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  • A61K40/17—Monocytes; Macrophages
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
  • A61K40/24—Antigen-presenting cells [APC]
• • A—HUMAN NECESSITIES
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  • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
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  • C12N2502/00—Coculture with; Conditioned medium produced by
  • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
  • C12N2502/1352—Mesenchymal stem cells
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  • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
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  • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
  • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
  • C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
  • C12N2506/1353—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from bone marrow mesenchymal stem cells (BM-MSC)
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  • C12N2510/00—Genetically modified cells
  • C12N2510/04—Immortalised cells

Definitions

• the present invention relates generally to macrophages and particularly, although not exclusively, to the establishment of GM-CSF dependent, self-renewing, nontransformed pig macrophages.
• Macrophages are specialised cells involved in the detection, phagocytosis and destruction of bacteria and other harmful organisms. In addition, they can also present antigens to T cells and initiate inflammation by releasing molecules (known as cytokines) that activate other cells.
• cytokines molecules
• Macrophages are crucial in defence against infection and studies with these cells in vitro are key to understanding host-pathogen interactions and to underpin vaccine development.
• tissue resident macrophages including alveolar macrophages (AMs) were believed to be bone-marrow hematopoietic stem cell (HSC) derived cells with a limited life-span.
• AMs alveolar macrophages
• HSC bone-marrow hematopoietic stem cell
• M-CSF macrophage colony-stimulating factor
• AMs granulocyte-macrophage colony-stimulating factor
• AMs have unique characteristics among macrophages including sensitivity and responses to pathogens (2).
• Macrophages are best investigated using primary cells because transformed cell lines may not accurately represent the primary cells. Macrophage pathogenesis studies mostly use cells isolated directly from organs or use ex vivo produced M-CSF derived macrophages from bone marrow progenitors or peripheral blood mononuclear cells (PBMC) (3). GM-CSF derived macrophages from human PBMCs can be used to model human lung AMs (4). Nevertheless, these methods provide cells with a limited life-span, restricted availability, are difficult to genetically manipulate and significant inter-donor variability limits their use. i Mouse models including macrophage systems are often used to study human and veterinary infections because of the relatively easy access to tissues and experiments in vivo. However, there are significant differences regarding macrophage responses and/or intracellular pathogen replication between different mammalian species, which limits the utility of murine studies (5).
• Macrophage studies in pigs are very important (5). Pigs are highly susceptible to various viral (e.g. African swine fever virus [ASFV], porcine reproductive and respiratory syndrome virus [PRRSV] and influenza A virus [IAV]) and bacterial (e.g. Mycobacterium avium, and Salmonella spp.) pathogens that replicate within macrophages and/or profoundly alter macrophage functions (7-9). Some of these pathogens spread from pigs to humans and cause serious diseases in them (e.g. IAV) (9). Furthermore, swine models are used increasingly in biomedical research as pig and human physiology, such as the anatomy and function of the airways and disease susceptibility are much closer to each other than to those of mice (10).
• viruses e.g. African swine fever virus [ASFV], porcine reproductive and respiratory syndrome virus [PRRSV] and influenza A virus [IAV]
• bacterial pathogens e.g. Mycobacterium avium, and Salmonella s
• M-CSF can be used to differentiate pig macrophages from monocytes and detailed analysis of LPS stimulated macrophages showed a close functional relationship between pig and human MCSF derived macrophages (5).
• these MCSF derived macrophages need to be differentiated freshly from bone marrow and similarly to mouse and human cells they have a limited life-span, which makes them unsuitable to produce viral vaccines and suitable, robust primary, non-transformed continuous pig macrophage cell lines are not available for research and vaccine production.
• Such macrophage lines would be required to study important pathogens such as IAV, ASFV and PRRSV.
