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One component of human lymphoblastoid interferon obtained from Namalwa cultures induced by Newcastle disease virus has been purified to a specific activity of 2.5 x 10(8) interferon units per mg of protein (protein content based on amino acid analysis). A single polypeptide species with an apparent molecular weight of 18,500 comigrating with the antiviral activity was observed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Preliminary amino-terminal sequencing results support the conclusion that the interferon species is essentially homogeneous.

Significant advances have been made in the last four years on the purification of interferons. Components of several human and mouse interferons have been purified to homogeneity. Partial characterization of these molecules has been achieved (i.e. amino acid composition, analytical maps of radiolabelled tryptic peptides, and amino terminal amino acid sequence). In addition, preliminary evidence has been accumulated which implies that the carbohydrate portion of the molecules may play a significant role in species specificity of interferons. At present, with minute quantities of homogenous interferons available, limited structural information has been obtained. Using current production and purification schemes, sufficient material can be obtained for further amino acid sequence studies and examination of the carbohydrate portion of the molecules. Additional structural characterization of the interferon gene(s). Furthermore, as sequence homologies have been observed between human and mouse interferons, all new structural information will help to establish the interrelationships in the family of interferon proteins.

The human lymphoblastoid cell line, Namalwa, can be cultured in serum-free media to cell densities of 3 to 4 x 10(6) cells per ml. These cultures produce up to 10 000 units of interferon per ml when induced with Newcastle disease virus, strain B1. Maximum accumulation of interferon was obtained at approx. 13 h post-induction.

A stable and predictable production system is described for pilot plant quantities (milligram) of human lymphoid interferon, using suspension culture of an African Burkitt's lymphoma derived cell line Namalva with induction by Newcastle disease virus, B-1 strain. Cell cultures were grown in impeller-driven 50-liter fermentors with dilution of the postinduction culture using serum-free medium. High levels of dissolved oxygen were necessary for optimum cell growth. A total of 4,207 liters of interferon culture was produced in a series of 116 fermentor runs. An average yield of 3.5 log(10) international units of interferon per ml was realized before processing. Trichloroacetic acid was used to precipitate the interferon. An average of 3.35 log(10) international units of interferon per ml was recovered in the final nonpurified product.

Homogeneous human lymphoblastoid interferon with an apparent molecular size of 18,500 daltons was characterized by its amino acid composition. Analysis of the amino terminal sequence by Edman degradation indicates that the sequence is unique.

The methodology for optimum Namalva cell growth and interferon production has been developed. Maximum yields of interferon were obtained following dilution of a saturation density suspension culture induced with NDV.

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 serum-free 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 10(5) to 9 X 10(5) 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.

Human lymphoblastoid interferon was produced on an 800-liter scale (2.6 X 10(9) units) by induction of Namalva cells with Newcastle disease virus, strain B1. The interferon was partially purified by anti-leukocyte interferon affinity chromatography, sulfopropyl Sephadex ion exchange chromatography, isoelectric focusing, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Recovery of interferon after gel electrophoresis varied from 11 to 33% based on the original crude material, with about 35,000-fold purification. The gel electrophoresis resolved the antiviral activity into two components with apparent molecular weights of 18,000 and 22,000; treatment with glycosidases resulted in all the activity being associated with the lower molecular weight species. Interferon activity could be completely (85 to 113%) recovered from the gels by elution into a buffer containing sodium dodecyl sulfate. The presence of sodium dodecyl sulfate did not appear to affect the assay of interferon. The protein could also be completely (75 to 106%) eluted from gels stained with coomassie blue, again with no loss in activity.

Other relevant areas of research regarding the structure and function of IFNs include the study of IFN fragments and hybrid IFN molecules prepared by recombinant DNA technology, and monoclonal antibodies against IFNs have also made major contributions to our knowledge of IFN structure and function. However, these topics have been addressed in a number of recent reviews (Zoon and Wetzel, 1984; Langer and Pestka, 1985). We are now awaiting the crystal structure of several human IFNs. Once this knowledge has been acquired, we can proceed to envisage at the three-dimensional level how these molecules interact with their receptors to produce their numerous biological activities.

Study Objective: To evaluate the toxicity and potential clinical efficacy of combined therapy with zidovudine and interferon-alpha for patients with Kaposi sarcoma and the acquired immunodeficiency syndrome (AIDS).Design: Nonrandomized, open trial study.Setting: Outpatient clinic of a government referral-based research hospital.Patients: Volunteer sample of 39 patients with human immunodeficiency virus (HIV) infection and Kaposi sarcoma.Interventions: Patients received zidovudine, 250, 100, or 50 mg orally every 4 hours; 6 weeks after interferon-alpha was begun at a dose of 5 million U/d, and the dose was increased every 2 weeks until a maximum tolerated dose was determined. Patients then received the maximum tolerated dose of the combination for a minimum of 12 weeks before formal efficacy evaluations.Measurements and Main Results: In the dose-escalation phase, the ability to tolerate interferon-alpha was clearly related to the zidovudine dose. Of the 13 patients receiving 250 mg of zidovudine, only 1 patient was able to tolerate at least 10 million U/d of interferon-alpha. Of the 12 patients receiving 100 mg of zidovudine, 8 tolerated 10 million U/d, 5 tolerated 15 million U/d, and none tolerated higher doses. Of the 12 patients receiving 50 mg of zidovudine, 8 tolerated 10 million U/d, 7 tolerated 15 million U/d, and 6 tolerated 20 million U/d or more. Dose-limiting toxicities included neutropenia (57%), fatigue (16%), thrombocytopenia (14%), and hepatic dysfunction (10%). Of the 22 patients who received a stable dose of both drugs for 12 weeks, 11 patients had a complete or partial tumor response and 8 showed an anti-HIV effect. Peak serum levels of interferon-alpha (32 to 250 U/mL) and zidovudine (0.40 to 3.85 microM) were in the ranges previously shown to be synergistic against HIV.Conclusions: Combination therapy with zidovudine and interferon-alpha can be administered to patients with HIV infection and Kaposi sarcoma in doses that effect antiviral and antitumor responses; it appears to have a potential role in managing such patients. [ABSTRACT FROM AUTHOR]

A novel assay was developed for Daudi cells in which the antiviral (AV) and antiproliferative (AP) activities of interferon (IFN) can be measured simultaneously. Using this novel assay, conditions allowing IFN AV protection but no growth inhibition were identified and selected. Daudi cells were treated under these conditions, and gene expression microarray analyses were performed. The results of the analysis identified 25 genes associated with IFN-α AV activity. Upregulation of 23 IFN-induced genes was confirmed by using reverse transcription-PCR. Of 25 gene products, 17 were detected by Western blotting at 24 h. Of the 25 genes, 10 have not been previously linked to AV activity of IFN-α. The most upregulated gene was IFIT3 (for IFN-induced protein with tetratricopeptide repeats 3). The results from antibody neutralizing experiments suggested an association of the identified genes with IFN-α AV activity. This association was strengthened by results from IFIT3-small interfering RNA transfection experiments showing decreased expression of IFIT3 and a reduction in the AV activity induced by IFN-α. Overexpression of IFIT3 resulted in a decrease of virus titer. Transcription of AV genes after the treatment of cells with higher concentrations of IFN having an AP effect on Daudi cells suggested pleiotropic functions of identified gene products. [ABSTRACT FROM AUTHOR]