Porcine cell line for virus production

Abstract

The present invention relates to a continuous porcine cell line that is capable of proliferating in medium free of animal-derived components. Further, the present invention relates to a method for producing a virus using said cell line and a virus obtainable by said method. Furthermore, the present invention relates to a method for accumulating a virus from an environmental sample using said cell line and a virus obtainable by said method.

Claims

1. A continuous and non-adherent kidney or testis porcine cell line that is capable of proliferating in medium free of animal-derived components, wherein the cell line comprises an endogenous protease for activation of virus infectivity, wherein the cell line is infected with a virus or transfected with a plasmid carrying (a) nucleic acid sequence(s) encoding a virus, and wherein the virus carries a protease cleavage site cleavable by said endogenous protease, and wherein the virus is a virus of the Orthomyxoviridae or Paramyxoviridae family.

2. The continuous porcine cell line of claim 1, wherein the cell line is porcine kidney-15S (PK-15S).

3. The continuous porcine cell line of claim 2, wherein the cell line is deposited at the DSMZ with the deposit number DSM ACC3307.

4. The continuous porcine cell line of claim 1, wherein the cell line is swine testis S (STS).

5. The continuous porcine cell line of claim 4, wherein the cell line is deposited at the DSMZ with the deposit number DSM ACC3308.

6. The continuous porcine cell line of claim 1, wherein the virus is an influenza virus or Newcastle disease virus.

7. A method for producing a virus comprising the steps of: providing a continuous and non-adherent porcine kidney or testis cell line that is capable of proliferating in medium free of animal-derived components and that is permissive for the virus, wherein the cell line comprises an endogenous protease for activation of virus infectivity, (ii) infecting said cell line with the virus or transfecting said cell line with a plasmid carrying (a) nucleic acid sequence(s) encoding the virus, and (iii) culturing the cell line infected or transfected in step (ii), thereby producing the virus, wherein the virus carries a protease cleavage site cleavable by said endogenous protease, and wherein the virus is a virus of the Orthomyxoviridae or Paramyxoviridae family.

8. The method of claim 7, which does not require the addition of an exogenous protease for activation of virus infectivity.

9. The method of claim 7, wherein the cell line is PK-15S.

10. The method of claim 9, wherein the cell line is deposited at the DSMZ with the deposit number DSM ACC3307.

11. The method of claim 7, wherein the cell line is STS.

12. The method of claim 11, wherein the cell line is deposited at the DSMZ with the deposit number DSM ACC3308.

13. The method of claim 7, wherein the virus is an influenza virus or Newcastle disease virus.

14. A continuous and non-adherent porcine cell line that is capable of proliferating in medium free of animal-derived components, wherein the cell line is deposited at the DSMZ with the deposit number DSM ACC3307.

15. A continuous and non-adherent porcine cell line that is capable of proliferating in medium free of animal-derived components, wherein the cell line is deposited at the DSMZ with the deposit number DSM ACC3308.

16. A method for producing a virus comprising the steps of: (i) providing a continuous and non-adherent porcine cell line that is capable of proliferating in medium free of animal-derived components and that is permissive for a virus, (ii) infecting said cell line with the virus or transfecting said cell line with a plasmid carrying (a) nucleic acid sequence(s) encoding the virus, and (iii) culturing the cell line infected or transfected in step (ii), thereby producing the virus, wherein the cell line is deposited at the DSMZ with the deposit number DSM ACC3307.

17. A method for producing a virus comprising the steps of: (i) providing a continuous and non-adherent porcine cell line that is capable of proliferating in medium free of animal-derived components and that is permissive for a virus, (ii) infecting said cell line with the virus or transfecting said cell line with a plasmid carrying (a) nucleic acid sequence(s) encoding the virus, and (iii) culturing the cell line infected or transfected in step (ii), thereby producing the virus, wherein the cell line is deposited at the DSMZ with the deposit number DSM ACC3308.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) The following Figures and examples are merely illustrative of the present invention and should not be construed to limit the scope of the invention as indicated by the appended claims in any way.

