are not incubated with the nonionic surfactant (e.g., polysorbate-20) for more than 30 minutes prior to the nuclease treatment step. 【0066】 In embodiments, a surfactant (e.g., polysorbate-20) forms part of the lysis buffer. Thus, in some embodiments, host cells are lysed using a lysis buffer containing at least one surfactant (e.g., polysorbate-20). An exemplary lysis buffer that may be used in the method of the present invention comprises about 500 mM Tris, about 20 mM MgCl2, about 50% (w / v) sucrose, and about 10% (v / v) polysorbate 20, and has a pH of about 8. The optimal concentration of the lysis buffer used to lyse a cell population may vary, for example, within the range of about 0.05 to 0.25 kg lysis buffer / kg container, about 0.10 to 0.20 kg lysis buffer / kg container, or about 0.11 to 0.16 kg surfactant / kg container. In a preferred embodiment, the concentration of the lysis buffer is about 0.13 kg lysis buffer / kg container. 【0067】 In some embodiments, the step of recovering adenovirus from the culture is performed at least about 48 hours after adding the adenovirus to the cell population, preferably at least about 96 hours after adding the adenovirus to the cell population, and most preferably at least about 120 hours after adding the adenovirus to the cell population. For example, in some embodiments, the step of recovering adenovirus from the culture is performed about 96 to 144 hours after adding the adenovirus to the cell population. 【0068】 In some embodiments, the step of recovering adenovirus from the culture is performed when the viability of cells in the cell population decreases, for example, when the viability of cells in the cell population decreases to less than about 99%, less than about 97%, less than about 95%, less than about 90%, or less than about 80%, preferably when the viability of the cell population decreases to less than about 95%. For example, in some embodiments, the step of recovering adenovirus from the culture is performed when less than about 99%, about 97%, about 95%, about 90%, or about 80%, preferably less than about 95%, of the cells in the cell population are viable. In some embodiments, the step of recovering adenovirus from the cell culture is performed when the oxygen consumption of cells decreases. Therefore, in some embodiments, the step of recovering adenovirus from the culture is performed when the viable cell density is about 1.5 × 10⁻⁶ 7 Less than cells / mL, approximately 1 × 10⁻⁶ 7 Cells / mL less than, approximately 6 × 10 6 Cells / mL less than 5.5 × 10⁻⁶ 6 Less than cells / mL, approximately 5 × 10 6 Less than cells / mL or approximately 4 × 10⁴ 6 This procedure is performed when the cell count drops below a certain level (cells / mL). 【0069】 In some embodiments, the host cell population is at least about 6 × 10 at harvest time. 5 Cells / mL, at least approximately 8 × 10⁴ 5 cells / mL, at least about 1 × 10⁶ 6 cells / mL, at least about 2 × 10⁶ 6 Cells, at least about 4 × 10 6 Cells, at least about 6 × 10 6 Cells, at least approximately 8 × 10 6 Cells or at least about 1 × 10⁻⁶ 7 The cells may have a cell density (e.g., viable cell density). In a preferred embodiment, the host cell population has at least about 4 × 10⁶ cells at harvest time. 6 The cells have a cell density (e.g., viable cell density). In some embodiments, the step of recovering adenovirus from the cell culture is performed when the oxygen consumption of the cells decreases. 【0070】 The host cell population was approximately 1 × 10⁶ at the time of harvesting. 9 cells / mL, up to approximately 1×10 8 cells / mL, up to approximately 8×10 7 cells / mL, up to approximately 6×10 7 cells / mL, up to approximately 4×10 7 cells / mL, up to approximately 2×10 7 cells / mL, up to approximately 1×10 7 cells / mL, up to approximately 8×10 6 Cells / mL, or up to approximately 6 × 10⁶ 6 It may have a cell density (e.g., viable cell density) of cells / mL. In some embodiments, the host cell population may have a maximum of approximately 8 × 10⁶ cells at harvest. 6 It has a cell density (e.g., viable cell density) of cells / mL. 【0071】 The host cell population was approximately 1 × 10⁶ at the time of harvesting. 5 cells / mL ~ approx. 1×10 9 cells / mL, approximately 8×10 5 cells / mL ~ approx. 1×10 8 cells / mL or approximately 1 × 10⁶ 6 cells / mL ~ approx. 1×10 7 It may have a cell density (e.g., viable cell density) of cells / mL. 【0072】 Upon recovery, the adenovirus-containing culture may contain at least one host cell protein (HCP). As used herein, the term "HCP" refers to a protein produced or encoded by a host cell population. 【0073】 Adenovirus-containing cultures may have HCP concentrations of at least about 20,000 ng / mL, at least about 30,000 ng / mL, at least about 40,000 ng / mL, at least about 50,000 ng / mL, at least about 60,000 ng / mL, at least about 70,000 ng / mL, at least about 80,000 ng / mL, at least about 90,000 ng / mL, or at least about 100,000 ng / mL. In a preferred embodiment, the adenovirus-containing culture has an HCP concentration of at least about 50,000 ng / mL. 【0074】 Adenovirus-containing cultures may have HCP concentrations of up to approximately 100,000 ng / mL, 90,000 ng / mL, 80,000 ng / mL, 70,000 ng / mL, 60,000 ng / mL, 50,000 ng / mL, 40,000 ng / mL, 30,000 ng / mL, or 20,000 ng / mL. In a preferred embodiment, the adenovirus-containing culture has an HCP concentration of up to approximately 75,000 ng / mL. 【0075】 Adenovirus-containing cultures may have HCP concentrations of approximately 20,000 ng / mL to 100,000 ng / mL, approximately 30,000 ng / mL to 90,000 ng / mL, or approximately 50,000 ng / mL to 80,000 ng / mL. 【0076】 In a preferred embodiment, the host cell population has a cell density (e.g., viable cell density) at the time of harvest and has the above-mentioned HCP concentration. For example, the host cell population has at least about 4 × 10⁻⁶ 6 The cells may have a cell density and an HCP concentration of at least approximately 50,000 ng / mL. 【0077】 In some embodiments of the method of the present invention, the step of recovering adenovirus from a cell culture is performed when the viral titer exceeds a threshold. For example, in some embodiments, the viral titer at recovery is at least about 0.5 × 10⁻⁶ 10 GC / mL, preferably at least about 1 × 10⁶ 11 GC / mL, at least about 2 × 10⁻⁶ 11 GC / mL, at least about 3 × 10⁻⁶ 11 GC / mL, or at least about 4 × 10⁻⁶ 11 The GC / mL ratio is preferred, and the most preferable is a viral titer of at least approximately 2 × 10⁶. 11 This is the case when the concentration is GC / mL. 【0078】 During adenovirus production, cultured cells may need to be supplied with fresh nutrients to ensure they remain viable. Therefore, in some embodiments, the method of the present invention includes the step of replacing or adding cell culture medium to the cell population after the adenovirus has been added. However, replacing or adding cell culture medium is costly and time-consuming. Therefore, in preferred embodiments, the method does not include the step of replacing or adding cell culture medium to the cell population after the adenovirus has been added. 【0079】 The inventors have demonstrated that the method of the present invention can be carried out on a large scale (see, for example, Example 7). For example, the method of the present invention may be suitable for recovering adenovirus-containing material (e.g., cell lysates and / or cell culture media, preferably cell lysates) in the range of up to about 5000 liters, for example, about 3 liters to about 3000 liters, preferably about 200 liters to about 2000 liters, from a single culture. Thus, in some embodiments of the method of the present invention, the culture process is carried out in a bioreactor. As used herein, “bioreactor” means a cell culture vessel suitable for a large-scale process. For example, in some embodiments, the bioreactor has a capacity of at least about 1 L, preferably at least about 1.2 L, about 3 L, about 50 L, about 1000 L, about 2000 L, about 3000 L, or about 5000 L, most preferably at least about 2000 L. In some embodiments, the bioreactor has at least about 7 × 10 9 10⁴ surviving T-REx® cells, preferably at least about 2.1 × 10⁴ 10 A number of surviving T-REx(trademark) cells, at least approximately 3.5 × 10⁶ 11 individual surviving T-REx(trademark) cells, at least approximately 5 × 10⁶ 12 10 surviving T-REx(trademark) cells or at least about 3 × 10⁶ 13 10⁴ surviving T-REx™ cells, most preferably at least about 5 × 10⁴ 12 It has the capacity of 100 surviving T-REx(trademark) cells. 【0080】 Adenovirus vector In preferred embodiments of the method of the present invention, the adenovirus is an adenovirus vector. As used herein, “adenovirus vector” means a form of adenovirus modified to insert a nucleotide sequence encoding a heterologous gene into a eukaryotic cell. As used herein, “heterologous gene” means a gene derived from an entity that is genotypically different from the rest of the entity being compared. Thus, heterologous gene refers to any gene that has not been isolated from, derived from, or based on a naturally occurring gene of an adenovirus. As used herein, “naturally occurring” means found in nature and not synthetically prepared or modified. 【0081】 In preferred embodiments of the method of the present invention, the adenovirus vector comprises a heterogene encoding a protein of interest, such as a therapeutic protein or an immunogenic protein. Alternatively, the heterogene may comprise a reporter gene that generates a detectable signal upon expression. Examples of such reporter genes include, but are not limited to, DNA sequences encoding fusion proteins, including, β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, membrane-bound proteins such as CD2, CD4, and CD8, influenza hemagglutinin protein, and other well-known proteins for which high-affinity antibodies exist or can be produced by conventional means, as well as, in particular, DNA sequences encoding fusion proteins, including membrane-bound proteins appropriately fused to an antigen-tagged domain derived from hemagglutinin or Myc. When these coding sequences associate with the regulatory elements that drive their expression, they provide signals detectable by conventional means including enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), and immunohistochemistry, as well as enzyme-, radioactive, colorimetric, fluorescence, or other spectroscopic assays, fluorescence-activated cell sorting assays, and immunological assays. 【0082】 In a preferred embodiment, the heterogene is a sequence encoding a product such as a biologically and medically useful protein, RNA, enzyme, or catalytic RNA, such as a therapeutic gene or immunogenic gene. The heterogene may be used, for example, for the treatment of gene deficiencies, as a cancer drug, as a vaccine, for the induction of an immune response, and / or for preventive purposes. In a preferred embodiment, the heterogene encodes an exogenous antigen, such as a naturally occurring form of an exogenous antigen, or a modified form thereof. As used herein, “exogenous antigen” means an antigen that induces a host immune response and is derived from an entity that is genotypically different from the host that induces the immune response. As used herein, a modified form of an exogenous antigen means a form of an exogenous antigen that induces a host immune response to a naturally occurring antigen and has at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the naturally occurring antigen. As used herein, induction of an immune response refers to the ability of a protein to induce a T cell and / or humoral immune response to a protein. The determination of the host immune response to the naturally occurring or modified forms of an exogenous antigen can be evaluated by any appropriate method, such as those described in Jeyanathan et al. 2020; Immunological considerations for COVID-19 vaccine strategies; Nature Reviews Immunology 20, 615-632 and Albert-Vega et al. 2018; Immune Functional Assays, From Custom to Standardized Tests for Precision Medicine; Frontiers in Immunology 9:2367. In some embodiments, modified forms of naturally occurring antigens induce a stronger host immune response than those induced by the naturally occurring antigen. In some embodiments, modified forms of naturally occurring antigens induce a weaker host immune response than those induced by the naturally occurring antigen. 