EP4731748A1

EP4731748A1 - A method of culturing lawsonia intracellularis bacteria

Info

Publication number: EP4731748A1
Application number: EP24735180.2A
Authority: EP
Inventors: Ad DE GROOF; Bartjan Willem SIMMELINK; Jelle STERK
Original assignee: Intervet International BV
Current assignee: Intervet International BV
Priority date: 2023-06-23
Filing date: 2024-06-18
Publication date: 2026-04-29
Also published as: WO2024260953A1; CN121399248A

Abstract

The invention pertains to a method of culturing Lawsonia intracellularis bacteria, the method comprising a) infecting a first portion of McCoy cells with Lawsonia intracellularis bacteria, b) growing the first portion of infected McCoy cells in a first growth medium at a dissolved oxygen concentration less than 18% to arrive at a first culture of McCoy cells infected with Lawsonia intracellularis bacteria, c) determining the concentration of pyroptotic McCoy cells in the first culture of McCoy cells, d) combining a second portion of the first culture of McCoy cells with a second growth medium, and passing a third portion of the first culture to a third growth medium containing fresh McCoy cells, such that the third growth medium is inoculated with 0.01-0.25 pyroptotic McCoy cells per fresh McCoy cell, e) growing the McCoy cells in the second growth medium at a dissolved oxygen concentration less than 18% and growing the McCoy cells in the third growth medium at a dissolved oxygen concentration less than 18% to arrive respectively at a second culture of McCoy cells infected with Lawsonia intracellularis bacteria and a third culture of McCoy cells infected with Lawsonia intracellularis bacteria, f) harvesting the McCoy cells infected with Lawsonia intracellularis bacteria from the second culture of McCoy cells and determining the concentration of pyroptotic McCoy cells in the third culture, and g) performing steps d)-f) for the third culture, wherein the third culture serves as the first culture of McCoy cells.

Description

A METHOD OF CULTURING LAWSONIA INTRACELLULARIS BACTERIA

Field of the invention

The present invention relates to a method of culturing Lawsonia intracellularis.

Background

Lawsonia intracellularis (also called Lawsonia) is the causative agent of proliferative enteropathy (also called enteritis or ileitis) in many animals, in particular pigs, and presents a clinical sign and pathological syndrome with mucosal hyperplasia of immature crypt epithelial cells, primarily in the terminal ileum. Other sites of the intestines that can be affected include the jejunum, caecum and colon. Weanlings and young adult pigs are principally affected with typical clinical manifestation of rapid weight loss and dehydration. Natural clinical disease in pigs occurs worldwide. The disease is consistently associated with the presence of intracellular curved bacteria, presently known as Lawsonia. Disinfectants are used to inactivate Lawsonia bacteria, while antibiotics are used for prophylaxis or treatment of ileitis. Furthermore, nutritional supplements are used to promote a beneficial gut microbiome. In general, however, vaccination against Lawsonia has shown to be an economically efficient measure to treat an infection with Lawsonia. This way ileitis can be controlled, allowing a better exploitation of the genetic growth potential of the pig.

It would therefore be desirable to have an efficient and economically attractive cultivation method for these intracellular bacteria for commercial production of substantive volumes of a vaccine to treat an infection with Lawsonia.

EP 843 818 describes a method to grow Lawsonia bacteria in McCoy cells adhered to a substrate (such as a flask bottom or micro-carriers). Indeed, McCoy cells have proven to be adequate host cells to cultivate Lawsonia bacteria. It is also known from this patent that for passage of a culture to actually grow the Lawsonia bacteria (i.e. to increase the net number of viable bacteria), one needs to incubate fresh uninfected McCoy cells at low oxygen, that is below 18%, whereafter these fresh cells are inoculated with already infected McCoy cells. Passage to fresh McCoy cells is believed to be necessary since the infected McCoy host cells are ultimately killed by the intracellular Lawsonia bacteria. Indeed, it has been described that a Lawsonia infection spreads via host cells that lyse and therewith release the intracellular bacteria such that they become available for infection of other cells.

EP 843 818 also describes to select the volume of infected culture to be used as an inoculum for fresh McCoy cells depending on the concentration of Lawsonia bacteria present in the medium of the culture of infected McCoy cells.