(2) FIG. 1: PK-15S and STS passage diagrams depicting cell densities (left) and viabilities (right). Generation of cell populations suitable for propagation in medium free of animal-derived components. Both cultures required an adaptation phase of at least 16 weeks. Especially generation of STS was complex and required shifts between semi-adherent and suspension culture formats. The semi-adherent culture format was achieved by supplementation of the CD-U4 medium with 0.1 volumes of DMEM. The letters in the chart denote: a, adaptation to animal-derived component free medium; r, rescue of adapted cell population; s, subcultivation and stabilization of final cell population; t, shift in culture format from semi-adherent to single-cell suspension; w, withdrawal of fetal calf serum; f, fetal calf serum. The right panel shows cell morphologies and size distribution of the final suspension cell lines.

(3) FIG. 2: Cell identities with expanded PCR analysis. MACF1 PCR described in [45] has been applied to different cell cultures. A BsrGI polymorphism allows differentiation of monkey (Marc-145) and swine (PK-15S and STS) that give otherwise similar amplification products. The simplicity of the MACF1 banding pattern allows identification of potential contaminations. The results presented here show that the cell lines are of expected identity and that no contamination has occurred, especially not between Marc-145 and PK-15S and not MDCK (another cell line permissive for AIV) and PK-15S. YNZ22 PCR furthermore allows to differentiate between cell lines of the same species origin, PK-15S and STS yield different signals.

(4) FIG. 3: Propagation of AIV in PK-15S suspension cultures in a stirred tank bioreactor. This experiment was performed with cells beyond passage 26 in single-cell suspension in chemically defined medium. Trypsin was used to activate the infectious activity of the LPAIVs and was added to 16 U/mL of culture volume per day. All LPAIVs were inoculated at a MOI of 10.sup.4. (A) Cell proliferation in the stirred tank bioreactor. Infection was usually performed at day 4 at typical cell densities between 3 and 510.sup.6 cells/mL. One bioreactor (open circle, red) was continued as reference without infection until day 6. Cell density was beyond 810.sup.6 at day 6 for this uninfected bioreactor. (B) Viability was above 90% in most experiments, also in this one. The cultures shown here correspond to those shown in panel A. (C) HAU units and (D) infectious TCID50 units of LPAIV H5N3, H7N7 and H9N2. (E) Combined chart depicting cell proliferation and infectious units in a bioreactor infected with vesicular stomatitis virus (VSV, Rhabdoviridae). MOI in this experiment was 10.sup.3.

(5) FIG. 4: PK-15 allows influenza virus propagation without exogenous protease addition. NDV also replicated in the here described porcine cells without addition of trypsin (data not shown, PK-15S). The bar graphs depict infectious titers at day 3.

(6) FIG. 5: Results obtained with the same viruses in MDCK suspension cultures in chemically defined medium CD-U5 supplemented for MDCK propagation. (A) Higher amounts of trypsin generally lead to higher infectious or hemagglutinating titers. Dependence on trypsin is confirmed further down. (B) Kinetic of LPAIV replication in MDCK suspension cultures. Note the strong decline of infectious units already 72 h post infection.

(7) FIG. 6: PK-15S and STS allows influenza virus propagation without exogenous protease addition. Replication of para- and orthomyxoviruses is inhibited in CR.pIX and MDCK cultures without addition of trypsin. () for no supplementation, (s) for addition of bovine serum to 2%, (t) for 16 U/mL trypsin per day. Shown are mean and standard deviation of at least 4 independent experiments. The chart depicts values obtained 3 days post infection.

EXAMPLES

(8) The examples given below are for illustrative purposes only and do not limit the invention described above in any way.