【0083】 In some embodiments, the exogenous antigen is derived from SARS-CoV-2, preferably the spike protein of SARS-CoV-2. SARS-CoV-2 is a newly emerging coronavirus that causes COVID-19, a severe acute respiratory illness. To date, no vaccine to prevent SARS-CoV-2 infection has been available globally. Because the virus uses its spike glycoprotein to interact with the cell receptor ACE-2 and the serine protease TMPRSS2 for entry into target cells, this spike protein is an attractive target for vaccine therapy. Therefore, in preferred embodiments, the heterogene encodes the naturally occurring form of the SARS-CoV-2 spike protein or a modified version thereof. The RNA, DNA, and amino acid sequences of the SARS-CoV-2 spike protein are known to those skilled in the art and can be found in many databases, for example, in the National Center for Biotechnology Information (NCBI) database, which has accession number NC_045512.2. For example, in some embodiments, the heterogene encodes the SARS-CoV-2 spike protein, which includes the amino acid sequence shown in Sequence ID No. 1. In another exemplary embodiment, a heterologous gene encodes a modified form of the SARS-CoV-2 spike protein containing the amino acid sequence shown in SEQ ID NO: 2. As is readily apparent, the amino acid sequence shown in SEQ ID NO: 2 contains the SARS-CoV-2 spike protein amino acid sequence having a signal peptide of the human tissue plasminogen activator gene (tPA) at its N-terminus. The presence of the N-terminal tPA sequence may enhance the immunogenicity of the SARS-CoV-2 spike protein. 【0084】 In addition to heterologous genes, vectors may also include conventional regulatory elements operably ligated to the heterologous genes to enable their transcription, translation, and / or expression in adenovirus-infected cells. As used herein, “operably ligated” includes both an expression regulatory sequence adjacent to the gene of interest and an expression regulatory sequence acting trans or at a distance to control the gene of interest. 【0085】 Expression regulatory sequences may include appropriate transcription start, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that enhance translation efficiency; sequences that enhance protein stability; and, if necessary, sequences that enhance the secretion of encoded products. As used herein, “promoter” is a nucleotide sequence that enables the binding of RNA polymerase and directs the transcription of a gene. Numerous expression regulatory sequences, including promoters that are internal, innate, constitutive, inducible, and / or tissue-specific, are known and available in the art. 【0086】 Adenovirus vectors may be derived from mammalian adenoviruses. In some embodiments of the method of the present invention, the adenovirus vector is derived from a human adenovirus. In some embodiments, the human adenovirus is a human adenovirus of serotype 5. In preferred embodiments, the human adenovirus is not a human adenovirus of serotype 5. 【0087】 In a preferred embodiment, the adenovirus vector is not derived from a human adenovirus. Therefore, the adenovirus vector may be derived from a non-human adenovirus, such as a chimpanzee adenovirus. In a particularly preferred embodiment, the adenovirus vector is derived from a chimpanzee adenovirus, such as ChAdOx1 (Antrobus et al. (2014) Mol.Ther. 22(3):668-674), ChAdOx2 (Morris et al. 2016 Future Virol. 11(9):649-659), ChAd3, or ChAd63. In a particularly preferred embodiment, the adenovirus vector is derived from ChAdOx1. 【0088】 In some embodiments of the method of the present invention, the adenovirus vector is for use in a vaccine and is derived from the same species as the species targeted by the vaccine. For example, in some embodiments, the vaccine targets a disease found in humans, and the adenovirus vector is derived from a human adenovirus. However, in preferred embodiments, the adenovirus vector is for use in a vaccine and is derived from a different species than the species targeted by the vaccine. For example, in some embodiments, the vaccine targets a disease found in humans, and the adenovirus vector is derived from a non-human adenovirus, such as chimpanzee adenovirus. The use of an adenovirus vector derived from a different species than the species targeted by the vaccine is thought to provide an improved vaccine with a lower incidence of existing anti-adenovirus immunity when administered. 【0089】 Adenovirus vectors can be engineered to be unable to replicate after administration to a host. Therefore, in some embodiments of the methods of the present invention, the adenovirus vector is a replication-deficient adenovirus vector (e.g., a replication-deficient adenovirus vector derived from chimpanzee adenovirus). As used herein, “replication-deficient adenovirus vector” means an adenovirus vector that cannot replicate in host cells lacking one or more adenovirus replication genes. In some embodiments, the adenovirus vector lacks the E1A gene. In some embodiments, the adenovirus vector is modified to prevent the host immune system from eliminating cells infected with the adenovirus vector. For example, in some embodiments, the adenovirus vector lacks the E1B gene and / or the E3 gene. In some embodiments, the adenovirus vector lacks the E1B gene. In some embodiments, the adenovirus vector lacks the E3 gene. In some embodiments, the adenovirus vector lacks both the E1B and E3 genes. In some embodiments, the adenovirus vector is a minimal adenovirus vector comprising an origin of replication (ori) and a packaging sequence. In some embodiments, the minimal adenovirus vector further comprises a heterologous gene encoding the protein of interest. 【0090】 cell population In preferred embodiments of the method of the present invention, the cell population is complementary to the adenovirus added to the cell population. As used herein, “cell population complementary to the adenovirus produced” is a cell population that has been engineered to express adenoviral factors not expressed by the adenovirus produced. For example, in some embodiments, the adenovirus added to the cell population does not express adenoviral DNA replication factors, while the cell population expresses adenoviral DNA replication factors. As used herein, “adenoviral DNA replication factors” are factors that naturally form part of the adenoviral DNA and are required for the adenovirus to replicate in a host cell. Therefore, in some embodiments, the adenovirus added to the cell population does not express E1A protein, E1B protein and / or E4 protein, while the cell population expresses E1A protein, E1B protein and / or E4 protein. 【0091】 The cell population may be a primary cell population newly isolated from a tissue. In some embodiments, the tissue is mammalian tissue. 【0092】 Alternatively, the cell population may be derived from a cell line suitable for culture. In some embodiments, the cell line is an immortalized cell line. In some embodiments, the cell line is a mammalian cell line. In some embodiments, the cell population includes mammalian cells. For example, in some embodiments, the cell population includes human fetal kidney (HEK) cells or is an HEK cell line. Mammalian cells may express adenovirus replication factors. For example, in some embodiments, the cell population expresses E1A protein, E1B protein, and / or E4 protein. In some embodiments, the cell population expresses tetracycline repressor protein. In preferred embodiments, the cell population includes T-REx® cells. In some embodiments, the cell population consists of T-REx® cells. In preferred embodiments, the cell population includes Expi293F-inducible cells (Thermofisher) or modified T-REX® cells. 【0093】 Vaccine production Adenoviruses containing heterologous genes can be administered in immunogenic compositions. As used herein, “immunogenic composition” is a composition comprising an adenovirus produced according to the method of the present invention that can induce an immune response, such as a humoral (e.g., antibody) and / or cell-mediated (e.g., cytotoxic T cell) response, to a heterologous gene product delivered by a vector after delivery to a mammal, preferably a human. Accordingly, adenoviruses produced according to the present invention may contain genes encoding a desired immunogen and can therefore be used in vaccines. Adenoviruses can be used as prophylactic or therapeutic vaccines against any pathogen for which antigens have been identified that are important for inducing an immune response and can limit the spread of the pathogen, and for which cDNA is available. 【0094】 Accordingly, in one embodiment, a method for producing a vaccine is provided, comprising producing an adenovirus according to the method of the present invention, purifying the adenovirus, and preparing a vaccine containing the purified adenovirus. Methods for purifying adenoviruses for use in vaccines are well known in the art, for example, as described in Vellinga et al. 2014; Challenges in Manufacturing Adenoviral Vectors for Global Vaccine Product Development; Human Gene Therapy 25:318-327. 【0095】 Such vaccines or other immunogenic compositions can be formulated in a suitable delivery vehicle. The level of immunity to heterologous genes encoded by adenoviruses can be monitored to determine the need for a booster, if present. After evaluation of serum antibody titers, any booster immunization may be desired. 【0096】 In some embodiments, the vaccine comprises an adjuvant. As used herein, “adjuvant” means a composition that enhances the immune response to an immunogen. Examples of adjuvants include inorganic adjuvants (e.g., inorganic metal salts such as aluminum phosphate or aluminum hydroxide), organic adjuvants (e.g., saponins, e.g., QS21, or squalene), oily adjuvants (e.g., Freund’s complete adjuvant and Freund’s incomplete adjuvant), cytokines (e.g., IL-1β, IL-2, IL-7, IL-12, IL-18, GM-CFS, and IFN-γ), and specific adjuvants (e.g., immunostimulatory complexes (ISCOMS), liposomes, or biodegradable microorganisms). Examples include, but are not limited to, crossfairs, virosomals, bacterial adjuvants (e.g., monophosphoryl lipid A, e.g., 3-de-O-acylated monophosphoryl lipid A (3D-MPL), or muramyl peptide), synthetic adjuvants (e.g., nonionic block copolymers, muramyl peptide analogs, or synthetic lipid A), synthetic polynucleotide adjuvants (e.g., polyarginine or polylysine), and immunostimulant oligonucleotides ("CpG") containing unmethylated CpG dinucleotides. 【0097】 In some embodiments, the adjuvant is formulated with a carrier such as a liposome, an oil-in-water emulsion, and / or a metal salt. 【0098】 In a preferred embodiment of the method of the present invention, the vaccine is a COVID-19 vaccine. For example, this disclosure provides the following embodiments. [1] A method for producing adenovirus for use in vaccines: (a) Adding adenovirus to the cell population in culture with a MOI that is insufficient to infect all cells in the cell population; (b) To provide a cell population containing adenovirus-infected cells by culturing the cell population under conditions that allow infection of the cell population with the adenovirus; (c) Culturing the cell population containing adenovirus-infected cells under conditions that allow for the replication of the adenovirus; and (d) A method comprising recovering the adenovirus from the culture. [2] The method according to claim 1, wherein step (a) is to add the adenovirus to the cell population at an MOI of about 0.01 to 1, preferably about 0.025 to 0.4, most preferably about 0.1. [3] The method according to claim 1 or 2, wherein step (a) includes adding the adenovirus to the cell population about 0 to 48 hours after inoculating the cell population into a cell culture medium. [4] The method according to claim 3, wherein step (a) includes adding the adenovirus to the cell population about 24 hours after inoculating the cell population into the cell culture medium. [5] The method according to any one of claims 1 to 4, characterized by a first infection and a second infection, wherein the first infection is induced by providing a first fraction of adenovirus-infected cells and adding the adenovirus to the cell population, and the second infection is induced by providing a second fraction of adenovirus-infected cells and by the adenovirus released into the culture by the first fraction of adenovirus-infected cells. [6] The method according to any one of claims 1 to 5, comprising switching the temperature to which the cell population is exposed from a first temperature to a second temperature, wherein the first and second temperatures allow infection of the cell population by the adenovirus. [7] The method according to item 6, wherein exposing the cell population to the second temperature increases the stability of the adenovirus in the culture. [8] The method according to claim 6 or 7, wherein exposing the cell population to the second temperature reduces the adenovirus particle:infected particle ratio in the culture. [9] The method according to any one of claims 6 to 8, wherein the second temperature allows the proliferation of the cell population.