• Humans and pigs are natural hosts for IAV and the disease can spread in both directions between these species (9). IAV is the leading infectious cause of human death. AMs and innate immune responses play key roles in the pathogenesis of IAV infection (11). Pigs are susceptible to a wide range of IAV strains and are considered to be the mixing vessel of these pathogens as both avian and human IAV strains replicate in pigs and coinfection may lead to new viruses such as the triple (bird- human-pig) reassortant H1 N1 strain responsible for the most recent pandemic in 2009.
• ASFV and PRRSV are pathogens of huge economic importance. These viruses replicate almost exclusively in macrophages, thus, for their efficient study and vaccine development establishment of a continuously growing permissive macrophage system would be necessary (7, 8).
• ASFV causes a devastating, Ebola disease-like haemorrhagic syndrome in pigs. It kills almost all infected animals and in 2018 and 2019 resulted in the death of almost 1 .5 million pigs and 10,000 wild boar.
• the lack of a vaccine limits disease control and research is directed to better understand how the virus interacts with and modulates macrophage functions. This is important, as potential vaccine strains deleted of anti-interferon viral genes elicit a vigorous innate response that restrict viral growth and prevent efficient vaccine production in normal macrophages (7).
• PRRSV is one of the most economically significant swine pathogens, endemic in the majority of pork producing countries, and lung AMs are primary targets of the virus in natural infections (8).
• PRRSV species There are two distinct PRRSV species which are rapidly diversifying and the emerging highly pathogenic strains spread with high speed and cause devastating effects.
• Vaccination is key to control the disease, however, available vaccines are not sufficiently protective and there is a great need to develop safer and more immunogenic vaccines to which a suitable in vitro system is crucially important.
• the present invention relates to the establishment of a GM-CSF derived, continuous, non-transformed pig macrophage system.
• a method for producing continuously replicating, non-transformed porcine macrophages comprising culturing a cell preparation from an organ obtained from a pig in culture medium to which GM-CSF has been added thereby differentiating the cell population into self-renewing, non-transformed macrophages.
• An aspect of the present invention provides a method for producing continuously replicating, non-transformed pig macrophages, comprising culturing a cell preparation from an organ obtained from a pig in culture medium to which GM-CSF has been added thereby differentiating the cell population into self-renewing, non-transformed macrophages.
• the cell preparation may be cultured with feeder cells such as mesenchymal feeder cells.
• Macrophages prepared using the method may be characterised by the expression of pig macrophage markers CD163 and CD172a.
• the cell preparation may be based on pig foetal spleen cells.
• the present invention also provides macrophages obtained or obtainable by the method as described and/or defined herein.
• the present invention also provides macrophages obtainable by the method described herein for use in medicine and/or medical/pharmaceutical research.
• the present invention also provides macrophages obtainable by the method described herein for use in the production of a vaccine.
• the present invention also provides a GM-CSF derived, continuously replicating, nontransformed pig macrophage cell line.
• the macrophages may be characterised by the expression of pig macrophage markers CD163 and CD172a.
• the present invention also provides a continuously replicating, non-transformed pig macrophage system.
• the system may be characterised by the expression of pig macrophage markers CD163 and CD172a.
• Some aspects and embodiments of the present invention are based on the principle or observation that continuously replicating non-transformed macrophages can be obtained from pig haematopoetic organs by using GM-CSF and suitable mesenchymal feeder cells.
• the following data has been obtained related to this system:
• the floating macrophage-like cells were tried to culture separately with GM-CSF. These cells while stayed alive for weeks did not proliferate further. Separated floating cells were also cultured with MCSF or GM-CSF and MCSF. These MCSF cultures showed initial robust cell proliferation; however, after a week of growth cells stop to multiply.
• the floating macrophage-like cells have been transferred to embryonic pig lung fibroblast cultures and similarly to the original cultures with the spleen derived fibroblasts, they attached and proliferated on the top the lung fibroblast feeder cells. They grew in clumps and many of these cells were floating in the medium. These cultures were maintained for more than year with regular media changes.