(9) 1.1 Stable Adaption to Single-Cell Suspension Cultivation

(10) Adaptation of adherent cells to proliferation in suspension and without dependence on serum is complex and unique for each cell line [46,47]. However, this step is important to obtain a system suitable for veterinary vaccine production that requires efficient, robust and highly cost-effective solutions. The significant advantages of single-cell suspension production systems suitable for cultivation in scalable bioreactors is briefly discussed in the context of peste des petits ruminants (PPR) vaccines [48], but the same considerations apply for all veterinary and human vaccines, especially if they are required at higher volumes and doses. The burden associated with bovine serum are discussed in the context of introduction of adventitious agents to pharmaceutical products [46].

(11) Successful adaptation to proliferation without serum and to proliferation in suspension induces profound changes in cell cultures. The affected cell lines must be guided through modulations in metabolic pathways, apoptotic cascades, cytoskeleton integrity, and intracellular vesicle targeting.

(12) Metabolic pathways shift because the wide spectrum of growth factors cannot be provided by chemically-defined medium or plant hydrolysates. Apoptosis needs to remain suppressed also in the absence of protective serum factors, and receptors responsible for anoikis induction must ignore absence of adhesion signals that are normally provided by the growth support and neighbouring cells. The cytoskeleton, a pervasive structure throughout the cell, must rearrange with the transition from anchored fusiforms to the artificially induced spheres. Finally, transport of cellular (and viral) components along actin filaments and microtubule tracks must be reorganized as cell polarity is lost in a suspension cell without basal and apical surfaces. The results are observable extensive and global shifts in the genome and proteome [49] so that parental cell and derived progeny are highly divergent and not interchangeable anymore. To avoid ambiguities due to this development, the cell lines that have shifted signalling and metabolic pathways towards a novel generation are denoted with the suffix S.fwdarw.G+(PK-15S.fwdarw.G+, STS.fwdarw.G+, or abbreviated PK-15S and STS).

(13) The process towards adaptation was characterized by phases with very low proliferation rates and viabilities that had to be alternated with subtle changes to the media and culture formats to prevent loss of the cultures.

(14) The experimental steps were the following: The adherent PK-15 cells were trypsinized and re-suspended to approx. 1510.sup.6 cells/mL in a cell culture medium based on CD-U4. CD-U4 cell culture medium is a modification of DMEM and can be obtained, for example, from GE Healthcare under catalogue T1313. Viability and cell density immediately decreased significantly (see FIG. 1). The cells were next adapted by continuous cultivation after dispersal of cell aggregates using cell-strainers and plant-derived trypsin during week 0 to 6. Suitable cell populations were rescued stepwise, by variation of medium composition during a passage, to a chemically-defined CD-U4-derivative over the course of further 10 weeks. Variations of the culture medium included supplementation with insulin-like growth factor between 10 and 150 ng/mL; glucose between 5 and 8 g/L; glutamine between 1 and 4 mM; bovine serum in a declining sequence from 1% to 0% in steps of 0.2%; CaCl.sub.2) from 21 to 27 mg/L; Fe(NO.sub.3).sub.3*9H.sub.2O from 0.008 to 0.010 mg/L; MgSO4 from 7.8 to 9.9 mg/L; KCl from 32 to 41 mg/L; NaHCO.sub.3 from 296 to 376 mg/L; NaCl from 512 to 650 mg/L; NaH.sub.2PO.sub.4 from 8.7 to 11.1 mg/L; L-arginine*HCl from 6.7 to 8.5 mg/L; L-cystine*2HCl from 5.0 to 6.4 mg/L; L-glutamine from 47 to 59 mg/L; glycine from 2.4 to 3.0 mg/L; L-histidine*HCl*H.sub.2O from 3.4 to 4.3 mg/L; L-isoleucine and L-leucine from 8.4 to 10.7 mg/L; L-lysine*HCl from 11.7 to 14.8 mg/L; L-methionine from 2.4 to 3.0 mg/L; L-phenylalanine from 5.3 to 6.7 mg/L; L-serine from 3.4 to 4.3 mg/L; L-threonine from 7.6 to 9.7 mg/L; L-tryptophan from 1.3 to 1.6 mg/L; L-tyrosine*2 Na*2H.sub.2O from 9.6 to 12.2 mg/L; soy, wheat and yeast hydrolysates at 0.5 to 3 g/L; dextrane sulfate between 20 and 80 mg/L; myo-inositol from 0.58 to 0.73 mg/L; riboflavin from 0.03 to 0.04 mg/L; any or all of choline chloride, folic acid, niacinamide, D-pantothenic acid* Ca, pyridoxal*HCl, pyridoxine*HCl, and thiamine*HCl from 0.32 to 0.41 mg/L; HEPES from 476.64 to 605.33 mg/L; and pyruvate*Na from 8.8 to 11.2 mg/L.