EP application 22197915.6 filed on September 27 2022 in the name of Intervet International BV describes a method of culturing Lawsonia bacteria, wherein McCoy cells are infected with Lawsonia bacteria to arrive at a first culture of McCoy cells infected with Lawsonia intracellularis bacteria. The number of pyroptotic McCoy cells in the first culture is then determined, and fresh McCoy cells are supplemented with pyroptotic McCoy cells in a ratio of 0.05-0.25 pyroptotic McCoy cells per fresh McCoy cell to arrive at a second culture of McCoy cells.

A disadvantage of the method according to EP 843 818 is however that the yield of the cultivation process varies substantially, even if the passaging is consistently based on the same ratio of Lawsonia bacteria and fresh McCoy cells (multiplicity of infection, MOI). This is inconvenient, since a reliable and stable supply yield is required for vaccine production and complying with vaccine demand.

A disadvantage of the method according to EP application 22197915.6 is that a compromise is struck between optimum conditions for antigen production in infected McCoy cells and optimum conditions for infecting a subsequent culture of fresh McCoy cells with Lawsonia bacteria.

Description of the invention

Thus, there is still a need for an improved cultivation technique for Lawsonia intracellularis bacteria.

To this end, a method of culturing Lawsonia intracellularis bacteria is provided, comprising the following steps: a) infecting a first portion of McCoy cells with Lawsonia intracellularis bacteria, b) growing the first portion of infected McCoy cells in a first growth medium at a dissolved oxygen concentration less than 18% to arrive at a first culture of McCoy cells infected with Lawsonia intracellularis bacteria, c) determining the concentration of pyroptotic McCoy cells in the first culture of McCoy cells, d) combining a second portion of the first culture of McCoy cells with a second growth medium, and passing a third portion of the first culture to a third growth medium containing fresh McCoy cells, such that the third growth medium is inoculated with 0.01-0.25 pyroptotic McCoy cells per fresh McCoy cell, e) growing the McCoy cells in the second growth medium at a dissolved oxygen concentration less than 18% and growing the McCoy cells in the third growth medium at a dissolved oxygen concentration less than 18% to arrive respectively at a second culture of McCoy cells infected with Lawsonia intracellularis bacteria and a third culture of McCoy cells infected with Lawsonia intracellularis bacteria, f) harvesting the McCoy cells infected with Lawsonia intracellularis bacteria from the second culture of McCoy cells and determining the concentration of pyroptotic McCoy cells in the third culture.

Steps d)-f) are then performed for the third culture as the first culture of McCoy cells. Thus, when steps d)-f) are performed for the third culture, the third culture is a second “first culture” in step d), of which a second “second portion” is passed to a second “second growth medium” and a second “third portion” is passed to a second “third growth medium”, and in step e) a second “second culture of McCoy cells infected with Lawsonia intracellularis bacteria” and the second “third culture of McCoy cells infected with Lawsonia intracellularis bacteria” are obtained.

Pyroptosis is a form of programmed cell death that is triggered by proinflammatory signals and involves activation of caspase-1. Pyroptosis is induced in McCoy cells infected with Lawsonia intracellularis bacteria.

It was found that when a portion of the first culture of McCoy cells was combined with growth medium, a relatively high and stable production of antigenic mass of Lawsonia intracellularis bacteria could be obtained in the resulting second culture of McCoy cells. Simultaneously, inoculating a growth medium containing fresh McCoy cells with a portion of the first culture in a ratio of pyroptotic McCoy celkfresh McCoy cell of (0.01 :1)-(0.25:1), resulted in an effective infection of the fresh McCoy cells with Lawsonia intracellularis bacteria, resulting in a third culture of McCoy cells infected with Lawsonia intracellularis bacteria. In this way, apart from a wider ratio between pyroptotic cells and fresh McCoy cells (down to 0.01), no compromise has to be made between using a culture of McCoy cells for the production of antigen mass of Lawsonia intracellularis bacteria, which is higher at a relatively high amount of pyroptotic McCoy cells, or for the propagation of Lawsonia intracellularis bacteria, which is more efficient at a relatively low amount of pyroptotic McCoy cells. The first culture of McCoy cells serves as a donor culture for the second or antigen culture and the third or further donor culture.