• Isolated floating cells from feeder containing cultures strongly express the pig macrophage markers CD163 and CD172a.
• Isolated floating macrophages from feeder containing cultures produce a strong cytokine response to bacterial endotoxin (LPS) stimulation.
• LPS bacterial endotoxin
• Isolated floating macrophages from feeder containing cultures can be serially passaged on Mitomycin treated STO fibroblast line feeder cells with pig GM-CSF for at least 4 passages. They grow continuously and have a doubling time of approximately 3-4 days.
• Isolated floating macrophages from the feeder cultures have been transduced with a 2 nd generation lentivirus expressing the SV40 Large T antigen tsA58 temperature sensitive mutant (Jat, P. S. & Sharp, P. A. (1989) Mol. Cell. Biol. 9, 1672-1681.).
• the tsA58 mutant of the SV40 Large T antigen is thermosensitive and can be used to generate conditionally immortalized cell lines (Jat, P. S. & Sharp, P. A. (1989) Mol. Cell. Biol. 9, 1672-1681.)
• the obtained cells (known as PLTA58 cells) grow robustly and without the need of feeder cells.
• PLTA58 cells are continuously growing without feeder cells and we have carried out at least 20 passages since their establishment in December 2021. We can regularly obtain at least 10 million cells from a T75 culture flask in one week of culture. We can freeze down the cells and culture them efficiently again after thawing.
• PLTA58 cells are factor dependent and their growth is enhanced by GMCSF and/or MCSF.
• PLTA58 cells express pig macrophage markers.
• PLTA58 cells can be efficiently stimulated with various innate response elicited ligands such as LPS (TLR4 ligand), Fsl-1 (TLR2 ligand), polyl:C (TLR3 ligand) and R848 (TLR7/8 ligand). 14. PLTA58 cells support the replication of the African Swine Fever virus.
• LPS TLR4 ligand
• Fsl-1 TLR2 ligand
• polyl:C TLR3 ligand
• R848 TLR7/8 ligand
• porcine GMCSF derived cells express markers characteristic for pig macrophages.
• Surface markers were detected on porcine macrophages with FACS using the antibodies for the scavenger receptor CD163 and signal regulatory protein alpha CD172a. Histograms show unstained (blue) and stained cells (red).
• the levels of pig macrophage produced TNFa is similar to those of human macrophages.
• TNF-a response in human GMDMs and pig macrophages is highly dependant on LBP present in Foetal Bovine Serum (FBS).
• FBS Foetal Bovine Serum
• Cells were stimulated with S-LPS (100 ng/ml) and R-LPS (100 ng/ml).
• S-LPS 100 ng/ml
• R-LPS 100 ng/ml
• TNF-a was measured by ELISA. Bars represent the mean from three sample ⁇ S.E.M.
• Pig macrophages produce an early TNF-a response after challenge with lipopolysaccharide.
• Pig cells were stimulated with IAV strain Perth/16/09 at MOI 3 and S-LPS (100 ng/ml).
• S-LPS 100 ng/ml.
• TNF-a cytokine production in pig GM-CSF-derived macrophages stimulated with S-LPS and IAV is time-dependent.
• Fig. 6 shows early TNF-a production in pig macrophages stimulated with S-LPS but not IAV. Additionally, the IAV responses are lower compared to those elicited by endotoxin.
• Pig and MPI macrophages transduced with RFP expressing lentivirus demonstrate that the pig macrophages can be efficiently targeted for recombinant protein expression by lentiviral vectors similarly to mouse MPI macrophages.
• Pig macrophage cells growing on lung fibroblast feeder cells (A) and transferred separated floating macrophages without feeder (B). Pig macrophages grown on the STO fibroblast cell line.
• PLTA58 cells express typical pig macrophage markers.
• PLTA58 cells stimulated with bacterial lipopolysaccharide (LPS)
• PLTA58 cells stimulated with poly l:C, Fsl-1 or R848.

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• 2022
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