Serum-free medium for poultry vaccine production and application thereof

Abstract

The present disclosure relates to a method for primary cell culture. The primary cells are cultured in a serum-free medium supplemented with a peptide derived from a plant or vegetable source and peptone. The method of primary cell culture may be one of the methods of expanding a virus, such as poxvirus.

Description

[0001] This divisional application is a divisional application based on the PCT invention patent application with application number 202080007328.X, which was filed on January 16, 2020 and entered the Chinese national phase, and the invention name is “Serum-free culture medium for poultry vaccine production and its use”.

[0002] Related information

[0003] This application claims priority to U.S. Provisional Patent Application No. 62 / 793,674, filed on January 17, 2019, and incorporates the entirety of the same by reference. Technical Field

[0004] The present disclosure generally relates to a cell culture medium formulation and a method for viral propagation and proliferation of cells in culture, particularly for the production of vaccines. Specifically, the present disclosure provides a serum-free, defined cell culture medium formulation that helps promote the in vitro culture of primary or secondary cell cultures for avian vaccine production, wherein the growth medium comprises a vegetarian diet. In addition, the present disclosure provides a method for viral propagation and proliferation of cells in culture using a serum-free growth medium. The culture medium of the present disclosure is particularly suitable for adherent culture of fibroblasts, such as chicken embryo fibroblasts. Background Art

[0005] Most viral vaccines are produced using cell culture systems. In cell culture-based vaccine production, virus yield is critical not only because higher virus yield enables more vaccine to be produced faster, but also because it is important in determining the economic feasibility of cell culture-based vaccine production methods.

[0006] The cell used for virus / vaccine production can be a cell line or a primary animal. Chicken embryo fibroblasts (CEF cells) are an example of primary cells used for virus production. Attenuated viruses are usually bred in CEF cell cultures. In vitro cell growth and propagation require culture conditions, which include a culture medium that can support cell growth and propagation. In general, the cell culture medium commonly used for culturing cells of mammalian origin comprises a rich salt solution containing vitamins, amino acids, essential trace elements and sugars. In addition, growth hormones, enzymes and bioactive proteins required for cell growth are usually added as supplements in the form of serum derived from animal blood or alternatively protein components isolated from serum derived from animal blood.

[0007] Examples of serum products derived from animal blood are fetal bovine serum, chicken serum, horse serum and porcine serum. These sera are derived from fractionated blood, from which red blood cells and white blood cells have been removed. Primary cells, such as CEF cells, are even more dependent on animal serum sources than cell lines. Therefore, primary cells are usually cultured in a cell culture medium containing 5% to 10% serum, which is fetal calf serum (FCS) in most cases.

[0008] Serum products derived from animal blood may contain exogenous pathogenic factors, such as viruses or prions. These pathogens may be transmitted to the animals / humans to be treated or vaccinated with vaccines or any other drug products produced in cell cultures supplemented with serum, and due to this potential risk, safety concerns about the use of serum have also been raised. Commonly used bovine serum supplements exemplify these concerns, and one of the many major problems / risks associated with their use is the possibility of transmitting factors causing bovine spongiform encephalopathy (bovine spongiform encephalopathy, BSE) to animals / humans in contact with products / vaccines produced in cell cultures using bovine serum supplements. Therefore, it is advantageous to use serum-free culture media to remove contaminants in serum, such as viruses or pathogenic prions, which must not be left in the final products of, for example, recombinant proteins or viral vaccine vectors.

[0009] Considering the possible risks associated with the use of animal serum in cell culture, it is obviously preferred to develop serum-free culture media that do not contain any animal-derived proteins or peptides. Specifically, replacing animal-derived proteins or peptides with proteins, peptides or lipids isolated from non-animal sources will reduce the safety concerns associated with serum.

[0010] Therefore, strategies to improve serum-free vaccine production are still needed. SUMMARY OF THE INVENTION

[0012] The present disclosure provides serum-free culture media that are capable of culturing a wide range of suspension and monolayer cells while being able to support avian vaccine production.