[10] The method according to any one of claims 6 to 9, wherein the conditions for allowing infection of the cell population with the adenovirus include culturing the cell population at a first temperature, and the conditions for allowing replication of the adenovirus include culturing the cell population at a second temperature.

[11] The method according to any one of claims 6 to 10, comprising switching the temperature to which the cell population is exposed from a first temperature to a second temperature about 48 to 96 hours after the adenovirus has been added to the cell population.

[12] The method according to claim 11, comprising switching the temperature to which the cell population is exposed from a first temperature to a second temperature about 72 hours after the adenovirus has been added to the cell population.

[13] The method according to any one of claims 6 to 12, wherein the first temperature is higher than the second temperature.

[14] The method according to any one of claims 6 to 13, wherein the first temperature is about 31 to 40°C, preferably about 35 to 38°C, and most preferably about 37°C.

[15] The method according to any one of claims 6 to 14, wherein the second temperature is about 27 to 40°C, preferably about 31 to 35°C, and most preferably about 33°C.

[16] The method according to any one of claims 6 to 15, wherein the cell population is cultured at the first temperature for at least about 72 hours.

[17] The method according to item 16, wherein the cell population is cultured at the first temperature for at least about 96 hours.

[18] The method according to any one of claims 6 to 17, wherein the cell population is cultured at the second temperature for at least about 48 hours.

[19] The method according to any one of claims 6 to 18, wherein the cell population is cultured at the second temperature until the adenovirus is recovered from the culture.