(15) A stable culture that proliferated in single-cell suspension without dependence on microcarriers could be maintained in chemically-defined medium at cell densities between 0.310.sup.6 and at least 1010.sup.6 cells/mL starting with week 16 (corresponding to passage 21). A stirred-tank bioreactor was inoculated out of this stable culture at passage 26, the culture was maintained for at least additional 8 weeks to demonstrate that adaptation has been accomplished. No animal-derived components and no trypsin was used after passage 5 and, therefore, was also not used towards inoculation of the bioreactor.

(16) A culture stably adapted to proliferation in suspension in chemically defined medium free of animal-derived components was also obtained of the ST cell line. This cell line transiently required supplementation with 30-60 mg/L putrescine, 30-50 mg/L spermine, 5-25 L/L TrypLE, 200 ng/mL insuline, 1-3 g/L soy hydrolysate, 0.05% methylcellulose, and 600-900 mg/L NaCl in addition to the components given above for full adaptation.

(17) A continuous passage history in media free of animal derived components is highly desirable. Animal derived components, especially bovine serum, can be contaminated with adventitious agents that can cause disease in vaccine recipients and that complicate trans-boundary dissemination of vaccine preparations [46]. Cryocultures of the PK-15S and STS cell lines were submitted according to the Budapest Treaty to the DSMZ on Sep. 21, 2016 and received the deposit numbers DSM ACC3307 and DSM ACC3308, respectively. Both cryocultures were obtained out of cultures that proliferate in animal-derived component free medium (the medium for PK-15S is even chemically-defined), they were passaged at least 10 times in such medium, and they are stored in preservation medium free of animal-derived components.

(18) In summary, a robust adaptation to continuous proliferation in medium free of animal derived components in true suspension is demonstrated for the PK-15 and ST cell lines for the first time.

(19) 1.2 Cell Line Identity and Purity

(20) Cell line cross-contamination is a frequent problem. To confirm that the here described PK-15S cell line is pure, PCR was performed to examine cell identity.

(21) Fehler! Verweisquelle konnte nicht gefunden werden. FIG. 2 shows a refined assay for identity of the cell lines with DNA isolated out of the cultures that are intended to be transferred back to the originating laboratory. Purity and identity of the cultures to within the detection limit of this sensitive method is confirmed. A MACF1-assay that has been expanded to include a BsrGI restriction fragment length polymorphism allows a clear distinction between porcine and simian cells. A microsatellite assay allows to differentiate different cell lines from the same species (PK15 and ST).

(22) The banding pattern in FIG. 2 was obtained as follows: DNA was isolated from 410.sup.6 cells with QIAmp DNA Blood Mini columns (Qiagen) according to the manufacturer's instructions. PCRs were performed with 100 ng of template DNA and 2 M of primer. Cycle parameters were 35 repeats of 55 C. annealing for 30 s, 72 C. extension for 180 s and 94 C. melting for 20 s. MACF1 primer sequences are CCATCTgCTgAgTATAAAgTggTgAA (SEQ ID NO: 1) and gCCTCCTTCTgCTTgAAgCA (SEQ ID NO: 2), single-primer PCR for amplification of YNZ22 minisatellites was performed with CTCTgggTgTCgTgC (SEQ ID NO: 3).

