Production of Human Lymphoblastoid Interferon by Namalva Cells

Transcription

1 /78/7-44$2./ JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1978, p 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 214 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 15 to 9 x 15 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. 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 18 U/mg of protein (8, 1, 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 16 to 1 x 16 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. t Present address: Department of Virology, Weizmann Institute of Science, Rehovot, Israel. 44 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 5% 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 164 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, 3 min) fetal bovine serum (FBS) as indicated. Penicillin (1 U/ml) and streptomycin (1,g/ml) were also added to the growth medium. Eagle minimum essential medium, supplemented with FBS and antibiotics, was also used in some studies. Cultural conditions. Cell cultures were established at an initial concentration of 2 x 15 to 3 x 15 cells per ml in growth medium containing 1% 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 6 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, to 16, hemagglutination (HA) units Downloaded from on April 28, 218 by guest

2 VOL. 7, 1978 PRODUCTION OF LYMPHOBLASTOID INTERFERON 45 per 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 1,25 x g for 1 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 -2 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 , 14-3 U/ml) titered 143 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 1:1 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 -2 C. The synthetic double-stranded ribonucleic acid (RNA) inducer of interferon, polyriboinosinic:polyribocytidylic acid and its complex with poly-l-lysine-carboxymethylcellulose (1) were obtained from Hilton B. Levy. [5,6-3H]uridine, 4 to 5 Ci/mmol, and U-_4C-labeled L-amino acid mixture, 1 mci/1 ml in.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 (2 ml) of culture were supplemented with 5,uCi of [3H]uridine and 2 yci of "4C-labeled L- amino acid mixture, incubated at 37 C for 3 min, and then centrifuged at 1,25 x g for 2 min at 5 C. The cells were washed with 1 ml of Earle balanced salt solution and suspended in 2.5 ml of phosphatebuffered saline (ph 7.4). The suspensions were made 1% (vol/vol) with perchloric acid (PCA), incubated for 1 min at C, and centrifuged at 1,25 x g for 1 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 1 ml of toluene scintillation fluid. Duplicate samples were prepared and counted for 1 min. RESULTS Cell growth kinetics. Namalva cell growth characteristics were studied to determine conditions yielding maximum rates of cell production (Fig. 1). RPMI 164 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 1% fresh medium a 1- to 2- day lag period in growth was observed. When cultures were made by a 1:2 dilution of growing (log phase) cells, there was less ofan interruption in the logarithmic portion of the cell growth curve. Using these conditions, it was possible to produce 5 to 5 liters of cells at a saturation density of 2 x 16 to 3 x 16 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 17 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 1% 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 15 cells per ml in one-half the desired volume of medium containing 1% FBS. After about 3 days, the culture volume was doubled by the addition of medium containing to 4% FBS. The cells were allowed to grow to final cell densities comparable to those obtained with 1% 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 (17 cells per ml) was compared with interferon production in similar cultures pretreated with 1 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 Downloaded from on April 28, 218 by guest