[20] The method according to any one of claims 1 to 19, wherein the cell population is cultured in a bioreactor having a capacity of at least about 1 L, preferably at least about 1.2 L, at least about 3 L, about 50 L, about 1000 L, about 2000 L, about 3000 L, or about 5000 L, most preferably at least about 2000 L. [twenty one] The method according to any one of claims 1 to 20, wherein the step of replacing or adding cell culture medium to the cell population after adding the adenovirus to the cell population is not included. [twenty two] The method according to any one of claims 1 to 21, comprising adding a cell culture additive to the cell population. [twenty three] The method according to claim 22, wherein the cell culture additive comprises DMSO, sodium butyrate, and / or CaCl2. [twenty four] The method according to item 22 or 23, wherein the cell culture additive is added to the cell population, and then the cell population is exposed to DMSO, preferably about 0.5% or 1% DMSO. [twenty five] The method according to any one of claims 22 to 24, wherein the cell culture additive is added to the cell population, and then the cell population is exposed to sodium butyrate, preferably about 1 mM sodium butyrate.

[26] The method according to any one of claims 22 to 25, wherein the cell culture additive is added to the cell population, and then the cell population is exposed to CaCl2, preferably about 2 mM CaCl2.

[27] The method according to any one of claims 22 to 26, comprising adding the cell culture additive to the cell population during step (c).

[28] The method according to any one of claims 22 to 27, comprising adding the cell culture additive to the cell population about 72 to 120 hours after adding the adenovirus to the cell population.

[29] The method according to any one of claims 1 to 28, comprising adding a feed to the aforementioned cell population.

[30] The method according to claim 29, comprising adding the feed to the cell population about 24 to 48 hours after adding the adenovirus to the cell population.

[31] The method according to any one of claims 29 to 30, comprising adding the feed to the cell population at least every 48 hours.

[32] The method according to claim 30 or 31, comprising adding the feed to the cell population at a final concentration of about 5% v / v.

[33] The method according to any one of claims 1 to 32, wherein the cell population is complementary to the adenovirus added to the cell population.

[34] The method according to any one of claims 1 to 33, wherein the cell population includes mammalian cells.

[35] The method according to item 34, wherein the mammalian cells express an adenovirus replication factor.

[36] The method according to claim 34 or 35, wherein the cell population includes HEK cells.

[37] The method according to any one of claims 34 to 36, wherein the cell population includes T-REx cells.

[38] The method according to item 34, wherein the cell population consists of T-REx cells.

[39] The method according to any one of items 1 to 38, wherein the adenovirus is a replication-deficient adenovirus.

[40] The method according to any one of items 1 to 39, wherein the adenovirus is a monkey adenovirus.

[41] The method according to item 40, wherein the salis adenovirus is a replication-deficient salis adenovirus.

[42] The method according to claim 41, wherein the replication-deficient sial adenovirus is ChAdOx1, ChAdOx2, ChAdOx3, or ChAd63, preferably ChAdOx1.

[43] The method according to any one of items 1 to 42, wherein the adenovirus is not a human adenovirus.

[44] The method according to any one of items 1 to 43, wherein the adenovirus encodes the nCoV-19 spike protein.

[45] The method according to any one of claims 1 to 44, wherein the step of recovering the adenovirus from the culture is performed approximately 96 to 144 hours after the adenovirus is added to the cell population.

[46] The method according to item 45, wherein the step of recovering the adenovirus from the culture is performed approximately 120 hours after the adenovirus is added to the cell population.

[47] The method according to any one of claims 1 to 46, wherein the step of recovering the adenovirus from the culture comprises lysing the cells of the cell population and recovering the adenovirus from the cell lysate of the cell population.

[48] The method according to any one of claims 1 to 47, wherein the step of recovering the adenovirus from the culture comprises recovering the adenovirus from the cell culture medium in which the cell population was cultured.

[49] The method according to any one of claims 1 to 48, comprising seeding cells into a cell culture vessel prior to step (a).

[50] Before step (a), at least about 0.5 × 10 6 Cells / mL, preferably at least about 0.8 × 10⁶ 6 cells / mL, most preferably at least about 1.2 × 10⁶ 6 The method according to claim 49, comprising seeding cells into a cell culture vessel at an initial cell density of cells / mL to provide the cell population in culture.

[51] At least approximately 1 × 10 6 The method according to any one of claims 1 to 50, comprising adding the adenovirus to a cell population in culture having a viable cell density of cells / mL.

[52] The method according to any one of claims 1 to 51, wherein the peak viral titer is approximately 6 to 8 days after the addition of the adenovirus to the cell population.

[53] The method according to any one of claims 1 to 52, wherein the conditions that allow infection of the cell population by the adenovirus are conditions that allow cell proliferation.

[54] The method according to any one of claims 1 to 53, wherein the conditions that allow replication of the adenovirus are conditions that allow cell proliferation.

[55] The method according to any one of items 1 to 54, wherein the vaccine is a COVID-19 vaccine.

[56] A method for producing an adenovirus for use in a vaccine, comprising culturing a cell population comprising a first fraction of adenovirus-infected cells under conditions that allow infection of a second fraction of the cell population with the adenovirus, wherein the second fraction of the cell population is infected with the adenovirus released into the culture by the first fraction of adenovirus-infected cells.

[57] The method according to claim 56, comprising culturing the cell population, which includes a first fraction of adenovirus-infected cells, under conditions that allow for the infection of the cells of the first fraction of the cell population with the adenovirus, before culturing the cell population under conditions that allow for the infection of the cells of the second fraction of the cell population with the adenovirus.

[58] The method according to claim 57, comprising adding the adenovirus to the cell population in culture before culturing the cell population under conditions that allow infection of the first fraction of cells in the cell population with the adenovirus.

[59] The method according to any one of claims 56 to 58, further comprising culturing the cell population, which includes adenovirus-infected cells, under conditions that allow for the replication of the adenovirus.

[60] The method according to any one of claims 56 to 59, further comprising recovering the adenovirus from the culture.

[61] The method described in any one of paragraphs 56 to 60: (a) Adding adenovirus to a population of cells in culture; (b) Culturing the cell population under conditions that allow infection of a first fraction of cells in the cell population with the adenovirus; (c) Culturing the cell population, including the first fraction of infected cells, under conditions that allow infection of a second fraction of cells in the cell population with the adenovirus, such that the second fraction of cells is infected with the adenovirus released into the culture by the first fraction of infected cells; (d) Culturing the cell population containing adenovirus-infected cells under conditions that allow for the replication of the adenovirus; and (d) A method comprising recovering the adenovirus from the culture.

[62] The method according to any one of claims 56 to 61, wherein the conditions for allowing infection of the first fraction of the cell population are conditions for allowing cell proliferation.

[63] The method according to any one of claims 56 to 62, wherein the conditions for allowing infection of the second fraction of the cell population are conditions for allowing cell proliferation.

[64] The method according to any one of claims 56 to 63, wherein the conditions for allowing infection of the first and second fractions of the cell population are the same.

[65] The method according to any one of claims 56 to 64, wherein the conditions for allowing infection of the first and second fractions of the cell population are different.

[66] The method according to any one of claims 56 to 65, further comprising culturing the cell population, which includes the first and second fractions of the infected cells, under conditions that allow infection of a third fraction of cells in the cell population with the adenovirus, wherein the third fraction of cells is infected with the adenovirus released by the first and / or second fractions of the infected cells.

[67] A method for producing adenovirus for use in vaccines: (a) Adding adenovirus to a population of cells in culture; (b) To provide a cell population containing adenovirus-infected cells by culturing the cell population under conditions that allow infection of the cell population; (c) Culturing the cell population containing adenovirus-infected cells under conditions that allow for the replication of the adenovirus; and (d) A method comprising recovering the adenovirus from the culture approximately 96 to 144 hours after adding the adenovirus to the cell population.

[68] A method for producing adenovirus for use in vaccines: (a) at least 0.5 × 10 6 To provide a cell population during culture by seeding cells into a cell culture vessel at an initial cell density of cells / mL; (b) Adding adenovirus to the cell population in culture; (c) To provide a cell population containing adenovirus-infected cells by culturing the cell population under conditions that allow infection of the cell population; (d) Culturing the cell population containing adenovirus-infected cells under conditions that allow for the replication of the adenovirus; and (e) A method comprising recovering adenovirus from the culture.