[0013] The present disclosure provides serum-free culture medium, and it comprises various inorganic salts, carbohydrates, amino acid, buffer, vitamin and compound to simulate natural cell environment.If desired, many of amino acid, carbohydrate, salt, vitamin and compound can be obtained from commercial synthetic culture medium without protein or growth promoter, such as EMEM or DMEM.

[0014] The present disclosure provides serum-free culture medium comprising tryptone (peptides and peptones) derived from plant or vegetable sources.

[0015] Additionally, the present disclosure provides methods for culturing cells and cell lines for viral propagation and proliferation using serum-free media. DETAILED DESCRIPTION OF THE INVENTION

[0017] One object of the present disclosure is to develop a serum-free medium that can culture a wide range of suspension and monolayer cells while supporting avian vaccine production.

[0018] The culture medium disclosed herein also includes various inorganic salts, carbohydrates, amino acids, buffers, vitamins and compounds to simulate the natural cell environment. If desired, many of the amino acids, carbohydrates, salts, vitamins and compounds can be obtained from commercially available synthetic culture media that do not contain proteins or growth promoters, such as EMEM or DMEM. In addition to the above-mentioned serum substitutes, various additional amino acids, salts, carbohydrates, vitamins and compounds, such as yeast extract and antibiotics, can also be added to the culture medium to form a complete serum-free culture medium disclosed herein.

[0019] Another object of the present disclosure relates to processes for culturing cells and cell lines for virus propagation and proliferation using serum-free media. Serum-free media include tryptones (peptides and peptones) derived from plant or vegetable sources.

[0020] The present disclosure provides a method for culturing primary cells, particularly primary avian cells, in a serum-free medium, and a method for producing viruses in primary cells under serum-free conditions. The method of the present invention for culturing primary cells may be characterized in that the cells are cultured in a serum-free medium supplemented with peptides and peptones derived from plant or vegetable sources.

[0021] Note that in this disclosure, especially in the claims, terms such as "comprising", "including", "comprising", etc. may mean "include", "include", "including", etc. The terms "consisting essentially of" and "consisting essentially of" allow the presence of elements not explicitly stated but exclude elements that are found in the prior art or that affect the basic or novel characteristics of the present invention.

[0022] Unless explicitly stated, technical terms are used in accordance with convention. Definitions of common terms in molecular biology are found in Genes V, edited by Benjamin Lewin, published by Oxford University Press in 1994 (ISBN 0-19-854287-9), The Encyclopedia of Molecular Biology, edited by Kendrew et al., published by Blackwell Science Ltd in 1994 (ISBN 0-632-02182-9), and Molecular Biology and Biotechnology: a Comprehensive Desk Reference, edited by Robert A. Meyers, published by VCH Publishers in 1995 (ISBN 1-56081-569-8).

[0023] The singular nouns "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. The word "or" refers to any one member of a particular list and includes any combination of members of that list.

[0024] The term "primary cell" used herein is well known to those skilled in the art. Not limited to the following definition, the term "primary cell" may refer to a cell freshly separated from an animal or human tissue, organ or organism, wherein the cell cannot continuously and infinitely replicate and divide. Typically, primary cells divide less than 100 times in cell culture, often less than 50 times, often less than 25 times. Therefore, primary cells do not undergo immortalization events. The example of primary cells is animal fibroblasts, such as but not limited to chicken embryo fibroblasts (CEF cells). The method of separating primary cells is well known. Typically, primary cell cultures are directly derived from tissues, organs or embryos by using protease to process tissues, organs or embryos to obtain single cells.

[0025] The methods of the present disclosure are not limited to cells that form a monolayer. According to an alternative embodiment, the methods of the present disclosure can be used for all other types of primary cells, such as cells that naturally grow in suspension culture (e.g., lymphocytes or other types of blood cells) or cells that naturally grow as adherent cells but have been adapted to grow in suspension culture.