(23) In summary, the here described PK-15S and STS cultures are pure and identifiable. Most importantly, the presence of simian or canine material was not observed, previously demonstrated to be highly permissive for influenza viruses, in PK-15S or STS cultures.

(24) 1.3 High Titers after Influenza Virus Infection

(25) Orthomyxoviruses depend on proteolytic activation of the viral receptor, the HA protein, very similar to the paramyxoviruses [33-35]. The embryonal tissues provide the required proteases if vaccines are produced in embryonated eggs. The proteases contained in embryonated eggs are replaced by exogenous trypsin for production of myxovirus vaccines in cell cultures.

(26) Influenza A viruses (AIVs) isolated with the help of cell lines have been proposed to be suitable as vaccine seeds [50]. Important parameters in the determination of vaccine properties are infectious units as an indicator for active viruses present in the preparations. Another important parameter is the haemagglutinating activity on erythrocyte preparations from chicken, horse or guinea pig as an indicator for the amount of potentially reactogenic antigen present in the vaccine harvest.

(27) The hemagglutinating and infectious activities of at least three different low pathogenic Influenza A viruses (LPAIVs) propagated in the suspension PK-15S cells in true suspension cultures in chemically-defined medium without microcarriers and at extremely low MOIs compare favourably, or are even superior to the results reported in MDCK cells [50,51] or obtained here as reference and that are shown in FIG. 5.

(28) The results shown in FIG. 3 were obtained as follows: Cells were maintained in CD-U4 (GE Healthcare #T1313) or CD-U5 medium (BD Biosciences #16ABP247) supplemented to 10 ng/mL with LONG R3IGF-I (Sigma #91590C) and 1 stable L-glutamine (such as GlutaMAX-I, Gibco #35050-038, alternatively 2 mM conventional glutamine). They were cultivated in 0.2 m-vented 125 mL-flasks (Corning #431143) with 25-62 mL working volume or vented 50 mL-tubes (Tubespin Bioreactor 50, TPP #87050) with 5-15 mL working volume. Incubation was performed in INFORS HT Multitron Cell shaking incubator set to 36.5 C. and 8% CO.sub.2, RPM is 150 for flasks and 180 for spin tubes, respectively, and amplitude or throw of the rotating platform is 5 cm.

(29) The cells were subcultivated between 810.sup.5 viable cells/mL and 610.sup.6 viable cells/mL for routine passaging, usually with two splits per week. Subpassage was performed by at least 5-fold dilution of the cell suspension without any centrifugation.

(30) Infectious titers of LPAIVs were determined on Vero cells. 1.510.sup.6 cells in DMEM:F12 medium containing 2 mM GlutaMAX I (both Gibco) and 5% fetal calf serum (Biochrom) were seeded into CellBIND 96-well plates (Corning) at 100 L of cell suspension. The medium was replaced on the following day against DMEM:F12 containing 2 mM GlutaMAX I and 1.5 g/mL trypsin (type IX-S, Sigma T0303), but no fetal calf serum. Serial dilutions in steps of 10 of LPAIV-containing virus samples were prepared in DMEM:F12 medium free of serum, and 10 L each of the dilutions were added to the Vero cultures. Virus replication was allowed at 37 C. for 72 h and detected by the cytopathic effect. Calculation of TCID.sub.50 values was performed according to Spearman and Krber using the implementation of the FAO [52, 53].

