0095-1137/78/0007-0044$02.00/0 JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1978, p. 44-51 Copyright i 1978 American Society for Microbiology

Vol. 7, No. 1 Printed in U.S.A.

Production of Human Lymphoblastoid Interferon by Namalva Cells KATHRYN C. ZOON,l* CHARLES E. BUCKLER,2 PAMELA J. BRIDGEN,' AND DALIA GURARI-ROTMANt Laboratory of Chemical Biology, National Institute ofArthritis, Metabolism, and Digestive Diseases,' and Laboratory of Viral Diseases, National Institute ofAllergy and Infectious Diseases,2 National Institutes of Health, Bethesda, Maryland 20014

Received for publication 14 July 1977

Optimum conditions for growth and interferon production by a human lymphoblastoid cell line, Namalva, have been studied. Adaptation to large-scale production is possible utilizing either Sendai virus or Newcastle disease virus. Priming of cultures before induction is unnecessary. The interferon produced has properties similar to human leukocyte interferon. The production of lymphoblastoid interferon per cell is increased two- to fourfold after dilution with serumfree medium of a saturation-density culture of Namalva induced with Newcastle disease virus. Maximum interferon yields were obtained 27 h after the addition of virus, using cultures diluted to 4 x 105 to 9 x 105 cells per ml. The presence of glutamine in the dilution medium was required for maximum interferon production. Newcastle disease virus appeared to inhibit the rates of RNA and protein synthesis more effectively in the diluted cultures. MATERIALS AND METHODS Cells. The human lymphoblastoid cell line, Namalva, established from a Burkitt lymphoma, was obtained from George Klein. These cells, a nonproducer line for the Epstein-Barr virus (7), are also incapable of Epstein-Barr virus production after induction with halogenated pyrimidines (11). A Namalva cell contains approximately three Epstein-Barr virus genome equivalents and only about 50% of the viral sequences, as determined by nucleic acid hybridization studies (2, 11). Media. Cell culture media were prepared by the National Institutes of Health Media Unit. RPMI 1640 medium containing 25 mM Hepes (N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid) was used for growth and interferon studies. The medium was supplemented with heat-inactivated (56°C, 30 min) fetal bovine serum (FBS) as indicated. Penicillin (100 U/ml) and streptomycin (100 ,g/ml) were also added to the growth medium. Eagle minimum essential medium, supplemented with FBS and antibiotics, was

Interferons are a group of glycoproteins capable of inhibiting viral multiplication in many different cell types. The biological specific activities of these antiviral agents are very high and, for human interferon, have been estimated to be at least 2 x 108 U/mg of protein (8, 10, 16). The minute quantities of interferon in crude preparations have hindered the successful purification and characterization of this antiviral agent and, thus, have stimulated investigation of production methods that might increase the yields of interferon in tissue culture systems. Recently, Strander et al. (12) described the production of interferon by the human lymphoblastoid cell line, Namalva, induced with Sendai virus. This continuous cell line is advantageous for the large scale production of interferon because it can be grown in large volumes in suspension and produces reasonably high titers of interferon. This paper describes the optimal growth and induction conditions of Namalva cultures and the kinetics of interferon production in cultures with cell densities of 1 x 106 to 10 x 106 cells per ml. In addition, a strategy is presented for the large-scale production of human interferon, which involves the dilution of saturation-density cultures, induced with Newcastle disease virus Bi (NDV-B1), with serumfree medium. The kinetics of accumulation of interferon have also been examined.

also used in some studies. Cultural conditions. Cell cultures were established at an initial concentration of 2 x 105 to 3 x 105 cells per ml in growth medium containing 10% FBS. Vessels of at least twice the fluid volume were used. The cultures were incubated at 37°C with or without agitation, which, when used, was produced by a magnetic bar rotating at 60 rpm placed on the bottom of the culture vessel. Cell counts were made using a Lang-Levy hemacytometer. Viability was determined by trypan blue exclusion. Viruses. Sendai virus, NDV Herts and Bl, and influenza virus NWS were grown in the allantoic cavity of embryonated eggs. Average working pools of virus contained 4,000 to 16,000 hemagglutination (HA) units

t Present address: Department of Virology, Weizmann Institute of Science, Rehovot, Israel.