3 46 ZOON ET AL. 3 J. CLIN. MICROBIOL. 25 V-4 2 x -J n -J LU. U 15 1 S e DAYS 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 1' cells per ml in RMPI % or 4% FBS. Cultures of2 ml were established in 5-ml bottles and incubated under spinner () (+) or stationary (A) conditions. Cell counts were made on a 1-ml sample removed each day. Viabilities remained between 9 and 95% during the experiment. a greater response than that obtained with Sendai virus was possible. Cultures were established at 17 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 17 cells per ml and at saturation density were induced with various amounts of virus (.6 to 12 HA units per 16 cells). Maximum interferon production was observed when >12 HA units per 16 cells TABLE 1. Induction of human lymphoblastoid interferon by various viruses Log1o interferon units Virus per ml 2 h postinductiona Chikungunyaa <.7 Sindbisb <.7 Influenza (NWS) b 1.4 Semliki Forestb 3.1 NDVc 3.7 Sendaic 3.9 anamalva cell cultures of 17 cells per ml. All cultures were incubated at 37 C. b Viral multiplicity, 1 infectious doses per cell. e 6 HA units per ml. were used. These results are comparable to those obtained by Strander et al. (12). No inhibition of yield was observed at the highest concentration of inducer virus. Ultraviolet inactivation of Downloaded from on April 28, 218 by guest

4 VOL. 7, 1978 PRODUCTION OF LYMPHOBLASTOID INTERFERON 47 Sendai infectivity neither stimulated nor significantly reduced the ability of the virus to induce interferon production over a wide range of ultraviolet dose. Synthetic double-stranded RNA was found not to induce detectable interferon when added to cultures of Namalva cells at 2,g/ml, a dose that will induce human foreskin fibroblasts. Likewise, the poly-l-lysine-carboxymethylcellulose complex, which has been found to be highly effective in primate cells (9), was ineffective. Neutralization of lymphoblastoid interferon. Sheep anti-human leukocyte interferon serum was found in two separate experiments to neutralize the Namalva cell interferon but with less efficiency than it neutralized leukocyte interferon. Antiserum in 1-fold excess of the minimum amount necessary to neutralize 99% of the homologous leukocyte interferon only neutralized 5% of the lymphoblastoid interferon. The same antiserum had no effect on the titer of human fibroblast interferon. When used as an immunoabsorbent (bound to Sepharose) reagent (3), this antibody is capable of binding >9% of Namalva interferon. Thus, in respect to specific antibody reactivity, lymphoblastoid interferon is similar to leukocyte interferon. Effect of variation in cultural conditions on interferon yields. In these cultures, increasing the concentration of amino acids (2x) or vitamins (2x) or altering the glucose concentration (O.lx to 5x) had no effect on the amount of interferon produced. Interferon yields were also unaffected by varying the serum concentration from to 1%. The optimum temperature for interferon production was examined with high-density cultures. Replicate cultures were incubated at 25, 33, 36, and 4C after induction with Sendai virus. Interferon yields at 33C were 1.5 to 2 times greater than at 36 C and 1 to 2 times greater than at 25 and 4C. Production of lymphoblastoid interferon after dilution of saturation-density cultures with serum-free medium. Saturationdensity cultures (1.5 x 11 to 3.4 x 1' cells per ml) grown in the presence of 1% FBS were induced with NDV-B1 at the concentration of 2.5 HA units per 15 cells. The culture was incubated for 1 h at 37C and stirred at 6 rpm on a Belico magnetic stirrer. The culture was then diluted with serum-free RPMI 164, supplemented with 25 mm HEPES and the antibiotics penicillin G (1 U/ml) and streptomycin sulfate (1,ug/ml), which had been warmed to 37C. After a 24- to 27-h incubation period at 37C with stirring at 6 rpm, the culture was centrifuged at 1,25 x g for 1 min at 25C. The superratant fluid was adjusted to ph 2 with concentrated HCI and allowed to stand at 5C for 24 h to inactivate the virus. Dilution of the cell culture with serum-free medium resulted in an increased production of interferon per cell (Table 2). Magimum yields of interferon were obtained with cultures diluted to 4 x 15 to 9 x 15 cells per ml. In this cell density range, interferon titers averaged 16 reference units per ml. Dilution of cultures to cell densities below 4 x 15 cells per ml yielded variable results, with interferon titers lower than 13 reference units per ml. Furthermore, it was