[69] A method for producing adenovirus for use in vaccines: (a) at least about 1 × 10 6 Adding adenovirus to a cultured cell population having a viable cell density of cells / mL; (b) To provide a cell population containing adenovirus-infected cells by culturing the cell population under conditions that allow infection of the cell population with the adenovirus; (c) Culturing the cell population containing adenovirus-infected cells under conditions that allow for the replication of the adenovirus; and (d) A method comprising recovering the adenovirus from the culture.

[70] A method for producing adenovirus for use in vaccines: (a) At an MOI insufficient to infect all cells in the cell population, at least about 1 × 10⁻⁶ 6 Adding adenovirus to the cell population in culture having a viable cell density of cells / mL; (b) To provide a cell population containing adenovirus-infected cells by culturing the cell population under conditions that allow infection of the cell population; (c) Culturing the cell population containing adenovirus-infected cells under conditions that allow for the replication of the adenovirus; and (d) Approximately 96 to 144 hours after the adenovirus was added to the cell population, the adenovirus is recovered from the culture. A method comprising switching the temperature to which the cell population is exposed from a first temperature to a second temperature, wherein the first and second temperatures allow the cell population to be infected by the adenovirus.

[71] A method for producing adenovirus for use in vaccines: (a) At an MOI insufficient to infect all cells in the cell population, at least about 1 × 10⁻⁶ 6 Adding adenovirus to the cell population in culture having a viable cell density of cells / mL; (b) Culturing the cell population under conditions that allow infection of a first fraction of cells in the cell population with the adenovirus; (c) Culturing the cell population, including the first fraction of infected cells, under conditions that allow infection of a second fraction of cells in the cell population with the adenovirus, such that the second fraction of cells is infected with the adenovirus released into the culture by the first fraction of infected cells; (d) Culturing the cell population containing adenovirus-infected cells under conditions that allow for the replication of the adenovirus; and (e) Approximately 96 to 144 hours after the adenovirus was added to the cell population, the adenovirus is recovered from the culture. A method comprising switching the temperature to which the cell population is exposed from a first temperature to a second temperature, wherein the first and second temperatures allow the cell population to be infected by the adenovirus.

[72] A method for preparing a vaccine containing adenovirus: (a) Adding adenovirus to the cell population in culture with a MOI that is insufficient to infect all cells in the cell population; (b) To provide a cell population containing adenovirus-infected cells by culturing the cell population under conditions that allow infection of the cell population; (c) Culturing the cell population containing adenovirus-infected cells under conditions that allow for the replication of the adenovirus; (d) Recovering the adenovirus from the culture; (e) Purifying the adenovirus; and (f) A method comprising preparing a vaccine containing the purified adenovirus.

[73] A method for preparing a vaccine containing adenovirus: (a) Culturing a cell population containing a first fraction of adenovirus-infected cells under conditions that allow infection of a second fraction of the cell population with the adenovirus, wherein the second fraction of the cell population is infected with the adenovirus released into the culture by the first fraction of infected cells; (b) Recovering the adenovirus from the culture; (c) Purifying the adenovirus; and (d) A method comprising preparing a vaccine containing the purified adenovirus.

[74] A method for preparing a vaccine containing adenovirus: (a) Adding adenovirus to a population of cells in culture; (b) To provide a cell population containing adenovirus-infected cells by culturing the cell population under conditions that allow infection of the cell population; (c) Culturing the cell population containing adenovirus-infected cells under conditions that allow for the replication of the adenovirus; (d) Approximately 96 to 144 hours after the adenovirus is added to the cell population, the adenovirus is recovered from the culture; (e) Purifying the adenovirus; and (f) A method comprising preparing a vaccine containing the purified adenovirus.

[75] A method for preparing a vaccine containing adenovirus: (a) at least 0.5 × 10 6 To provide a cell population during culture by seeding cells into a cell culture vessel at an initial cell density of cells / mL; (b) Adding adenovirus to the cell population in culture; (c) To provide a cell population containing adenovirus-infected cells by culturing the cell population under conditions that allow infection of the cell population; (d) Culturing the cell population, including adenovirus-infected cells, under conditions that allow for the replication of the adenovirus; (e) Recovering adenovirus from the culture; (f) Purifying the adenovirus; and (g) A method comprising preparing a vaccine comprising the purified adenovirus.

[76] A method for preparing a vaccine containing adenovirus: (a) at least about 1 × 10 6 Adding adenovirus to a cultured cell population having a viable cell density of cells / mL; (b) To provide a cell population containing adenovirus-infected cells by culturing the cell population under conditions that allow infection of the cell population with the adenovirus; (c) Culturing the cell population containing adenovirus-infected cells under conditions that allow for the replication of the adenovirus; (d) Recovering the adenovirus from the culture; (e) Purifying the adenovirus; and (f) A method comprising preparing a vaccine containing the purified adenovirus.

[77] A method for preparing a vaccine containing adenovirus: (a) At an MOI insufficient to infect all cells in the cell population, at least about 1 × 10⁻⁶ 6 Adding adenovirus to the cell population in culture having a viable cell density of cells / mL; (b) To provide a cell population containing adenovirus-infected cells by culturing the cell population under conditions that allow infection of the cell population; (c) Culturing the cell population containing adenovirus-infected cells under conditions that allow for the replication of the adenovirus; (d) Approximately 96 to 144 hours after the adenovirus is added to the cell population, the adenovirus is recovered from the culture; (e) Purifying the adenovirus; and (f) preparing a vaccine containing the purified adenovirus, A method comprising switching the temperature to which the cell population is exposed from a first temperature to a second temperature, wherein the first and second temperatures allow the cell population to be infected by the adenovirus.

[78] A method for preparing a vaccine containing adenovirus: (a) At an MOI insufficient to infect all cells in the cell population, at least about 1 × 10⁻⁶ 6 Adding adenovirus to the cell population in culture having a viable cell density of cells / mL; (b) Culturing the cell population under conditions that allow infection of a first fraction of cells in the cell population with the adenovirus; (c) Culturing the cell population, including the first fraction of infected cells, under conditions that allow infection of a second fraction of cells in the cell population with the adenovirus, such that the second fraction of cells is infected with the adenovirus released into the culture by the first fraction of infected cells; (d) Culturing the cell population containing adenovirus-infected cells under conditions that allow for the replication of the adenovirus; and (e) Approximately 96 to 144 hours after the adenovirus is added to the cell population, the adenovirus is recovered from the culture; (f) Purifying the adenovirus; and (g) preparing a vaccine containing the purified adenovirus, A method comprising switching the temperature to which the cell population is exposed from a first temperature to a second temperature, wherein the first and second temperatures allow the cell population to be infected by the adenovirus.

[79] A method for increasing the yield of adenovirus during adenovirus production, comprising culturing a population of cells in culture at a first temperature in the presence of adenovirus, and switching the temperature to which the cell population is exposed to a second temperature, wherein the first and second temperatures allow infection of the cell population by the adenovirus.

[80] Adenovirus for use in vaccines, which can be obtained by the method described in any one of items 1 to 71.