The second growth medium that is combined with the second portion of the first culture of McCoy cells provides nutrients and allows the McCoy cells and Lawsonia intracellularis bacteria in the second culture of McCoy cells to grow for a longer time. This results in a higher production of antigenic mass of Lawsonia intracellularis bacteria. Furthermore, the complete second culture of McCoy cells can be used for harvesting. Thus, also the harvest yield of the antigenic mass of Lawsonia intracellularis bacteria is increased. This also contributes to a reduction in the production costs of e.g. vaccines requiring the cultivation of Lawsonia intracellularis bacteria.

By inoculating the third growth medium with 0.02-0.25 pyroptotic McCoy cells per fresh McCoy cell, the Lawsonia intracellularis bacteria are effectively propagated in the third culture of McCoy cells. Since the first culture is not used for the harvesting of McCoy cells for antigenic mass of Lawsonia intracellularis bacteria, the third growth medium can be inoculated with a portion of the first culture of McCoy cells at a moment that the concentration of pyroptotic McCoy cells in the first culture of McCoy cells is suitable for passing a second portion of the first culture of McCoy cells to the third growth medium containing fresh McCoy cells, such that the third growth medium is inoculated with 0.02- 0.25 pyroptotic McCoy cells per fresh McCoy cell.

Furthermore, a culture of McCoy cells infected with Lawsonia intracellularis bacteria that is used as donor culture, such as the first culture of McCoy cells and the third culture of McCoy cells, can be grown in a relatively small volume. This saves medium and allows for the use of a relatively small reactor, and contributes to a further reduction in the production costs of e.g. vaccines requiring the cultivation of Lawsonia intracellularis bacteria. The volume of the first and/or third culture of McCoy cells can for example be half or a third of the volume of the second culture of McCoy cells.

It is noted that many media are known that support the growth of McCoy cells as well as Lawsonia bacteria. It is commonly known how to constitute a medium that supports growth of cells or bacteria. For cells, classical culture media were originally developed by Eagle, Ham and others in the 1950’s and 60’s. They found that a medium which fulfils the basic needs for growth should comprise inorganic salts, a nitrogen source (for example in the form of nitrogen containing compounds such as peptides or proteins), a carbon source and vitamins. The media are advantageously buffered to prevent them from becoming either too acidic or too alkaline. Within this basic recipe, many different constitutions are available. For example, one could opt for animal derived components to provide the amino acids, but one could also choose for chemically defined amino acids. For the other compounds numerous variations are possible as well. To cultivate bacteria even less requirements are necessary. In short, to constitute a medium that supports growth of cells or bacteria is relatively simple.

However, optimization of growth and/or metabolite production can take some time, in particular when a medium is preferred that is free of serum or other animal derived components. However, the type of work is routine: strategies for improving medium performance however are commonly known in the art and elaborately described in literature (see for example a review article by Kennedy and Krouse in the Journal of Industrial Microbiology & Biotechnology (1999) 23, 456-475). A medium as simple as the commonly known DMEM may be sufficient to apply the present invention, optionally supplemented with e.g. glucose, one or multiple growth factors or essential amino acids such as L-glutamine or cysteine, and/or antibiotics.

The first, second and third growth medium may have the same composition but may also be of a different composition, for example when the first and/or second and/or third cultures of McCoy cells are to be grown for a different number of days.

The environment is also not very critical, although a dissolved oxygen concentration below 18% (that is below the dissolved oxygen concentration in balance with normal atmospheric oxygen) is necessary to obtain adequate growth of Lawsonia intracellularis bacteria. Preferably the dissolved oxygen concentration is as low as 0, 1 , 2, 2.5, 3, 4, 5,6, 7, 7.5, 8, 9, 10, 11 , 12, 12.5, 13, 14, 15, 16, 17, or 17.5%.

Definitions

McCoy cell: a fibroblast of mouse origin (e.g. ATCC CRL-1696).

Fresh McCoy cells: McCoy cells that are not infected by Lawsonia intracellularis bacteria. To culture: to maintain a population of cells.

To pass: to re-seed cultured cells into fresh medium, typically containing fresh culture cells, in order to support growth of the cells.

To grow: to net increase the amount of viable cells.

To harvest: to collect cells for further processing to arrive at an end product containing the harvested (processed) cells.

Growth medium: medium suitable to maintain viable cells and to support growth of the cells.

Suspension: a state wherein cells are present in a medium without being adhered to a surface.