observed that the yields of interferon produced by the diluted cultures were independent of the serum concentrations of the dilution media over the range of to 1% FBS (data not shown). Using this dilution procedure in conjunction with the methods previously described for the growth of Namalva cells in 5 to 7% FBS, it may be possible to obtain a crude interferon preparation containing only 1 to 2% FBS. Effect of glutamine on the production of TABLE 2. Production of lymphoblastoid interferon in saturation density and diluted culturesa Ex Culture volume Cell density Interferon ti- Exptb Cutr oue(cells per nml ter (log1o IF (ml) x 1-5) unilts per 15 cells) , a Cultures were diluted with serum-free medium containing glutamine 1 h after induction with NDV- B1. IF, Interferon. b Cultures were incubated at 35 to 37'C for 24 h in experiments 1-5, and for 27 h in experiments 6 and 7. Downloaded from on April 28, 218 by guest

5 48 ZOON ET AL. lymphoblastoid interferon in undiluted and diluted cultures. Table 3 shows that the production of interferon in diluted cultures is dependent on the presence of glutamine. A 6- to 18-fold decrease in interferon per 15 cells was observed when glutamine was omitted from the dilution medium. Doubling or tripling the normal glutamine concentration in the dilution medium did not further increase the yield of interferon. However, the addition of glutamine to saturation cultures to a final concentration of.27% did not alter the production of interferon. Thus, the glutamine-dependent increase in interferon yield per cell was seen only in the diluted cultures. Kinetics of interferon production. Cells resuspended at 17 cells per ml were induced with Sendai virus, and cumulative interferon production at various times from to 2 h was determined. Figure 2 shows the results of three such determinations. Although the final titers obtained varied over a fivefold range, maximum titers were generally found by 1 h postinduction. The kinetics of cumulative interferon production appeared to be essentially complete 22 h after induction with NDV in saturation-den- TABLE 3. Effect ofglutamine on the production of lymphoblastoid interferon in undiluted and diluted culturesa Inter- Cell feron titer density Exptb (cells/ Dilution medium" (logi ml x It 1-5) uniots/ cells) Undiluted Serum-free + glutamine Serum-free - glutamine Undiluted Undiluted + glutamined Serum-free + glutamine Serum-free - glutamine Undiluted Serum-free + glutamine Serum-free - glutamine 1.9 a Cultures were diluted 1 h after induction with NDV-B1. IF, Interferon. b Culture volume in experiments 1 and 2 was 1 ml. Culture volume in experiment 3 was 2 ml. In experiment 1, cultures were incubated for 24 h after induction; in experiments 2 and 3, for 27 h after induction. c RPMI 164 (without glutamine) supplemented with 25 mm HEPES and the antibiotics penicillin G and streptomycin sulfate. d L-glutamine was added to a final concentration of.27%. z LL_ HOURS POST INDUCTION FIG. 2. Kinetics of interferon production by Namalva cells. Cell cultures (1O7 cells per ml) were induced with Sendai virus (6 HA units per ml). Replicate cultures were removed at times indicated and processed for interferon assay. Three separate experiments are shown. sity cultures and 27 h after induction with NDV in cultures diluted to 4 x 15 cells per ml. Incubation of the diluted culture up to 54.5 h after induction did not result in a greater yield of interferon. The kinetics of interferon production in cultures diluted to cell densities of 1 x 15 to 3 x 15 cells per ml were also examined. Maximum cumulative yields of interferon were again 27 h postinduction. These studies suggest that the rates of interferon production after the addition of NDV-B1 or Sendai are dependent on cell numbers. The effect of actinomycin D on the production of Namalva interferon. Figure 3 shows the effect of actinomycin D, added at various times after induction with Sendai virus, on the production of lymphoblastoid interferon. The production of an actinomycin D-sensitive product, presumably messenger RNA (mrna), appears to be required during the first 4 h after interferon induction. Thereafter, actinomycin D does not inhibit the continuing synthesis