[0026] As shown below, the cells can also be used for serum-free amplification of viruses useful as vaccines.

[0027] In general, viruses used as vaccines, including wild-type viruses, attenuated viruses and recombinant viruses, are grown and amplified under serum-containing conditions. However, as mentioned above, there is a potential risk that serum contains pathogenic factors that may be transmitted to animals / humans treated or vaccinated with vaccines. In order to reduce the risk of contaminants in vaccines, another aspect of the present disclosure is to passage and / or culture those viruses that have been previously proliferated under serum-containing conditions and are planned to be used as vaccines under serum-free conditions.

[0028] Unexpectedly, primary cells that grow naturally as adherent cells can effectively attach to the surface of the cell culture container without forming an unacceptable amount of aggregates, and can grow in the logarithmic phase in the absence of serum, because it is generally believed that primary cells rely on a variety of different factors and components contained in serum. In addition, it is generally believed that when cultivated in serum-free medium, adherent cells form non-viable aggregates that do not adhere to the surface of the cell culture container. Thus, unexpectedly, it is sufficient to supplement the serum-free medium with peptides and peptones derived from plants or vegetable sources to obtain the attachment and growth of adherent primary cells. In addition, it is also unexpected that primary cells cultivated in suspension culture can be cultivated with the medium used in the method according to the present disclosure.

[0029] In particular, it is surprising that primary avian cells, such as chicken embryo fibroblasts (CEF) of the present invention, can be cultured in serum-free medium supplemented with peptides and peptones derived from plant or vegetable sources to adhere to the surface of a cell culture vessel without forming unacceptable amounts of cell aggregates. However, it is understood that avian cells grow poorly in serum-free medium that does not contain growth factors or attachment factors found in serum, i.e., it is unexpected that the poor growth characteristics of primary avian cells can be significantly improved by supplementing serum-free medium with peptides and peptones from plant or vegetable sources.

[0030] The term "cell culture" in a serum-free medium in the context of adherent primary cells refers to the inoculation of cells in a culture vessel in a serum-free medium, the growth of cells in the logarithmic phase until a monolayer is formed and / or the maintenance of cells in a serum-free medium after the formation of a monolayer. The term "cell culture" in a serum-free medium also refers to a method in which all of the above steps are performed in a serum-free medium, so that no animal serum products are present during the entire cell culture process. Therefore, in a more general sense, the term "culturing cells in a serum-free medium" refers to that all of the culture media during the formation of a monolayer are serum-free media. The culture media used in all of the above steps may be supplemented with peptides and peptones derived from plant or vegetable sources.

[0031] The term "cell culture in serum-free medium" in the context of cells grown in suspension culture refers to the seeding of cells in a culture vessel in serum-free medium, the maintenance of cells in serum-free medium after the cells have grown in logarithmic phase and / or have reached a saturation density where replication no longer occurs. The term "cell culture in serum-free medium" refers to a method in which all of the above steps are performed in serum-free medium, such that no animal serum products are present during the entire cell culture. The culture media used in all of the above steps may be supplemented with peptides and peptones derived from plant or vegetable sources.

[0032] The term "serum-free" medium refers to any cell culture medium that does not contain serum from animal or human sources. Suitable cell culture media are well known to those skilled in the art. These media contain salts, vitamins, buffers, carbohydrates, amino acids, and other compounds, such as yeast extract and antibiotics.

[0033] The culture medium used according to the methods of the present disclosure, in particular for adherent cells such as CEF cells, comprises peptides and peptones derived from plant or vegetable sources. Examples of peptides and peptones are vegetable tryptone.

[0034] The serum-free medium may also include one or more additives selected from microbial extracts, plant extracts, and extracts from non-mammalian animals. The microbial extract may be a yeast extract or a yeast powder ultrafiltrate. The plant extract may be a rice extract or a soybean extract. The extract from non-mammalian animals may be a fish extract.