Pyroptosis: inflammatory form of programmed cell death involving activation of caspase- 1.

Pyroptotic cell: cell in the process of undergoing pyroptosis.

Viable cell: a cell capable of growing and dividing.

Viability: ratio of viable cells over the sum of viable and not-viable cells.

Viable cell density: number of viable cells per unit of volume.

Embodiments of the invention

The optimum ratio of pyroptotic McCoy cells and fresh McCoy cells when passing the third portion of the first culture to the third growth medium containing fresh McCoy cells depends i.a. on the number of days the third culture is to be grown, for example whether the third culture is to be used as a donor culture and passing a portion of the third culture to fresh medium containing fresh McCoy cells on a predetermined day. The ratio of pyroptotic McCoy cells to fresh McCoy cells may for example be 0.02:1 , 0.025:1 , 0.03:1 , 0.04:1 , 0.05:1 , 0.06:1 , 0.07:1 , 0.075:1 , 0.08:1 , 0.09:1 , 0.1 :1 , 0.12:1 , 0.15:1 , 0.2:1 , 0.225:1 or 0.25:1 , in order to adapt to a specific combination of number of fresh McCoy cells, volume of the fresh medium and number of days the third culture is planned to be grown. Those variabilities fall in the routine ambit of any person skilled in the art of cell culture.

Depending on the concentration of pyroptotic McCoy cells in the first culture, the concentration of Lawsonia intracellularis bacteria in the first culture, and the number of days the second culture is to be grown, the second portion of the first culture that is combined with the second growth medium may be any amount ranging from 1%-99% of the volume of the first culture, such as 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%.

Depending on the concentration of pyroptotic McCoy cells in the first culture, the concentration of fresh McCoy cells in the third growth medium, and the number of days the third culture is to be grown, the third portion of the first culture that is passed to the third growth medium may be any amount ranging from 1%-99% of the volume of the first culture, such as 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%.

In an embodiment of the invention, the second growth medium is free of fresh McCoy cells. In this way, most of the nutrients provided by the second growth medium is used for the production of antigenic mass of Lawsonia intracellularis bacteria. This results in a higher yield of antigenic mass of Lawsonia intracellularis bacteria in the second culture of McCoy cells.

In an embodiment, the McCoy cells of the first culture of McCoy cells and/or the second culture of McCoy cells and/or the third culture of McCoy cells are grown in suspension. By maintaining McCoy cells in a suspended state during incubation, maximum growth of the McCoy cells, and thus Lawsonia intracellularis bacteria, is achieved by increasing each individual cell’s exposure to growth media and the proper mixture of oxygen and carbon dioxide. Furthermore, suspension cultivation allows for a continuous cultivation of McCoy cells, as opposed to cultivation in batches.

The cells can be agitated and maintained in suspension by a variety of methods known in the art, including, for example, culture flasks, roller bottles, membrane cultures and spinner flasks. The cells may be kept in suspension during incubation by incubating the cells in a reactor employing a paddle, propeller or other means to agitate the culture and keep the cells contained therein in suspension. Using suspension cultures greatly facilitates keeping the cells actively growing and permits continuous culture expansion and scale-up.

In an embodiment, the first culture is grown for a period of 2, 3, 4, 5, 6, or 7 days. In this way, sufficient time is allowed for the McCoy cells and Lawsonia intracellularis bacteria to grow before combining the second portion of the first culture of McCoy cells with the second growth medium and combining the third portion of the first culture of McCoy cells with the third growth medium.

In an embodiment, the second culture is grown for a period of 2, 3, 4, 5, 6, or 7 days. In this way, sufficient time is allowed for the McCoy cells and Lawsonia intracellularis bacteria to grow before harvesting.

In an embodiment, the third culture is grown for a period of 2, 3, 4, 5, 6 or 7 days. In this way, sufficient time is allowed for the McCoy cells and Lawsonia intracellularis bacteria to grow before combining a portion of the third culture with a second “second growth medium” and a second “third growth medium”.

Preferably, the first culture is grown for a period of 2, 3, 4 or 5 days, the second culture is grown for a period of 4, 5, 6 or 7 days, and the third culture is grown for a period of 2, 3, 4 or 5 days. More preferably, the first culture is grown for a period of 3 or 4 days, the second culture is grown for a period of 5, 6 or 7 days, and the third culture is grown for 3 or 4 days.