of interferon. This suggests that the interferon mrna is fairly stable, and that this substance is synthesized de novo in Namalva cultures. Simila J. CLIN. MICROBIOL. results have been observed in chicken embryo and mouse L cells (21) and in Krebs 2 cells (22). Effect of NDV-B1 on the rates of RNA and protein synthesis in saturation density and diluted cultures. To investigate the increase in interferon yields in the diluted cultures, studies were initiated to examine the effects of NDV-B1 on the rates of RNA and protein synthesis in saturation density and diluted cultures. In these experiments, cultures were labeled with Downloaded from on April 28, 218 by guest

6 VOL. 7, 1978 PRODUCTION OF LYMPHOBLASTOID INTERFERON 49 radioactive precursors as described above. Rates of RNA and protein synthesis (Fig. 2) were monitored by measuring the ratio of the specific activity of the PCA precipitate, in disintegrations per minute (dpm) per optical density unit at 26 nm (OD2), to the specific activity of the PCA-soluble fraction (dpm/od2w). Examining rates of RNA and protein synthesis in this manner normalizes changes in the macromolecular synthesis resulting from changes in the transport of precursors into the cell. Figure 4A shows that NDV-B1 did not appear to affect the rate of RNA synthesis in saturationdensity cultures. However, in the diluted cultures (Fig. 4B), a 5% inhibition in the rate of RNA synthesis was observed in the presence of NDV-B1 27 h after induction. The rate of protein synthesis was inhibited by NDV-B1 in both saturation-density and diluted cultures (Fig. 4C and D). In two experiments, incubation of the cultures with virus resulted in a 75% inhibition in the rate of protein synthesis in the saturationdensity culture and an 85% inhibition in the diluted culture, as compared with their respective uninduced cultures between 22 and 27 h. Therefore, these results suggest that NDV-B1 is more effective in reducing the rates of RNA and protein synthesis in the diluted cultures than in the undiluted cultures. DISCUSSION The present studies were concemed with optimiing conditions for cell growth and human 4 -J rx. "I- 3 z D L HOUR OF ACTINOMYCIN-D ADDITION FOLLOWING INTERFERON INDUCTION FIG. 3. Inhibition of interferon production by Sendai virus-induced Namalva cultures by actinomycin D. Replicate cultures (17 cells per ml) were established and induced by Sendai virus (6 HA units per ml). At, 1, 2, 4, 7, and 2 h after induction, 1 pg of actinomycin D per ml was added to separate cultures. All cultures and the uninhibited control were harvested at 24 h postinduction and processed for interferon assay. interferon production, with the intent of extending these conditions to large-scale suspension cultures. The cells selected for study, Namalva, have been shown to produce human interferon after stimulation with Sendai virus (12). This report has extended these observations and has determined that lymphoblastoid cells are suitable for use in the large-scale production of human interferon. Starting with initial culture densities of 2.5 x 15 cells per ml, saturation densities were routinely achieved in 4 days under the conditions used in this study. The lack of an effect of priming the cells by pretreatment with human interferon was not surprising, since it has been reported that Namalva cells spontaneously produce interferon (1). However, detectable amounts of interferon were not found in crude culture supernatants in the present study. About.5 U/ml of interferon was detected after a 1-fold concentration of fluids from saturation-density cultures. The lack of a requirement for priming in this system is also an advantage in large-scale production, since none of the interferon product needs to be utilized for the priming of additional batches. The dilution procedure described offers several advantages for the large-scale production of lymphoblastoid interferon. First, the yield of interferon per cell is increased two- to fourfold. In addition, purification is simplified by increasing the specific activity of interferon in the crude preparation, thus permitting the crude material to be concentrated more effectively. Finally, production costs are decreased by reducing