[0035] Serum-free media can also be used with the addition of serum products derived from animal blood (eg, 0.5% to 3%) as a means of reducing the overall serum concentration (5% to 10%) normally required to support the growth of primary adherent cells or cell lines.

[0036] The amplification of the virus may include the following steps: the first step is to culture primary cells according to the above method, that is, to culture primary cells in a serum-free medium supplemented with peptides and peptones derived from plant or vegetable sources. All conditions and definitions described above in the primary cell culture method are also applicable to the definition of the first step of the virus amplification method according to this embodiment of the present disclosure. The second step is to infect primary cells with viruses. The third step is to incubate the infected cells in a serum-free medium until progeny viruses are produced. Finally, in the fourth step, the virus is separated from the infected cells, or the cells carrying the virus are collected to keep these infected cells alive.

[0037] The method for infecting primary cells according to the second step of the method for virus amplification of the present invention is known. For example, the virus can be simply added to the culture medium. Alternatively, the culture medium can be removed and the virus can be added to a fresh culture medium, which is then added to the cells. In order to obtain an effective infection, the amount of the virus / culture medium suspension should be as low as possible to have a high virus concentration. After the virus attaches, additional culture medium can be added.

[0038] In the third step of the method of the present invention, the infected cells are cultured in a serum-free medium until progeny viruses are produced.

[0039] Progeny viruses can be concentrated and purified according to methods well known to those skilled in the art.

[0040] The method disclosed in the present disclosure relates to a method for amplifying poxvirus, which comprises the following steps: (I) culturing primary cells according to the above method, i.e., culturing primary cells in a serum-free medium supplemented with polypeptides and peptones derived from plant or vegetable sources; (II) infecting primary cells with poxvirus; (III) culturing the infected cells in a serum-free medium until progeny viruses are produced; and (IV) isolating viruses from the infected cells. The viruses isolated according to the method of the present invention do not contain any products and / or infectious factors contained in animal serum.

[0041] The process of passaging, culturing and / or purifying viruses that have been previously amplified under serum-containing conditions under serum-free conditions is called "derivation". This derivatization can also be used if it is not entirely clear how to obtain the master seed for vaccine production. Derivatization performed under serum-free conditions greatly reduces the risk of contamination in the vaccine, especially undesirable infectious agents.

[0042] The method according to the present disclosure relates to culturing primary avian cells and amplifying viruses, wherein the viruses are the viruses to be re-derived. For example, the method of the present invention is useful for the re-derivation of viruses and viral stocks, particularly for stocks that have been, or may have been, previously passaged in a serum-containing culture medium.

[0043] A single passage of the starting material (i.e., the virus to be re-derived) under serum-free conditions may be sufficient for the re-derivation of the virus. The term "passage" refers to the step of culturing cells under serum-free conditions, infecting cells with the virus to be re-derived, and obtaining the virus produced in the infected cells. Although a single passage may be sufficient, it may be preferred that the virus be passaged several times under serum-free conditions. In this case, fresh cells are infected again with the virus obtained from the first passage step. The passage carried out under serum-free conditions can be combined with one or more methods of purifying viruses under serum-free conditions. The total number of passages may optionally be 1 to greater than 10, such as 3 to 8 or 4 to 6, by including purification. The technique of purification is a standard virology method mastered by those skilled in the art.

[0044] As previously mentioned, it is well known to those skilled in the art that primary avian cells do not grow well under serum-free conditions. The additional stress associated with poxvirus infection may cause the already stressed cells to die before significant poxvirus expansion occurs. Surprisingly, according to the methods of the present invention, avian cells cultured in serum-free medium supplemented with polypeptides and peptones derived from plant or vegetable sources effectively support poxvirus viral replication and expansion.

[0045] The poxvirus is preferably an orthopox virus. Examples of orthopox viruses are avipoxvirus and vaccinia virus.