In an embodiment, step g) is repeated 2-40 times, such as 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13 ,14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, or 39 times.

Because for vaccine production large quantities of Lawsonia bacteria are required, one repetition of step g) is generally not sufficient to provide enough Lawsonia intracellularis bacteria to fulfill the demand for vaccine production. Thus, by performing multiple repetitions, the culturing of Lawsonia intracellularis bacteria can be performed as a substantially continuous process and a higher yield can be obtained.

Furthermore, the first culture of McCoy cells is generally established from a frozen stock or seed of McCoy cells. Reviving these cells and scaling up to a volume suitable for growing McCoy cells and Lawsonia bacteria for vaccine production requires time and resources. Thus, by repeating step g), a continuous process of growing Lawsonia intracellularis bacteria, harvesting Lawsonia intracellularis bacteria and inoculating a culture with pyroptotic McCoy cells can be performed from a single initial starting culture.

The duration of each repetition of step g) may vary and is selected individually. Preferably, the duration of each repetition of step g) is individually chosen such that steps d)-f) are repeated in a rhythm in days of 5-5-4, 5-4-5, 4-5-5, 4-3, or 3-4. In this way, the McCoy cells and Lawsonia intracellularis bacteria can be grown in a continuous fashion while for example avoiding having to pass and/or harvest in weekends. For example, a rhythm of 5-5-4 means that the first “second culture” is grown for five days, the second “second culture” is grown for five days and the third “second culture” is grown for four days. This rhythm may then be repeated. It may also be followed by a different rhythm.

Preferably, the first portion of the first culture is in each repetition of step g) chosen such that the second culture of McCoy cells reaches a concentration of pyroptotic cells of 50- 90% on a predetermined day. In this way, the second culture of each repetition is suitable for passing pyroptotic cells to fresh McCoy cells for a subsequent repetition of passing and growing the McCoy cells.

In an embodiment, the concentration of fresh McCoy in the third growth medium is 1x10⁵- 1x10⁷ cells/mL. Thus, sufficient cells are provided to develop into the third culture of McCoy cells infected with Lawsonia intracellularis bacteria. Preferably, the fresh McCoy cells are provided in a volume of 50-2500 L of fresh medium, such as 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1250, 1300, 1400, 1500, 1600, 1700, 1750, 1800, 1900, 2000, 2100, 2200, 2300, or 2400 L. 5x10⁵- 5x10⁶ cells/mL, 6x10⁵-2.5x10⁶ or 7.5x10⁵-1x10⁶ fresh McCoy cells are for example provided in a volume of 2000 L of third growth medium.

1. Measuring the yield of the cultivation process

1. 1. Method

The yield of the cultivation process is determined using an enzyme-linked immunosorbent assay (ELISA), measuring antigenic mass being a polysaccharide associated with the outer cell membrane of Lawsonia intracellularis, as described in EP2268308. The wells of a polystyrene microtitre plate are coated with a monoclonal antibody (MoAb) against Lawsonia intracellularis. Serial three-fold dilutions of samples are incubated in the plates. Subsequently, the bound antigens are quantified using a second MoAb against Lawsonia intracellularis labelled with horse radish peroxidase and using 3, 3’, 5,5’- tetramethylbenzidine as substrate. The calibration line is drawn from the values of a reference standard with known antigenic mass which is included in each test. The antigenic mass of test samples in Units (U)/mL is determined against the antigenic mass of the reference standard.

1.2. Results

Fig. 1A shows the average yield of antigenic mass from a culture of McCoy cells infected with Lawsonia intracellularis bacteria according to a method of culturing Lawsonia intracellularis bacteria from the prior art, where the growth medium was inoculated with 125 Lawsonia intracellularis bacteria per fresh McCoy cell.