the quantities of FBS, virus, and cells required to prepare crude interferon. The presence of glutamine in the dilution medium is essential for the maximum yield of interferon per cell using the described method. Studies using mouse LS cells (5) have shown that glutamine is generally the first amino acid exhausted from the medium not only due to its central role in metabolic processes but also because of its instability in the culture medium. Although glutamine does appear to be necessary for the production of interferon, depletion of glutamine in the saturation-density culture is not the factor responsible for lower yields. It may be that, in diluting the culture, we are also reducing the concentration of an inhibitor or toxic substance that reduces interferon production. The inhibition of interferon production in cultures treated with actinomycin D implies that de novo mrna transcription is necessary for Namalva cell interferon production. The cellfree synthesis of interferon utilizing mrna extracted from Sendai-induced Namalva cells has been observed (S. Peska, personal communication; P. Pitha, personal communication). Downloaded from on April 28, 218 by guest

7 5 ZOON ET AL. J. CLIN. MICROBIOL. E Cl, &.4 u 2 A.25 3.o Saturation Density - RNA -i A +NDV - -NV -NDV - II Saturation Density - Protein- - -NDV~ X ~ o +NDV I _ Diluted Cells - RNA I Diluted Cells - Protein o-- -o '4,~~~~ \ NDV n B +NDV - _ ;-NDV C D HOURS FIG. 4. Effect of NDV-BI on the rates of RNA and protein synthesis in undiluted and diluted cultures. Saturation-density cultures (16.5 x 1' cells per ml) were induced iwth NDV-Bl as described. The cells were diluted with serum-free medium to 4.1 x 1' cells per ml. Samples of culture (1 ml) were removed at the time points indicated and were radioactively labeled as described in the text. (A) Saturation density culture. RNA, zero time rate of incorporation into PCA precipitate: uninduced, 79,954 dpm/od2w; induced, 78,96 dpm/od2,. RNA, zero time rate of incorporation into the PCA-soluble fraction: uninduced, 1,227,126 dpm/od2wp,e; induced, 1,14,42 dpm/od2we. (B) Diluted culture. RNA, zero time rate of incorporation into PCA precipitate: uninduced, 26,86 dpm/od2wo; induced, 28,295 dpm/od26o. RNA, zero time rate of incorporation into PCA-soluble fraction: uninduced, 832,311 dpm/od26; induced, 929,16 dpm/od26o. (C) Saturation density culture. Protein, zer.o time rate of incorporation into PCA precipitate: uninduced, 1,725 dpm/od2,; induced, 9,86 dpm/od26o. Protein, zero time rate of in corporation into PCA-soluble fraction: uninduced, 122,67 dpm/od26; induced, 117,75 dpm/od26. (D) Diluted culture. Protein, zero time rate of incorporation into PCA precipitate: uninduced, 1,611 dpm/od26o; induced, 1,594 dpm/od26o. Protein, zero time rate of incorporation into PCA-soluble fraction: uninduced, 137,576 dpm/od26o; induced, 13,763 dpm/od2eo. +NDV I I- Cl wc,, -4 ae 9 C.) a: 3 1- Downloaded from The cessation of interferon production about 12 h postinduction, when culture densities of 17 cells per ml were used, may be related to an inhibition of cell macromolecular synthesis by Sendai virus. Some inhibition of the rates of RNA and protein synthesis has been observed in saturation-density and diluted cultures induced with NDV. In addition, the rates of interferon production in these lower cell-density Namalva cultures are slower than those reported for other cell types (6, 2). Thus, the prolongation of the rate of interferon production (in these lower cell-density cultures) may be accompanied by a delay in appearance of the regulatory mechanisms that are believed to terminate interferon synthesis (13, 18-2). Based on the model proposed by Tomkins (14, 15), it has been suggested that initiation of repressor mrna transcription and repressor protein synthesis occurs shortly after the de novo synthesis of the interferon mrna. In the current studies, NDV may function in a manner similar to metabolic inhibitors that enhance interferon production by selectively suppressing repressor formation. Thus, the greater inhibition of protein and RNA synthesis in diluted Namalva cultures may include a more efficient inhibition of repressor fornation. Production of milligram quantities