[0046] The term "avian pox virus" refers to any avian pox virus, such as Fowlpox virus, Canarypox virus, Uncopox virus, Mynah pox virus, Pigeon pox virus, Psittacine pox virus, Quail pox virus, Peacock pox virus, Penguin pox virus, Sparrow pox virus, Starling pox virus and Turkey pox virus. Preferred avian pox viruses are Canarypox virus and Avian pox virus.

[0047] The term "Marek's disease virus" refers to any of the alphaherpesvirus serotypes used in the production of Marek's disease vaccines, including but not limited to virus serotypes 1, 2, and 3.

[0048] One of the objectives of the present disclosure is to develop a method for the production of a vaccine, in particular a poxvirus vaccine obtained by the above method. According to a preferred embodiment the poxvirus is a fowlpox (FP) virus.

[0049] Another object of the present disclosure is to develop a method for vaccine production using Marek's disease vaccine (serotypes 1, 2 and 3) production in serum free medium.

[0050] Another object of the present disclosure is to develop a composition comprising a virus, in particular a recombinant Marek's serotype 3 virus (HVT) produced according to the method of the present disclosure. Due to the method of virus amplification, the composition does not contain any products and / or infectious agents contained in animal serum. In contrast, virus-containing compositions produced according to traditional methods contain residual compounds derived from animal serum. This is particularly true for compositions containing Marek's virus vaccines produced according to traditional methods, such as HVT virus serotypes.

[0051] Another object of the present disclosure is to develop a method for vaccine production using infectious bursal disease vaccine production in serum free medium.

[0052] Another object of the present disclosure is to develop a method for treating or immunizing an animal (including a human) in need thereof, which comprises administering to the animal or human a virus as defined above or a composition as defined above.

[0053] The present disclosure will be better understood in conjunction with the following embodiments, which are intended to illustrate the present disclosure rather than to limit the scope of the present disclosure. Example

[0054] Example 1: Primary chicken embryo fibroblast (CEF) cell culture

[0055] Cell culture preparation

[0056] Enzyme preparation: Use GIBCO's TrypLE Select Enzyme (1X) without further dilution.

[0057] Growth medium preparation: Prepare a culture medium comprising EMEM medium and VEGITONE (1X) (from SIGMA), wherein the VEGITONE in the EMEM medium is approximately 5% v / v.

[0058] Digestion of embryos: Embryos were washed three times in 50 mL of phosphate buffered saline (PBS) from GIBCO and then digested four times in TrypLE solution, each digestion period lasting five minutes.

[0059] The cells were collected in a beaker containing a 50 / 50 solution of VEGITONE and EMEM and washed with cell preparation medium (EMEM medium containing 5% VEGITONE v / v). The cells were then centrifuged at 750 RPM for twelve minutes and stored at 2-7°C for up to 72 hours.

[0060] The total volume of cells collected was 110 mL, and the cell density was 6.0x10 6 cells / mL.

[0061] Prepare each primary cell culture vessel (6 in total) according to Table 1 below.

[0062] Table 1. Primary cell culture vessel conditions

[0063]

[0064] After 48 hours of incubation at 35-39.0°C and 0.2 RPM, the spinner flasks were 100% confluent.

[0065] Passaging of HVT virus in cell culture

[0066] 1x10 per bottle 8 Primary (1°) CEF cell cultures were inoculated into five spinner flasks at a ratio of 1:1 cells. 1X VEGITONE was used at a ratio of approximately 5% in the culture medium (EMEM) to provide additional nutrients for the CEF cells. After incubation at 39.0°C and 0.2 RPM for 48 hours, the spinner flasks were 100% confluent.

[0067] CEF cells were inoculated with 1 ampoule of HVT SR-3, then incubated at 35-39°C and 0.2RPM, and the cytopathic effect (CPE) was determined to be about 60% after about 46 hours. The first passage (X+1) of these HVT-infected CEF cells was harvested by trypsin digestion, then centrifuged and resuspended in a vial containing 6 different cryopreservation medium formulations. The vials were then frozen to about -110°C using a controlled rate freezer. The resuspended vials were labeled as X+1 (1) for condition 1, X+1 (2) for condition 2, X+1 (3) for condition 3, X+1 (4) for condition 4, X+1 (5) for condition 5, and X+1 (6) for condition 6. Cryopreservation medium and freezing data are shown in Table 2 below.