[81] A vaccine containing an adenovirus that can be obtained by the method described in any one of paragraphs 72 to 79. 【0099】 Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in which this disclosure pertains. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994) and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide many common dictionaries of the terms used herein. 【0100】 This disclosure is not limited to the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of embodiments of this disclosure. 【0101】 Unless otherwise specified, nucleic acid sequences are written from left to right in the 5' to 3' direction, and amino acid sequences are written from left to right in the amino to carboxyl direction. 【0102】 The headings provided herein are not intended to limit the various aspects or embodiments of this disclosure. Furthermore, it will be understood that any embodiment described herein is applicable to any aspect described herein. 【0103】 Concentrations, quantities, volumes, percentages, and other numerical values ​​may be expressed herein in range form. Numerical ranges include the number defining the range. Where a range of values ​​is given, unless otherwise explicitly indicated in the context, each intermediate value between the upper and lower limits of that range, up to one-tenth of the lower limit, is also understood to be specifically disclosed. Each smaller range between any stated value or intermediate value within a stated range and any other stated value or intermediate value within that stated range is included in this disclosure. The upper and lower limits of these smaller ranges may be independently included in or excluded from their ranges, and each range in which either, neither, or both limits are included in the smaller range, according to any specifically excluded limit in the stated range, is also included in this disclosure. Where a stated range includes one or both limits, a range excluding one or both of the included limits is also included in this disclosure. Such range formats are used merely for convenience and brevity, and should be flexibly interpreted to include not only the numerical values ​​explicitly detailed as limits to the range, but also all individual numerical values ​​or subranges contained within that range, as if each numerical value and subrange were explicitly detailed. 【0104】 When used herein and in the appended claims, it should be noted that unless otherwise clearly indicated in the context, the singular forms "a," "an," and "the" refer to multiple objects. For example, a reference to "an agent" includes multiple such agents, and a reference to "the agent" includes one or more agents and their equivalents known to those skilled in the art. 【0105】 "Approximately" may generally mean an acceptable degree of error in the quantity being measured, given the nature or precision of the measurement method. Exemplary degrees of error are within 20 percent (%) of a given value or range of values, typically within 10%, and more typically within 5%. Preferably, the term "approximately" is understood herein to mean ±(±)5%, preferably ±4%, ±3%, ±2%, ±1%, ±0.5%, or ±0.1% of the numerical value of the number used. 【0106】 Embodiments described herein as "including" one or more features may also be deemed to be disclosures of corresponding embodiments that "essentially consist of" or "consist of" such features. 【0107】 The embodiments described above should be understood as illustrative examples. Further embodiments are conceivable. Any feature described in any one embodiment may be used alone or in combination with other features described, and may also be used in combination with one or more features of any other embodiment or any combination of any other embodiments. Furthermore, equivalents and modifications not described above may also be used without departing from the scope of the invention as defined in the appended claims. 【0108】 All references herein, including all data, tables, figures, and original texts presented therein, are incorporated herein in their entirety by reference. [Examples] 【0109】 Example 1 - Adenovirus infection using high and low MOI T-REx(trademark) cells, 0.5 × 10 6 Viable cells / mL were seeded in a 3L bioreactor and subjected to high-MOI or low-MOI adenovirus infection regimes based on those shown in Figure 1 or Figure 2. Briefly, in the high-MOI infection regime, T-REx® cells were approximately 3-5 × 10⁶ cells. 6Grow until the cell density reaches cells / mL, then dilute 1:1 at that point and infect with adenovirus at MOI 10. Approximately 24 hours after infection, when the cell density reaches 1×10 6 viable cells / mL, a commercially available feed for HEK293 cells was given to the infected cells. Separate cultures were harvested 24, 48, and 72 hours after infection to assess the virus titer. 【0110】 In contrast, for the low MOI regime, T-REx™ cells were seeded into a 3 L bioreactor at 0.7×10 6 viable cells / mL and infected with adenovirus at MOI 0.075 approximately 24 hours after seeding. The infected cells were fed on day 4, and separate cultures were harvested 72, 96, 120, 148, and 196 hours after seeding to assess the virus titer. 【0111】 During the process, the viable cell density was determined using Vi-Cell, and the cell viability was measured using Vi-Cell. 【0112】 After collection, qPCR was performed to determine the viral genome titer, and the result was obtained as the number of gene copies per 1 mL of sample. The method of performing qPCR to determine the viral genome titer is well known in the art. Briefly, viral DNA is extracted from viral particles, and the copy number of viral DNA is determined by PCR using primers specific to the transgene. In some cases, infectivity titer was determined using a plaque assay, and the result was obtained showing the number of infectious units per 1 mL of sample. The method of performing a plaque assay to determine infectivity titer is well known in the art. In some cases, viral particle titer was measured using HPLC, and the result was obtained showing the number of viral particles per 1 mL of sample. The method of using HPLC to quantify adenovirus is well known in the art, for example, as described in Blanche et al. 2000; An improved anion-exchange HPLC method for the detection and purification of adenovirus particles; Gene Therapy 7, 1055-1062. 【0113】 As shown in Figure 3A, in cells infected with a high MOI, the viable cell density increased until the day of infection, at which point a significant decrease in peak cell density was observed. The viable cell density was approximately 2–3 × 10⁶ at about 24–48 hours after infection. 6 After recovering to cells / mL, approximately 72 hours after infection, the number of cells increased to 1-2 × 10⁶. 6 The level decreased to cells / mL. 【0114】 Surprisingly, in cultures infected with a low MOI, the viable cell density increased until 5-6 days after seeding, reaching approximately 4-6 × 10⁶ cells. 6 A peak cell density of cells / mL was reached. A slight decrease in peak cell density was observed for these cells on day 7. Interestingly, cells supplied on day 3, or on days 2 and 4, showed a higher peak cell density than cells supplied only on day 4, suggesting that the timing of cell supply may influence peak cell density. 【0115】 As shown in Figure 3B, cell viability was not affected by any of the treatment regimens and remained above 90% for all regimens tested up to 7 days of culture. Surprisingly, Figure 3C shows that cells infected at low MOI had a higher viral titer than cells infected at high MOI. In particular, Figure 3C shows that cells infected at low MOI had a peak viral titer of approximately 3×10 11 GC / mL on day 6 of culture (5 days after infection), whereas cells infected at high MOI had a peak viral titer of approximately 1 - 1.5×10 11 GC / mL on day 6 of culture (2 days after infection). Importantly, the product derived from the low MOI process had equivalent quality to that derived from the high MOI process, as shown in Figure 3D. 【0116】 In summary, as shown in Figure 4A, the use of the low MOI process results in a higher viral titer, characterized by an increase in viral genome concentration, increase in infectious units / mL, and increase in viral particle titer, compared to the use of the high MOI process. As shown in Figure 4B, the use of the low MOI process resulted in a similar viral genome to infectious unit ratio and similar product quality as the high MOI process. 