of human lymphoblastoid interferon seems from these studies to be a possibility. Adjustment of cultural and induction conditions has not, however, produced a 1- to 2-fold increase in the amount of interferon per cell over that reported by Strander et al. (12). The effort required to produce 19 to 11 units of interferon per batch is still formidable, but the engineering technology for the very large scale production of Namalva cells and the induction of human interferon is being developed. ACKNOWLEDGMENTS We thank David L. Rogerson, Jr., Douglas L. Johnson, and Henry Powell for their expert technical assistance. We are grateful to Sikta Bose, Urs Th. Ruegg, Lila Corley, Christian B. Anfinsen, Jon A. Green, and Hilton B. Levy for their helpful discussions and assistance during this study. LlTERATURE CITED 1. Adams, A., B. Lidin, IL Strander, and K. Canteli Spontaneous interferon production and Epstein- Barr virus antigen expression in human lymphoid cell lines. J. Gen. Virol. 28: Andersson, M Amounts of Epstein-Barr virus on April 28, 218 by guest

8 VOL. 7, 1978 PRODUCTION OF LYMPHOBLASTOID INTERFERON 51 DNA in somatic cell hybrids between Burkitt lymphoma-derived cell lines. J. Virol. 16, Anfinsen, C. B., S. Bose, L Corley, and D. Gurari- Rotman Partial purification of human interferon by affinity chromatography. Proc. Natl. Acad. Sci. U.S.A. 71: Armstrong, J. A Semi-micro, dye-binding assay for rabbit interferon. Appl. Microbiol. 21: Griffiths, J. B., and S. J. Pirt The uptake of amino acids by mouse cells (strain LS) during growth in batch culture and chemostat culture: the influence of cell growth rate. Proc. R. Soc. London Ser. B. 168: Havell, E. A., and J. Vilcek Production of hightitered interferon in cultures of human diploid cells. Antimicrob. Agents Chemother. 2: Klein, G., L Dombos, and B. Gothoskar Sensitivity of Epstein-Barr virus (EBV) producer and nonproducer human lymphoblastoid cell lines to supennfection with EB-virus. Int. J. Cancer 1: Knight, E. J Interferon: purification and initial characterization from human diploid cells. Proc. Natl. Acad. Sci. U.S.A. 73: Levy, IL B., G. Baer, S. Baron, C. E. Buckler, G. J. Gibbs, M. J. Iadarola, W. T. London, and J. Rice A modified polyriboinosinic-polyribocytidylic acid complex that induces interferon in primates. J. Infect. Dis. 132: Ng, ML H., and J. Vilcek Interferons: physiochemical properties and control of cellular synthesis. Adv. Protein Chem. 26: Pritchett, IL, ML Pedersen, and E. Keiff Complexity of EBV homologous DNA in continuous lymphoblastoid cell lines. Virology 74: Strander, H., K. E. Mogensen, and K. Cantell Production of human lymphoblastoid interferon. J. Clin. Microbiol. 1: Tan, Y. H., J. A. Armstrong, Y. H. Ke, and M. Ho Regulation of cellular interferon production: enhancement by antimetabolites. Proc. Natl. Acad. Sci. U.S.A. 67: Tomkins, G. M., T. D. Gelehrter, D. Granner, D. Martin, Jr., H. H. Samuels, and E. B. Thompson Control of specific gene expression in higher organisms. Science 166: Tomkins, G. M., E. B. Thompson, S. Hayashi, T. Gelehrter, D. Granner, and B. Peterkofaky Tyrosine transaminase induction in mammalian cells in tissue culture. Cold Spring Harbor Symp. Quant. Biol. 31: Torma, E. T., and K. Pauker Purification and characterization of human leukocyte interferon components. J. Biol. Chem. 251: Valle, M. J., G. W. Jordan, S. Haahr, and T. C. Merigan Characteristics of immune interferon produced by human lymphocyte culture compared to other human interferons. J. Immun. 115: Vilcek, J Metabolic determinants of the induction of interferon by a synthetic double-stranded polynucleotide in rabbit kidney cells. Ann. N. Y. Acad. Sci. 173: Vil&ek, J., and M. H Ng Post-transcriptional control of interferon synthesis. J. Virol. 7: Vilcek, J., T. G. Roonsan, and F. Varacalli Differential effects of actinomycin D and puromycin on the release of interferon induced by double-stranded RNA. Nature (London) 222: Wagner, R. R Inhibition of interferon biosynthesis by actinomycin D. Nature (London) 24: Wagner, R., and A. S. Huang Inhibition of RNA and interferon synthesis in Krebs-2 cells infected with vesicular stomatitis virus. Virology 28:1-1. Downloaded from on April 28, 218 by guest