[0068] Table 2: Cryopreservation medium and freezing data

[0069]

[0070]

[0071] 1 RMBio's irradiated calf serum, 2 1x LB VEGITONE by SIGMA, 3 1% methylcellulose (4000 CP) (from SIGMA) in 1% PBS-solution, and 4Samples were diluted at a ratio of 0.5 mL of formulated vaccine in 49.5 mL of Marek's diluent and then diluted 1:500 for titration and viable cell counts (1:1 Trypan blue).

[0072] Example 2: Primary chicken embryo fibroblast (CEF) cell culture

[0073] Cell culture preparation

[0074] Enzyme preparation: Use GIBCO's TrypLE Select Enzyme (1X) without further dilution.

[0075] Growth medium preparation: Prepare a culture medium comprising RS-EMEM medium and VEGITONE (1X) (from SIGMA), wherein the VEGITONE in the EMEM medium is approximately 5% v / v.

[0076] Digestion of embryos: Embryos were washed three times in 1000 mL of phosphate buffered saline (PBS) from GIBCO and then digested four times in 50 mL of TrypLE solution, each digestion period lasting five minutes.

[0077] The cells were collected in a beaker containing 20 mL of EMEM (containing 20% ​​VEGITONE) and washed with 10 mL of cell preparation medium (EMEM medium containing 5% VEGITONE v / v). The cells were then centrifuged at 750 RPM for twelve minutes and stored at 2-7°C for up to 72 hours.

[0078] Collect cells in a total volume of 200 mL, which is 28x10 6 cells / mL.

[0079] Prepare each primary cell culture vessel (12 in total) according to Table 3 below.

[0080] Table 3. Primary cell culture vessel conditions

[0081]

[0082] Second passage of HVT virus in cell culture

[0083] 4.5x10 per spin bottle 8 Primary (1°) CEF cells were seeded in ten 850 cm 2 The cells were placed in a spinner flask and incubated at 35-39.0°C and 0.15 RPM. 1X VEGITONE was used at a ratio of approximately 5% in the culture medium (EMEM) to provide nutrition to the CEF cells.

[0084] A spinner bottle containing a CEF cell suspension was inoculated with 1 mL of X+1 (2) from Table 2 and incubated at 35-39.0°C and 0.15 RPM. After about 50 hours of inoculation, the cytopathic effect (CPE) was determined to be about 60%. The second passage (X+2) of these HVT-infected cells was collected using 1xTrypLESelect and centrifuged at 750x g for 15 minutes to precipitate the cells. The precipitated cells were resuspended in PBS containing 5% 1X LB VEGITONE and 0.1% methylcellulose. 2.0 mL of this cell suspension was added to the remaining 9 spinner bottles to produce the third passage (X+3) of HVT-infected CEF cells.

[0085] The third passage of HVT virus in cell culture

[0086] Nine spinner bottles containing CEF cell suspensions were inoculated with 2mL of second passage cells (X+2), and incubated at 35-39.0°C and 0.15RPM. After inoculation for about 48 hours, it was determined that the cytopathic effect (CPE) was about 75-80%. Each spinner bottle was collected using 1xTrypLESelect and contained the third passage cells (X+3), and was centrifuged for 15 minutes at 750x g to precipitate cells. Before centrifugation, approximately 20mL of EMEM containing 10% VEGITONE was used and TrypLE Select was used. The precipitated cells were resuspended in two different cryopreservation culture media, and a controlled speed freezer was used to freeze. Cryopreservation culture media and freezing data are shown in Table 4 below.

[0087] Table 4: Cryopreservation medium and freezing data

[0088]

[0089] 1 1x LB VEGITONE by SIGMA, 2 1% methylcellulose (4000 CP) (from SIGMA) in a 1% PBS solution.