【0117】 In summary, these results show that cells infected at low MOI reach a higher peak density and produce a higher viral titer than cells infected at high MOI. As can be readily appreciated, the use of low MOI infection significantly reduces the required amount of viral seed compared to high MOI infection. Thus, due to the lower starting material required, infection of cells at low MOI represents a much more scalable method for adenovirus production than infection of cells at high MOI. 【0118】 Example 2 - Effect of Infection at Low MOI on Viral Titer Next, the inventors tested the effect of a range of low MOIs on peak viable cell density, cell viability, and viral titer. Briefly, T-REx™ cells were seeded at 0.7×10 6Viable cells were seeded in a 3L bioreactor at a concentration of viable cells / mL and infected with adenovirus at an MOI of 0.026–0.270 approximately 24 hours after seeding. Infected cells were supplied on days 2 and 4, and separate cultures were harvested approximately 5, 6, and 7 days after seeding. As described above, viable cell density and viability were measured daily for each culture. 【0119】 As shown in Figure 5A, cells infected with a lower MOI had a higher peak viable cell density. Specifically, cells infected with an MOI of 0.026 to 0.030 had a peak viable cell density of approximately 7 to 8 × 10⁶. 6 Cells with a peak cell density of cells / mL were found to be approximately 3 × 10⁶ cells, while cells infected with an MOI of 0.232–0.270 had a peak cell density of approximately 3 × 10⁶ cells / mL. 6 It had a peak cell density of cells / mL. 【0120】 As shown in Figure 5B, cell viability tended to decrease with increasing MOI. Therefore, cells infected with an MOI of 0.026–0.030 had a viability of 95% even on day 7 of culture, while cells infected with an MOI of 0.232–0.270 showed a significant decrease in viability from about 4 days after infection, reaching a viability of about 75% on day 7 of culture. 【0121】 Surprisingly, as shown in Figure 5C, the viral titer on day 7 (6 days after infection) was indirectly proportional to the MOI. Therefore, the viral titer on day 7 was highest in cells infected with the lowest MOI tested and lowest in cells infected with the highest MOI tested. A similar pattern was observed for viral titers on day 6 (5 days after infection), with a tendency for higher viral titers to be observed in cells infected with lower MOIs. In contrast, viral titers on day 5 (4 days after infection) were almost equal across all MOIs tested, except for the lowest MOI, which clearly showed the lowest viral titer on day 5. Notably, the highest viral titer was observed on day 6, and the highest viral titer on day 5 was lower than the lowest viral titer observed on either day 6 or 7. 【0122】 Example 3 - Effect of cell seeding density on viral titer The inventors then evaluated viral titers at different initial cell seeding densities infected on either day 0 or day 1. Briefly, T-REx® cells were seeded at 0.5–1.2 × 10⁶ 6 Cells were seeded in ambr 250 containers at a concentration of cells / mL and infected with adenovirus at a target MOI of 0.025 or 0.075 on day 0 or day 1 after seeding. Cells were cultured for up to 7 days after infection, and cell cultures were harvested to assess viral titer. 【0123】 As shown in Figure 6A, the increase in cell density surprisingly increases the viral titer of infected cultures 0 days after cell seeding. Specifically, 0.5 × 10⁻⁶ 6 The cell seeding density of cells / mL is 1 × 10⁻¹⁴ when the culture is infected with an MOI of 0.025. 11 This resulted in viral titers of less than VG / mL, but 1.2 × 10⁻⁶ 6 The cell seeding density of cells / mL is approximately 4.5 × 10⁶ when the culture is infected with the same MOI. 11 This resulted in dramatically high viral titers in VG / mL. Similar effects were observed in cultures infected with an MOI of 0.075. Figure 6B shows similar results for cultures infected on day 1 after cell seeding. 【0124】 Example 4 - Effect of cell dilution on viral titer The inventors then evaluated whether cell dilution during infection affects viral titer. Briefly, T-REx® cells were divided into 0.8 × 10⁶ cells. 6 Cells were seeded at a concentration of 250 cells / mL in ambr containers and infected with adenovirus at a target MOI of 0.025 or 0.075 on day 0 or day 1 post-seeding. Several cultures were diluted at the time of infection. Cells were cultured for up to 5 days post-infection, and cell cultures were harvested to assess viral titer. 【0125】 As shown in Figures 7A and 7B, diluting cells at the time of infection dramatically reduces the viral titer. Specifically, Figure 7A shows that when cells are diluted at the time of infection on day 0 after cell seeding, the viral titer is approximately 1.5 × 10⁻⁶. 11 VG / mL to 5×10 10 This shows a decrease to VG / mL. Similarly, Figure 7B shows that when cells are diluted at the time of infection on day 1 after cell seeding, the viral titer is approximately 2-2.5 × 10⁶. 11 VG / mL to 5×10 10 ~1 × 10 11 This indicates a decrease in VG / mL. 【0126】 Example 5 - Effect of cell culture additives on viral titer The inventors then evaluated whether the cell culture additives DMSO, sodium butyrate, or CaCl2 could alter the viral titer. Briefly, they evaluated whether T-REx® cells could alter the viral titer. 6 Cells were seeded in ambr 250 containers at a viable cell / mL and infected with adenovirus at a target MOI of 0.075 on day 1 after seeding. Cell culture additives were added on day 4 or 5 after seeding, as shown in Figure 10. Cells were cultured for up to 6 days after infection, and the cell cultures were harvested to evaluate viral titer. 【0127】 As shown in Figure 8, the addition of 0.5% DMSO on day 4 or 1% DMSO on day 4 or 5 increased viral titers compared to the control. Similarly, the addition of 1 mM sodium butyrate on day 4 also increased viral titers compared to the control. Finally, the addition of 1 mM CaCl2 on day 4 increased viral titers compared to the control, but the addition of 2 mM CaCl2 on day 4 or 5 did not. 【0128】 Example 6 - Effect of temperature shift on viral titer The inventors then evaluated whether temperature shifts during the infection process could alter peak cell density, cell viability, and viral titer. Therefore, cells were cultured at 37°C until infection, and then the temperature was shifted to 31°C, 33°C, or 35°C approximately 3 hours after the adenovirus was added to the culture. As shown in Figure 9A, the cell density of all samples closely mimicked at least one of the control cultures cultured at 37°C throughout the entire process. 【0129】 Surprisingly, Figure 9B shows that cell viability was higher in cultures subjected to temperature shifts, indicating that larger temperature shifts are associated with increased cell viability. In particular, cultures shifted to 31°C showed approximately 90% cell viability even after 192 hours of incubation, cultures shifted to 33°C showed approximately 65% ​​cell viability, and cultures shifted to 35°C showed approximately 55% cell viability, similar to that observed in cultures not subjected to temperature shifts. 【0130】 Finally, Figure 9C shows that the viral titer on day 2 post-infection was highest in cultures that were not subjected to temperature shifts. However, the viral titer in these cultures decreased dramatically between days 2 and 3 post-infection. In one control culture, the viral titer was approximately 2.5 × 10⁶ on day 2 post-infection. 11 From VG / mL to 1.5 × 10⁻¹⁴ on day 3 after infection 11 The VG / mL level decreased to less than VG. Surprisingly, the viral titer in cultures subjected to temperature shifts increased between 2 and 3 days post-infection. In particular, the viral titer in cultures subjected to a 33°C temperature shift increased to 2 × 10⁶ on day 3 post-infection. 11 The VG / mL ratio increased to over 50%. 【0131】 Example 7 - Scalability To demonstrate that the low MOI process is scalable, the inventors then compared the process in 1000L and 3L bioreactors. As shown in Figures 10A and 10B, the peak cell density and cell viability in the 1000L bioreactor reached acceptable levels by day 5 of culture, similar to those observed in the 3L bioreactor. Surprisingly, as shown in Figure 10C, the viral titer was approximately three times higher in the cultures from the 1000L bioreactor at day 5 compared to those from the 3L bioreactor. 【0132】 In summary, these results indicate that the use of low-MOI infections provides a highly scalable and efficient process for adenovirus production.