McCoy cells were grown in a 2000 L reactor. The concentration of Lawsonia intracellularis bacteria present in the medium of a first culture of McCoy cells infected with Lawsonia intracellularis bacteria was determined after growing the first culture of McCoy cells for 4 or 5 days by means of dark field microscopy, and a portion of the first culture to be passed to a second growth medium containing 6x10⁵ fresh McCoy cells was chosen such that the second growth medium was inoculated with a multiplicity of infection (MOI) of 125. In other words, the second growth medium was inoculated with 125 Lawsonia intracellularis bacteria per fresh McCoy cell. The McCoy cells infected with Lawsonia intracellularis bacteria in the second growth medium were grown to arrive at a second culture of McCoy cells infected with Lawsonia intracellularis bacteria. The yield of the antigenic mass of Lawsonia intracellularis bacteria in the second culture of McCoy cells infected with Lawsonia intracellularis bacteria was determined by ELISA after 5 days of growing.

Fig. 1A shows the variability in yield of antigenic mass between repeats of this method.

Fig. 1 B shows the average yield of antigenic mass from a culture of McCoy cells infected with Lawsonia intracellularis bacteria according to the prior art, where the growth medium was inoculated with 0.1 pyroptotic McCoy cell per fresh McCoy cell.

McCoy cells were grown in a 2000 L reactor. The concentration pyroptotic McCoy cells in a first culture of McCoy cells infected with Lawsonia intracellularis bacteria was determined after growing the first culture of McCoy cells for 4 or 5 days, and a portion of the first culture to be passed to a second growth medium containing 6x10⁵ fresh McCoy cells was chosen such that the second growth medium was inoculated with 0.1 pyroptotic McCoy cell per fresh McCoy cell. The McCoy cells infected with Lawsonia intracellularis bacteria in the second growth medium were grown to arrive at a second culture of McCoy cells infected with Lawsonia intracellularis bacteria. The yield of the antigenic mass of Lawsonia intracellularis bacteria in the second culture of McCoy cells infected with Lawsonia intracellularis bacteria was determined by ELISA after 5 days of growing.

Fig. 1 B shows a smaller variability in antigenic mass between repeats and a higher average antigenic mass yield compared to Fig. 1A.

Fig. 2 and 3 show the yield of antigenic mass from an antigen culture of McCoy cells infected with Lawsonia intracellularis bacteria according to a method of culturing Lawsonia intracellularis bacteria according to the invention.

McCoy cells were grown in a 2 L reactor. The concentration pyroptotic McCoy cells in a first culture of McCoy cells infected with Lawsonia intracellularis bacteria was determined after growing the first culture of McCoy cells for 4 days. A second growth medium was in duplo combined with a second portion of 667 mL of a first culture of McCoy cells infected with Lawsonia intracellularis bacteria was combined with 1333 mL of a second growth medium free of fresh McCoy cells, and a third portion of the first culture to be passed to a third growth medium containing 6x10⁵ fresh McCoy cells was chosen such that the third growth medium was inoculated with 0.1 pyroptotic McCoy cell per fresh McCoy cell. The McCoy cells infected with Lawsonia intracellularis bacteria in the second growth medium and the third growth medium were grown to arrive respectively at a second and third culture of McCoy cells infected with Lawsonia intracellularis bacteria. The yield of the antigenic mass of Lawsonia intracellularis bacteria in the second culture of McCoy cells infected with Lawsonia intracellularis bacteria was determined by ELISA after 4 and 7 days of growing. The concentration pyroptotic McCoy cells in the third culture of McCoy cells infected with Lawsonia intracellularis bacteria was determined and the third culture of McCoy cells infected with Lawsonia intracellularis bacteria was used as a second “first culture”.

Fig. 2 shows the yield of antigenic mass from duplicate I and duplicate II of a second culture of McCoy cells infected with Lawsonia intracellularis bacteria grown for 5 days. The yield of antigenic mass of each duplicate is higher than the yield obtained according to the methods of culturing Lawsonia intracellularis bacteria from the prior art as shown in Fig. 1A and 1 B.

Fig. 3 shows the average yield of antigenic mass from duplicates of a second culture of McCoy cells infected with Lawsonia intracellularis bacteria grown for 5 days (Fig. 3A) or 7 days (Fig. 3B). The average yield of antigenic mass after 5 days of growing the second culture of McCoy cells infected with Lawsonia intracellularis bacteria as shown in Fig. 3A was higher than the average yield of antigenic mass obtained according to the methods of culturing Lawsonia intracellularis bacteria from the prior art as shown in Fig. 1A and 1 B. Furthermore, the average yield of antigenic mass after 7 days of growing the second culture of McCoy cells infected with Lawsonia intracellularis bacteria (Fig 3B) was higher compared to the average yield of antigenic mass after 7 days of growing the second culture of McCoy cells infected with Lawsonia intracellularis bacteria (Fig. 3A).