[0090] An exemplary medium formulation for CEF cell culture is shown in Table 5, and an exemplary medium formulation for cryopreservation of CEF cells or CEF cells infected with HVT virus is shown in Table 6.

[0091] Table 5: CEF cell culture medium recipe. The amounts required to prepare 1 L of serum-free culture medium are shown.

[0092] Components Volume / L EMEM powder (Sigma M1018) 10-12 g LB Broth,VEGITONE(Sigma 28713) 0.5-2.0 g Sodium bicarbonate 0.35-1.0 g antibiotic solution 0-20mL Sterile DI water Add appropriate amount to make up the total volume to 1000mL

[0093] Table 6: CEF / Virus Cryopreservation Medium. Quantities to prepare 1 L of medium are shown.

[0094] Components Volume / L EMEM powder (Sigma M1018) 10-12 g Sodium bicarbonate 0.35-1.0 g Dimethyl sulfoxide (DMSO) 50mL antibiotic solution 0-20mL Sterile DI water Add appropriate amount to make up the total volume to 1000mL

[0095] HVT virus yields produced using serum-free medium according to Table 5 were comparable to batches produced under similar growth / incubation conditions using conventional growth medium containing approximately 6% calf serum (Table 7).

[0096] Table 7: Recombinant HVT virus strain production data

[0097]

[0098] *Batch efficiency is the total PFU recovered per spinner flask divided by the total number of CEF cells seeded into the spinner flask.

[0099] Example 3: Fowlpox vaccine virus, bursal disease vaccine virus and Marek's disease vaccine virus (serotypes 1, 2 and 3) Growth in serum-free medium

[0100] CEF cells infected / inoculated with various original virus strains according to Table 8 below were cultured in serum-free medium according to Table 5 and placed under similar growth conditions, resulting in observed CPE of at least 50% and virus replication (as shown in Table 8 below).

[0101] Table 8: Vaccine virus and resulting yield data

[0102]

[0103] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description.

Claims

1. A culture medium comprising a basal culture medium and a plant protein source, wherein the basal culture medium does not contain human or animal protein and the plant protein source is tryptone of plant origin.

2. The culture medium of claim 1, wherein the basal culture medium is EMEM.

3. The culture medium of claim 1, wherein the basal culture medium is DMEM.

4. The culture medium of claim 1, wherein the plant-derived tryptone is VEGITONE.

5. The culture medium of claim 1, further comprising an antibiotic.

6. The culture medium of claim 1, further comprising yeast extract.

7. The culture medium of claim 1, further comprising salts.

8. The culture medium of claim 1, further comprising a carbohydrate.

9. The culture medium of claim 1 further comprising additional amino acids.

10. The culture medium of claim 1, further comprising one or more metal ions, vitamins, and cofactors.

11. The culture medium of claim 1, further comprising a serum product derived from animal blood.

12. The culture medium of claim 11, wherein the serum product of animal origin comprises 0% to, but not more than, 7% v / v of the culture medium.

13. The culture medium of claim 1, further comprising at least one buffer selected from the group consisting of CaCl2.2H2O, MgSO4.7H2O, NaH2PO4.2H2O, sodium pyruvate, HEPES and MOPS, present in an amount sufficient to maintain the pH of the culture medium in the range of 6.5-7.

5.

14. A method for virus propagation, the method comprising: a. Cultivating avian embryonic cells in the culture medium of claim 1, b. Establish adherent or non-adherent cell cultures that can proliferate in serum-free basal medium, c. When the cell density reaches at least 1.0x10 6 cells / mL, infecting the avian embryonic cells with a virus selected from poxvirus or poxvirus vector, infectious bursal disease virus, Rispens, SB-1, HVT or any Marek's serotype 1, 2 or 3 virus vector, d. culturing the infected avian embryonic cells in the medium of claim 1 until the CPE reaches at least 50%, and e. Collecting the virus.

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