Claims

[Claim 1] A method for producing adenovirus for use in vaccines: (a) Adding adenovirus at an MOI of 0.01 to 1 to a cell population in culture having a viable cell density of at least 1 × 10⁶ cells / mL, thereby causing a first infection of a first fraction of the cell population; (b) To provide a cell population containing adenovirus-infected cells by culturing the cell population under conditions that allow infection of the cell population with adenovirus without replacing or adding cell culture medium to the cell population; (c) Culturing the cell population containing adenovirus-infected cells under conditions that allow for the replication of the adenovirus; and (d) recovering the adenovirus from the culture, A method comprising switching the temperature to which the cell population is exposed from a first temperature to a second temperature 48 to 96 hours after adding the adenovirus to the cell population, wherein the first temperature is higher than the second temperature. [Claim 2] The method according to claim 1, wherein step (a) includes adding the adenovirus to the cell population at an MOI of 0.025 to 0.4, preferably MOI of 0.

1. [Claim 3] The method according to claim 1 or 2, wherein step (a) includes adding the adenovirus to the cell population 0 to 48 hours after inoculating the cell population into a cell culture medium. [Claim 4] The method according to claim 3, wherein step (a) includes adding the adenovirus to the cell population 24 hours after inoculating the cell population into the cell culture medium. [Claim 5] The method according to any one of claims 1 to 4, comprising switching the temperature to which the cell population is exposed from the first temperature to the second temperature 72 hours after the adenovirus has been added to the cell population. [Claim 6] The method according to any one of claims 1 to 5, wherein the first temperature is 31 to 40°C, preferably 35 to 38°C, and most preferably 37°C. [Claim 7] The method according to any one of claims 1 to 6, wherein the second temperature is 27 to 40°C, preferably 31 to 35°C, and most preferably 33°C. [Claim 8] The method according to any one of claims 1 to 7, wherein the cell population is cultured at the first temperature for at least 72 hours. [Claim 9] The method according to claim 8, wherein the cell population is cultured at the first temperature for at least 96 hours. [Claim 10] The method according to any one of claims 1 to 9, wherein the cell population is cultured at the second temperature for at least 48 hours. [Claim 11] The method according to any one of claims 1 to 10, wherein the cell population is cultured at the second temperature until the adenovirus is recovered from the culture. [Claim 12] The method according to any one of claims 1 to 11, wherein the cell population is cultured in a bioreactor having a capacity of at least 1 L, preferably at least 1.2 L, at least 3 L, 50 L, 1000 L, 2000 L, 3000 L or 5000 L, most preferably at least 2000 L. [Claim 13] The method according to any one of claims 1 to 12, comprising adding a cell culture additive to the cell population. [Claim 14] The cell culture additive is DMSO, sodium butyrate and / or CaCl ２ The method according to claim 13, including the method described in claim 13. [Claim 15] The method according to claim 13, wherein after adding the cell culture additive to the cell population, the cell population is exposed to DMSO, preferably 0.5% or 1% DMSO. [Claim 16] The method according to any one of claims 13 to 15, wherein the cell culture additive is added to the cell population, and then the cell population is exposed to sodium butyrate, preferably 1 mM sodium butyrate. [Claim 17] After adding the cell culture additive to the cell population, the cell population is treated with CaCl ２ Preferably 2 mM CaCl ２ The method according to any one of claims 13 to 16, wherein exposure to the following is required. [Claim 18] The method according to any one of claims 13 to 17, comprising adding the cell culture additive to the cell population during step (c). [Claim 19] The method according to any one of claims 13 to 18, comprising adding the cell culture additive to the cell population 72 to 120 hours after adding the adenovirus to the cell population. [Claim 20] The method according to any one of claims 1 to 19, comprising adding a feed to the cell population. [Claim 21] The method according to claim 20, comprising adding the feed to the cell population 24 to 48 hours after adding the adenovirus to the cell population. [Claim 22] The method according to any one of claims 20 to 21, comprising adding the feed to the cell population at least every 48 hours. [Claim 23] The method according to claim 21 or claim 22, comprising adding the feed to the cell population at a final concentration of 5% v / v. [Claim 24] The method according to any one of claims 1 to 23, wherein the cell population is complementary to the adenovirus added to the cell population. [Claim 25] The method according to any one of claims 1 to 24, wherein the cell population includes mammalian cells. [Claim 26] The method according to claim 25, wherein the mammalian cells express an adenovirus replication factor. [Claim 27] The method according to claim 25 or claim 26, wherein the cell population includes HEK cells. [Claim 28] The method according to any one of claims 25 to 27, wherein the cell population comprises T-Rex cells. [Claim 29] The method according to claim 25, wherein the cell population consists of T-Rex cells. [Claim 30] The method according to any one of claims 1 to 29, wherein the adenovirus is a replication-deficient adenovirus. [Claim 31] The method according to any one of claims 1 to 30, wherein the adenovirus is monkey adenovirus. [Claim 32] The method according to claim 31, wherein the aforementioned sali adenovirus is a replication-deficient sali adenovirus. [Claim 33] The method according to claim 32, wherein the replication-deficient sial adenovirus is ChAdOx1, ChAdOx2, ChAdOx3, or ChAd63, preferably the replication-deficient sial adenovirus is ChAdOx1. [Claim 34] The method according to any one of claims 1 to 33, wherein the adenovirus is not a human adenovirus. [Claim 35] The method according to any one of claims 1 to 34, wherein the adenovirus encodes the nCov-19 spike protein. [Claim 36] The method according to any one of claims 1 to 35, wherein the step of recovering the adenovirus from the culture is performed 96 to 144 hours after the adenovirus is added to the cell population. [Claim 37] The method according to claim 36, wherein the step of recovering the adenovirus from the culture is performed 120 hours after the adenovirus has been added to the cell population. [Claim 38] The method according to any one of claims 1 to 37, wherein the step of recovering the adenovirus from the culture comprises lysing the cells of the cell population and recovering the adenovirus from the cell lysate of the cell population. [Claim 39] The method according to any one of claims 1 to 38, wherein the step of recovering the adenovirus from the culture comprises recovering the adenovirus from the cell culture medium in which the cell population was cultured. [Claim 40] The method according to any one of claims 1 to 39, comprising seeding cells into a cell culture vessel before step (a). [Claim 41] Before step (a), at least 0.5 × 10 ６ cells / mL, preferably at least 0.8 × 10⁶ ６ Cells / mL, most preferably at least 1.2 × 10⁶ ６ The method according to claim 40, comprising seeding cells into a cell culture vessel at an initial cell density of cells / mL to provide the cell population in culture. [Claim 42] The method according to any one of claims 1 to 41, wherein the peak viral titer is 6 to 8 days after the addition of the adenovirus to the cell population. [Claim 43] The method according to any one of claims 1 to 42, wherein the conditions that allow infection of the cell population by the adenovirus are conditions that allow cell proliferation. [Claim 44] The method according to any one of claims 1 to 43, wherein the conditions that allow the replication of the adenovirus are conditions that allow cell proliferation. [Claim 45] The method according to any one of claims 1 to 44, wherein the vaccine is a COVID-19 vaccine. [Claim 46] A method for producing adenovirus for use in vaccines: (a) at least 1 × 10 ６ Add adenovirus at an MOI of 0.01 to 1 to a cultured cell population having a viable cell density of cells / mL; (b) To provide a cell population containing adenovirus-infected cells by culturing the cell population under conditions that allow infection of the cell population without replacing or adding cell culture medium to the cell population; (c) Culturing the cell population containing adenovirus-infected cells under conditions that allow for the replication of the adenovirus; and (d) The process includes recovering the adenovirus from the culture 96 to 144 hours after adding the adenovirus to the cell population, A method comprising switching the temperature to which the cell population is exposed from a first temperature to a second temperature, wherein the first and second temperatures allow the cell population to be infected by the adenovirus. [Claim 47] A method for producing adenovirus for use in vaccines: (a) at least 1 × 10 ６ Add adenovirus at an MOI of 0.01 to 1 to a cultured cell population having a viable cell density of cells / mL; (b) Culturing the cell population under conditions that allow infection of a first fraction of cells in the cell population with the adenovirus, without replacing or adding cell culture medium to the cell population; (c) Culturing the cell population, including the first fraction of infected cells, under conditions that allow infection of a second fraction of cells in the cell population with the adenovirus, wherein the second fraction of cells is infected with the adenovirus released into the culture by the first fraction of infected cells; (d) culturing the cell population containing adenovirus-infected cells under conditions that allow for the replication of the adenovirus; and (e) The process includes recovering the adenovirus from the culture 96 to 144 hours after adding the adenovirus to the cell population. A method comprising switching the temperature to which the cell population is exposed from a first temperature to a second temperature, wherein the first and second temperatures allow the cell population to be infected by the adenovirus. [Claim 48] A method for preparing a vaccine containing adenovirus, which includes: (a) adding an adenovirus to a cell population in culture having a viable cell density of at least 1×10 ６ cells / mL at an MOI of 0.01 to 1; (b) To provide a cell population containing adenovirus-infected cells by culturing the cell population under conditions that allow infection of the cell population without replacing or adding cell culture medium to the cell population; (c) Culturing the cell population containing adenovirus-infected cells under conditions that allow for the replication of the adenovirus; (d) Recovering the adenovirus from the culture 96 to 144 hours after adding the adenovirus to the cell population; (e) Purifying the adenovirus; and (f) preparing a vaccine containing the purified adenovirus, A method comprising switching the temperature to which the cell population is exposed from a first temperature to a second temperature, wherein the first and second temperatures allow infection of the cell population by the adenovirus, and the first temperature is higher than the second temperature. [Claim 49] A method for preparing a vaccine containing adenovirus, which includes: (a) at least 1 × 10 ６ Adding adenovirus at an MOI of 0.01 to 1 to the aforementioned cell population in culture having a viable cell density of cells / mL; (b) Culturing the cell population under conditions that allow infection of a first fraction of cells in the cell population with the adenovirus, without replacing or adding cell culture medium to the cell population; (c) Culturing the cell population, including the first fraction of infected cells, under conditions that allow infection of a second fraction of cells in the cell population with the adenovirus, wherein the second fraction of cells is infected with the adenovirus released into the culture by the first fraction of infected cells; (d) culturing the cell population containing adenovirus-infected cells under conditions that allow for the replication of the adenovirus; and (e) Recovering the adenovirus from the culture 96 to 144 hours after adding the adenovirus to the cell population; (f) Purifying the adenovirus; and (g) preparing a vaccine containing the purified adenovirus, A method comprising switching the temperature to which the cell population is exposed from a first temperature to a second temperature, wherein the first and second temperatures allow infection of the cell population by the adenovirus, and the first temperature is higher than the second temperature.