(31) Haemagglutination units (HAU) were determined with chicken erythrocytes and serial dilutions of infected cell suspensions in PBS in steps of 2. Erythrocytes from 7 mL of a 25% complete blood suspension in Alsever buffer (E200, Labor Dr. Merk & Kollegen GmbH, Germany) were washed thrice in cold PBS and diluted in PBS to a concentration that yields an OD of 2.9 to 3.1 at 576 nm. This suspension was stored for up to one week at 4 C. Centrifugation for the washing steps was performed at room temperature with 500g, brake set to low, for 5 min each.

(32) The assay was performed in round-bottom 96-well microtiter plates. The first row of the plate was filled with 200 L of the crude culture supernatant and all other rows received 100 L of PBS. Next, 100 L of virus sample was added to the second row, the pipetting tips were replaced and the suspension mixed with fresh tips. This two-fold dilution was continued serially into the next row, and, for samples with high potency, continued into a second plate. Each well thus contained 100 L of diluted virus sample and was subsequently supplemented with 100 L of the erythrocyte suspension. This volume was mixed by pipetting and the turbid solution allowed to stand undisturbed for 60 min at ambient temperature (22 C.). HAU/100 L are given by the highest dilution where formation of the characteristic dot was not yet visible.

(33) The bioreactor experiments were conducted in DASBox (DASGip, Eppendorf) bioreactor units. These stirred-tank reactors are equipped with a Marine impeller with 3 blades and 60-250 mL working-volume vessels. Gas mixing was performed with N2, air, CO.sub.2 and O.sub.2. The pH was adjusted with CO.sub.2 and 1 M Na.sub.2CO.sub.3. Inoculation was performed to 0.810.sup.6 PK-15S and 110.sup.6 BHK cells/mL in CD-U4 medium and the culture was allowed to proliferate for 4 days to approximately 310.sup.6 cells/mL. Glutamine was usually added to 2 mM at day 4 and glucose to 6 g/L at day 5. (see FIG. 3).

(34) The parameters for the cell proliferation phase were 37 C. culture temperature, 60% DO (dissolved oxygen) saturation in the medium, 180 rpm for the impeller, and a pH 7.1. The pH was increased to 7.5 units as a gradient over 6 h after infection.

(35) Trypsin (type IX-S, Sigma T0303) was added automatically to the infected culture to 16 U/mL of reactor volume per day in 4 burst of 4 mL each separated by 6 hours. This particular trypsin is provided as 1 g lyophylisate that was resuspended to 1 mg/mL in PBS. Specific activity depends on the lot and was 18500 U/mg in our experiments. Addition of 16 U/mL therefore corresponds to less than 0.9 g/mL of trypsin which is well below the toxic level in the range of 2.5 g/mL.

(36) In summary, for the first time, a PK-15S cell line was demonstrated to allow production of LPAIV seed viruses to high infectious and haegglutinating titers in a stirred tank bioreactor in chemically defined medium. The results were at least as good as those obtained with MDCK.

(37) 1.4 Independent of Trypsin

(38) The virions of many viruses such as ortho- and paramyxoviruses must be proteolytically processed for activation of the infectious units. The proteases contained in embryonated eggs are sufficient to activate influenza and Newcastle disease viruses. The addition or ectopical expression [54] of exogenous trypsin or other proteases is required for production of these viruses in cell cultures.

(39) It would be highly advantageous, and very unusual, if a continuous cell line would allow production of influenza virus isolates independent of exogenously added trypsin and only with a natively inherent cognate protease activity. Such an inherent property may help in the identification and production of viruses, e.g. of low-pathogenic field isolates. Such a property is clearly different from the introduction of selected non-cognate proteases by genetic recombination.

(40) To test for such a property requires a cell line that robustly proliferates in a medium as the here described chemically-defined cell proliferation medium with a very low protein content (10 ng/mL of recombinant IGF) and without other substances that may interfere with the enzymatic activity of proteases.