2. Determining a third portion of a culture of McCoy cells infected with Lawsonia bacteria based on caspase- 1 activation

2. 1. Caspase- 1 activation assay

Pyroptosis is triggered by caspase-1 activation. Pyroptotic cells are therefore characterized by active caspase-1. In order to measure caspase-1 activity and determine the concentration of pyroptotic McCoy cells in a culture, the FAM-FLICA™ (Bio-Rad, Hercules, USA) was used. Fluorescent labeled inhibitors of caspase-1 (FLICA) easily diffuse into the cell and bind covalently to active caspase-1.

According to the manufacturer’s manual, 290 pL of 5x10⁵ cells/mL were plated in a 12- well plate. 10 pL 30x reconstituted FLICA solution was added to the wells and incubated in the dark for 30 minutes at 37 °C, 5% CO2 at 150 rpm. After incubation, cells were washed using 2 mL 1x apoptosis wash buffer, followed by centrifugation at 200 g for 5 minutes at room temperature. Cells were washed a second time with 1 mL 2x apoptosis wash buffer, followed by centrifugation at 200 g for 5 minutes at room temperature. The cell pellet was fixed using the fixative provided with the kit, and the cells were analyzed by flow cytometry using BD Facsverse™ (BD Biosciences, USA) and a 527/32 filter. 10,000 cells were acquired for each sample.

2.2. Selecting the third portion of the culture of McCoy cells infected with Lawsonia bacteria based on caspase- 1 activation The exact ratio of pyroptotic McCoy cells per fresh McCoy cells is determined depending on the volume of the vessel containing the fresh McCoy cells and the number of days the McCoy cells are to be grown in the third growth medium. The required volume of the third portion of the culture of McCoy cells infected with Lawsonia intracellularis bacteria is then calculated based on the concentration of pyroptotic McCoy cells in the culture of McCoy cells infected with Lawsonia intracellularis bacteria and the exact ratio of pyroptotic McCoy cells per fresh McCoy cell with which the third growth medium is to be inoculated. In this way, the method can be adapted to culturing at different scales and to a desired length of a growth period, for example in order to avoid work on weekend days or to avoid depletion of medium.

3. Determining the third portion of the culture of McCoy cells infected with Lawsonia bacteria based on McCoy cell viability

3. 1. Measuring cell viability

It was experimentally found that there was a correlation between the concentration of not- pyroptotic cells and the viability of the McCoy cells in the first culture, as shown in Fig. 4 for the strain of McCoy cells used.

Viability can be determined by directly measuring number of viable cells and the total number of cells. Cell viability can be measured e.g. using an assay measuring the protease activity of cells. CellTiter-Fluor™ Cell Viability Assay (Promega) makes use of a cell permeable fluorogenic protease substrate (GF-AFC) which is cleaved by proteases in live cells, resulting in a fluorescent signal proportional to the number of viable cells.

Viability can also be determined by measuring the number of not-viable cells and the total number of cells. In this case, a measure of viable cells can then be derived by subtracting the number of not-viable cells from the total number of cells. Not-viable cells can for example be stained using 4',6-diamidino-2-phenylindole (DAPI) or propidium iodide (PI), and acridine orange can be used to stain the total of cells present in a sample. Commercially available systems, such as a Nucleocounter or ViCELL analyser can then be used to establish the viability of the first culture of infected McCoy cells. The fraction of not-viable cells consists of cells still in the process of dying, and cells that have already died. Thus, the number of viable cells (and the viability) may even be obtained by determining the total number of cells, the dead cells and the cells undergoing cell death. In this case, the number of viable cells is obtained by subtracting the number of dead cells and the number of cells undergoing cell death from the total number of cells.

3.2. Selecting the portion of the culture of McCoy cells infected with Lawsonia bacteria based on McCoy cell viability

The concentration of not-pyroptotic cells can be determined quickly and conveniently by measuring the viability of the McCoy cells in a culture of McCoy cells infected with Lawsonia intracellularis bacteria and deriving the number of not-pyroptotic cells present in the that culture based on the total number of cells, the number of (not-)viable cells in that culture, and the correlation shown in Fig. 4. The number of pyroptotic cells is then calculated by subtracting the number of not-pyroptotic cells from the total number of cells.