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PRODUCTION OF LYMPHOBLASTOID INTERFERON

VOL. 7, 1978

ml. In some of these studies, NDV-B1 was used rather than Sendai virus because of the potential hazard of the Sendai virus to local mouse colonies. Chikungunya virus was a mouse brain preparation. Sindbis virus and Semliki Forest virus were propagated in chicken embryo tissue culture. Interferon. Culture fluids were collected, and Namalva cells were removed by centrifugation at 1,250 x g for 10 min. The supernatant fluid was adjusted to pH 2 by the addition of hydrochloric acid and kept at 4°C overnight. This procedure destroyed residual viral infectivity as well as the ability of the virus to induce interferon. The acidic fluids were then adjusted to pH 5 to 6.5 by the addition of sodium hydroxide. Interferon samples were stored at 4°C or -20°C until assayed. The reference standard, human leukocyte interferon, was obtained from the Research Resources Branch, National Institute of Allergy and Infectious Diseases. Interferon assays were performed as previously described (4), using a vesicular stomatitis virus cytopathic effect inhibition system and HSF-4 human foreskin fibroblasts. In this assay, the National Institutes of Health human reference interferon (G-023901-527, 104-3 U/ml) titered 1043 U/nil. Interferon neutralization. The neutralization of various interferons was examined by a modification of the procedure of Valle et al. (17). Dilutions of interferon (1:3) were incubated with 1:10 dilutions of anti-human leukocyte interferon serum at 37°C for 1 h. The mixtures were then assayed for residual interferon activity. Chemicals. Actinomycin D was obtained from the Drug Research and Development Branch, National Cancer Institute. It was dissolved in Eagle minimum essential medium and stored at -20°C. The synthetic double-stranded ribonucleic acid (RNA) inducer of interferon, polyriboinosinic:polyribocytidylic acid and its complex with poly-l-lysine-carboxymethylcellulose (10) were obtained from Hilton B. Levy. [5,6-3H]uridine, 40 to 50 Ci/mmol, and U-_4C-labeled L-amino acid mixture, 1 mCi/10 ml in 0.1 N HCl, were obtained from New England Nuclear Corp., Boston, Mass. NCS solubilizer was purchased from Amersham/Searle, Arlington Heights, Ill. Rates of incorporation of [3HJuridine and 14Clabeled L-amino acids into lymphoblastoid cultures. Samples (20 ml) of culture were supplemented with 5 ,uCi of [3H]uridine and 20 yCi of "4C-labeled Lamino acid mixture, incubated at 37°C for 30 min, and then centrifuged at 1,250 x g for 20 min at 5°C. The cells were washed with 10 ml of Earle balanced salt solution and suspended in 2.5 ml of phosphatebuffered saline (pH 7.4). The suspensions were made 10% (vol/vol) with perchloric acid (PCA), incubated for 10 min at 0°C, and centrifuged at 1,250 x g for 10 min at 5°C. The PCA precipitates were resuspended in 1 ml of phosphate-buffered saline. NCS solubilizer (1 ml) was added to portions of both the PCA-resuspended precipitates and soluble fractions, followed by 10 ml of toluene scintillation fluid. Duplicate samples were prepared and counted for 10 min. per

RESULTS

Cell growth kinetics. Namalva cell growth characteristics were studied to determine con-

45

ditions yielding maximum rates of cell production (Fig. 1). RPMI 1640 medium was found to be superior to Eagle miniimum essential medium. The rate of growth was found to increase with the concentration of serum. Agitation of the cell suspension produced a more rapidly growing culture than was obtained in stationary cultures. When new cultures were established by suspending cells in 100% fresh medium a 1- to 2day lag period in growth was observed. When cultures were made by a 1:2 dilution of growing (log phase) cells, there was less of an interruption in the logarithmic portion of the cell growth curve. Using these conditions, it was possible to produce 5 to 50 liters of cells at a saturation density of 2 x 106 to 3 x 106 cells per ml in commercially available tissue culture vessels. In initial studies, following the procedures reported by Strander et al. (12), the cells were concentrated to 107 cells per ml by centrifugation before interferon induction. This procedure was found to be impractical if very large volumes of cells were to be used. Additionally, the presence of 10% FBS in the crude interferon prepared from saturation-density cultures was undesirable for starting material for purification. Therefore, a growth procedure was developed that resulted in a lower final serum concentration. Cultures were established at 2 x 105 cells per ml in one-half the desired volume of medium containing 10% FBS. After about 3 days, the culture volume was doubled by the addition of medium containing 0 to 4% FBS. The cells were allowed to grow to final cell densities comparable to those obtained with 10% serum cultures. Thus, cultures for interferon induction at final serum concentrations of 5 to 7% could be obtained. In the large-scale production of interferon, the reduction of the serum concentration would result in a crude interferon preparation with an increased specific activity (interferon units per milligrams of protein). In addition, since the serum is an expensive component in the production of interferon, this procedure would reduce the cost considerably. Priming. In many interferon production systems, exposure of cells to interferon before induction (priming) results in an increase in the amount of interferon induced. The effect of priming Namalva cells was examined. Production by unprimed cultures (107 cells per ml) was compared with interferon production in similar cultures pretreated with 100 U of human leukocyte interferon per ml 2 h before induction with virus. No difference in the amount of interferon produced at the priming concentration examined was observed, and therefore priming was not employed for the remaining studies. Inducers. Other viral inducers of interferon production were examined to determine whether

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FIG. 1. Kinetics of Namalva cell growth. Initial inocula were obtained from a saturation-density culture by resuspending cells after centrifugation at 2.5 x 10' cells per ml in RMPI 1640 + 10% or 4% FBS. Cultures of 200 ml were established in 500-ml bottles and incubated under spinner (0) (+) or stationary (A) conditions. Cell counts were made on a 1-ml sample removed each day. Viabilities remained between 90 and 95% during the experiment.

greater response than that obtained with Sendai virus was possible. Cultures were established at 107 cells per ml in 4% FBS. Different viruses were added to separate cultures, and the fluids were harvested 24 h later. After the culture fluid was processed to remove residual virus (pH 2), interferon assays were performed (Table 1). Sendai virus and NDV produced comparable amounts of interferon under these conditions. Semliki Forest virus induced about fivefold less interferon than Sendai, and influenza (NWS strain) induced a very small amount. Neither Chikungunya nor Sindbis viruses induced detectable amounts of interferon. The dose response effect of both Sendai virus and NDV was studied. Cultures at 107 cells per ml and at saturation density were induced with various amounts of virus (0.6 to 120 HA units per 106 cells). Maximum interferon production was observed when >12 HA units per 106 cells a

TABLE 1. Induction of human lymphoblastoid interferon by various viruses Virus

Chikungunyaa Sindbisb Influenza (NWS) b Semliki Forestb NDVc Sendaic

Log1o interferon units per ml 20 h postinductiona