(41) Infectious titers and HA titers of LPAIVs were determined as described above. Detection of NDV replication in the titration plates was facilitated by immunostaining: the Vero indicator cells were fixed in methanol for 10 min, allowed to dry to completion, and rehydrated with PBS containing 0.05% Tween-20. NDV antiserum (GD Animal Health) was added to a dilution of 1:2000 in PBS containing 1% fetal calf serum and incubated for 1 h at room temperature. After two washes with PBS, secondary antibody (anti-chicken, Alexa Fluor 488 labelled, host rabbit, Dianova, 303-545-003 at 1 g/L) was added at a dilution of 1:2000 for 2 h at ambient temperature or overnight at 4 C. Infected wells were identified by fluorescence after two washes with PBS.

(42) The HA units were determined as described for LPAIVs above with one modification, titration was performed with sonicated, rather than crude cell lysate. Sonication was performed using a Vial Tweeter (set to 20 s of 100% cycle and 90% amplitude) that allows handling of closed sample caps to avoid cross-contamination (Hielscher, Germany).

(43) The results are shown in FIGS. 4 and 5.

(44) In summary, a cell culture system that does not require exogenous trypsin, and that allows propagation of LPAIVs to very high hemagglutinating and infectious titers has been developed.

(45) 1.5 the Protease in the Porcine Cell Lines is Endogenous

(46) It has further been quantified whether the protease of the porcine cell lines is active intracellularly as has been described for human airway epithilium [55,56]. Serum contains a wide spectrum of high-molecular weight protease inhibitors [57] that would interfere with exogenous proteases (such as trypsin) or proteases that are being secreted by the host cell or contained in the vaccine preparation.

(47) The data shown in FIG. 6 was obtained as follows: suspension cells were provided at a cell density of 210.sup.6 cells/mL in chemically-defined medium. Bovine serum was added to a concentration of 2% 2 h prior to infection. Next, the CR.pIX and MDCK cell lines that were previously shown to allow propagation of influenza viruses only in presence of trypsin were given the protease, the porcine cell lines were examined always in absence of trypsin. LPAIV H5N3 was added to a MOI of 10.sup.4 and titers were determined 3 days PI. CR.pIX and MDCK positive controls consisted of reactions without serum but with trypsin, and negative controls of reactions without serum and trypsin.

(48) FIG. 6 demonstrates that exogenous trypsin is required for propagation of influenza viruses in the avian and canine cell lines. Furthermore, this trypsin supplementation is being quenched by addition of serum. This observation is in stark contrast to the phenomenon that influenza viruses replicate to high titers also without trypsin and in the presence of bovine serum in PK-15S and STS. The high-molecular weight protease inhibitors present in serum cannot enter the cell. This experiment, therefore, demonstrates the activity of an endogenous intracellular protease capable of activating LPAIVs in cultures of PK-15S and STS.

(49) That the endogenous activity is unusually high in the PK-15S and STS cell lines in combination with the suspension culture format and in medium free of animal derived components can be inferred from the high susceptibility and high permissivity. For example, the infectious yield for any of the tested strains (H5N3, H7N7 or M9N2) in PK-15S was at least 10.sup.6 TCID.sub.50/mL, the MOI was 10.sup.4 viruses/cell, and cell density was 210.sup.6 cells/mL. The input was, thus, of (210.sup.6 cells/mL)(10.sup.4 TCID.sub.50/cell)=200 TCID.sub.50/mL. The burst size was (10.sup.6 TCID.sup.50/mL): (200 TCID.sub.50/mL)=5000. A burst size higher than 100 demonstrates high permissivity and that productive infection with such a burst size is obtained with a MOI of only 10.sup.4, 10-fold below 0.001, demonstrates high susceptibility.

(50) An unusually broad investigation has been presented that includes industrially relevant avian, canine and porcine cell lines. The data shown in FIG. 6 clearly demonstrates that only the hitherto undescribed advantageous combination of chemically defined medium, metabolically or genomically shifted porcine cell line and suspension culture format allows for robust production of myxoviruses without addition of exogenous trypsin and animal-derived supplements.

REFERENCES

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