The exact ratio of pyroptotic McCoy cells per fresh McCoy cells is determined depending on the volume of the vessel containing the fresh McCoy cells and the number of days the McCoy cells are to be grown in the third growth medium. The required volume of the portion of the culture of McCoy cells infected with Lawsonia bacteria is then calculated based on the viability of the McCoy cells in the culture of McCoy cells infected with Lawsonia intracellularis bacteria and the exact ratio of pyroptotic McCoy cells per fresh McCoy cell with which the third growth medium is to be inoculated. In this way, the method can again be adapted to culturing at different scales and to a desired length of a growth period, for example in order to avoid work on weekend days or to avoid depletion of medium.

4. Long-term experience with the donor-antigen culturing approach

The culturing method for the production of L. intracellularis antigen on McCoy cells, according to the invention, has now been applied at large scale for several more manufacturing runs, and its unexpected advantages have been consistently obtained.

Figure 5 presents a graphical representation, similar as in Figure 1 panels A and B, of L. intracellularis antigenic mass yields produced in 2000 L scale bioreactors, using the donor-antigen culturing approach of the present invention. When compared to prior art procedures (panel A), cultures according to the invention (panels B and C) consistently produce more antigen, with higher average values, and with substantially increased consistency and reduced variability.

Claims

1. A method of culturing Lawsonia intracellularis bacteria, the method comprising: a) infecting a first portion of McCoy cells with Lawsonia intracellularis bacteria, b) growing the first portion of infected McCoy cells in a first growth medium at a dissolved oxygen concentration less than 18% to arrive at a first culture of McCoy cells infected with Lawsonia intracellularis bacteria, c) determining the concentration of pyroptotic McCoy cells in the first culture of McCoy cells, d) combining a second portion of the first culture of McCoy cells with a second growth medium, and passing a third portion of the first culture to a third growth medium containing fresh McCoy cells, such that the third growth medium is inoculated with 0.01-0.25 pyroptotic McCoy cells per fresh McCoy cell, e) growing the McCoy cells in the second growth medium at a dissolved oxygen concentration less than 18% and growing the McCoy cells in the third growth medium at a dissolved oxygen concentration less than 18% to arrive respectively at a second culture of McCoy cells infected with Lawsonia intracellularis bacteria and a third culture of McCoy cells infected with Lawsonia intracellularis bacteria, f) harvesting the McCoy cells infected with Lawsonia intracellularis bacteria from the second culture of McCoy cells and determining the concentration of pyroptotic McCoy cells in the third culture, g) performing steps d)-f) for the third culture, wherein the third culture serves as the first culture of McCoy cells.

2. The method according to claim 1 , wherein the second growth medium is free of fresh McCoy cells.

3. The method according to claim 1 or 2, wherein in step d) the third portion of the first culture is passed to the third growth medium such that the third growth medium is inoculated with 0.05-0.15 pyroptotic McCoy cells, preferably with 0.07- 0.12 pyroptotic McCoy cells.

4. The method according to any of the previous claims, wherein the McCoy cells of the first culture of McCoy cells and/or the McCoy cells of the second culture of McCoy cells and/or the McCoy cells of the third culture of McCoy cells are grown in suspension.

5. The method according to any of the previous claims, wherein the first culture and/or the second culture and/or the third culture of McCoy cells is grown for a period of 2-7 days.

6. The method according to claim 5, wherein the first culture is grown for a period of 3-4 days, the second culture is grown for 5-7 days, and the third culture is grown for 3-4 days.

7. The method according to any of the previous claims, wherein the first and/or second and/or third culture of McCoy cells is grown at a dissolved oxygen concentration of 0-5%.

8. The method according to any of the previous claims, wherein step g) is performed 2-40 times.

9. The method according claim 8, wherein step g) performed three consecutive times, and the second culture is two times out of said three times grown for 5 days and one time out of said three times grown for 4 days.

10. The method according to claim 8, wherein step g) is performed two consecutive times, and the second culture is one time out of said two times grown for 3 days and one time out of said two times grown for 4 days.

11. The method according to any of the previous claims, wherein the viability of the McCoy cells contained in the third portion of the first culture of McCoy cells in step d) is 80-98%.