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The gene coding for bovine interferon-omega1 (BoIFN-omega1) was recently cloned and expressed at high levels in the yeast Pichia pastoris. The recombinant BoIFN-omega1 protein shows antiviral activity in different cell lines and has an antiluteolytic effect in cyclic ewes. In this article, we describe a method for purification of BoIFN-omega1 expressed in the methylotrophic yeast P. pastoris and characterization of its activity in vivo. The recombinant protein secreted to the culture medium had low activity because of self-aggregation. BoIFN-omega1 was solubilized using urea and desalting and finally purified by ion exchange chromatography on Q-Sepharose Fast Flow. The yield of purified product was approximately 300 mg/L of fermentation culture, with a specific antiviral activity of 10(8) IU/mg. Its purity was at least 80%. The biologic characterization of purified BoIFN-omega1 was determined by induction of an antiviral state on ewes challenged with 100 lethal doses (LD) of Aujeszky virus and by the extension of the corpus luteum life span and interestrous interval in cyclic cows. Ewes treated with 2 x 106 IU/kg BoIFN-omega1 were protected from Aujeszky virus infection. In cows receiving an intrauterine infusion of 1 mg BoIFN-omega1, equally distributed between the two uterine horns, twice daily from day 14 to day 22 of the experimental estrous cycle, the lifespan of the corpus luteum (25 vs. 19 days) and the interestrous intervals (26 vs. 21 days) were extended when compared with a control group (p < 0.05). We show that recombinant BoIFN-omega1 purified from P. pastoris has high antiviral activity and is an effective antiluteolytic agent in cattle.

Interferon-tau (IFN-tau) is a novel type I IFN that was originally identified as a pregnancy recognition hormone. IFN-tau shares all of the biological properties of other type I IFNs including antiviral activity and antiproliferative activity through induction of the cell cycle inhibitor gene product p21WAF1. It is a promising therapy for cancers, viral infections, and for autoimmune disorders such as multiple sclerosis, without the adverse side effects associated with IFN-alpha and IFN-beta. Here, we describe novel growth and induction conditions for the expression of functionally active and uniformly 15N-labeled IFN-tau from Pichia pastoris in a minimal media for use in initial 2D- and 3D-NMR studies in solution. Purified 15N-IFN-tau was homogenous, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and MALDI-TOF mass spectrometer (MS), and retained full biological activity. MS analysis confirmed uniform isotopic labeling of IFN-tau with 15N incorporation exceeding 99%. Circular dichroism (CD) as well as 1D-NMR and 15N-1H heteronuclear single quantum coherence (HSQC) spectra confirmed that purified 15N-labeled IFN-tau has a stable secondary structure. Besides providing a route for isotope labeling of IFN-tau, our procedure may be useful for the expression and purification of other proteins that are difficult to obtain in Pichia pastoris grown in minimal media.

The early conceptus (embryo and associated membranes) of domestic ruminats signals its presence to the maternal uterus through production of interferon-tau (IFN-tau). Production of IFN-tau ensures continued production of progesterone, the hormone of pregnancy, by the ovarian corpus luteum. This paper reports the high-level expression and efficient secretion of biologically active recombinant ovine IFN-tau (rOvIFN-tau) by Pichia pastoris. The developed method produces more than 80% pure recombinant ovine IFN-tau, obviating the need for further purification for many purposes. Initial fermentation studies produced IFN-tau at 280 mg/liter and demonstrate the potential of this system for large-scale production of IFN-tau.

Ovine trophoblast protein-1 (oTP-1) is a unique, Type I, trophoblast interferon (IFN) that possesses potent antiviral activity and is thought to be primarily responsible for maternal recognition of pregnancy in sheep. To provide sufficient amounts of protein for detailed studies, a synthetic gene for oTP-1 was designed and assembled in Escherichia coli, subcloned into a yeast expression plasmid, and used to overproduce recombinant oTP-1 in Saccharomyces cerevisiae. Recombinant oTP-1 was purified from soluble yeast extract using sequential ion-exchange and molecular sieve chromatography. Recombinant oTP-1 purified in this fashion exhibited potent antiviral activity (0.6 x 10(8) U/mg) similar to native oTP-1. This expression system will enable production of large quantities of soluble, biologically active, and correctly processed recombinant oTP-1. Furthermore, the synthetic gene construct facilitates introduction of mutations for ongoing structure/function studies of this unique, Type I, trophoblast IFN.

Equine interferon-beta 1 (EqIFN-beta 1) was purified from extracts of recombinant Escherichia coli by sequential chromatography on hydroxylapatite, anion-, and cation-exchangers. The resulting protein was greater than 98% pure as determined by sodium dodecylsulfate gel electrophoresis, gel permeation HPLC, and reverse-phase HPLC. Amino-terminal amino acid sequencing revealed that essentially all molecules contained an additional amino-terminal methionine. The specific antiviral activity of EqIFN-beta 1 determined on equine dermal fibroblasts challenged with vesicular stomatitis virus (VSV) was approximately 5 X 10(8) U/mg. Less than 0.001% of this activity was observed in antiviral assays using human (A549), murine (L-M), ovine (SCP), or bovine (MDBK and BT) cells challenged with VSV or encephalomyocarditis virus. A series of monoclonal murine IgG antibodies were developed which neutralize the antiviral activity of EqIFN-beta 1. None of these antibodies nor rabbit antiserum to EqIFN-beta 1 were able to neutralize human IFN-beta; antiserum to human IFN-beta did not neutralize EqIFN-beta 1. Two of the monoclonal antibodies were used to establish a rapid one-step solid-phase enzyme immunoassay for EqIFN-beta 1.

The use of a cascade filtration system for production and recovery of human leukocyte-derived alpha interferon (HuIFN-alpha) has been investigated. Included in the investigation were mass transfer studies of HuIFN-alpha across ultrafiltration membranes, design and evaluation of a laboratory-scale production system and determination of IFN production kinetics in both batch and filtration systems. The cascade filtration system allows separation of interferon from leukocytes and virus during production with simultaneous concentration and complete recovery. Kinetic studies indicate that HuIFN-alpha production can be enhanced by appropriate timing of the filtration procedure. While the current study is concerned with HuIFN-alpha, it is expected that the techniques developed will be applicable to the production and recovery of both natural and recombinant IFNs and lymphokines in general.

We report a simple procedure for the isolation of a mouse interferon beta (beta IFN) from Ehrlich ascites tumor cells infected with Newcastle disease virus. The procedure consists of four chromatographic steps (two on controlled pore glass, one on CM-Sephadex, and one on phosphocellulose) with stepwise elution. The product purified is a 35-40 kD beta IFN with a specific activity of 10(9) units/mg protein.

Human leukocyte-derived interferon (HuIFN-alpha) can be displaced from controlled-pore glass (CPG) with alkylamines at 0.1-0.3 M concentration. The eluting power of an alkylamine correlates positively with the extent of its alkylation: CH3NH3 + less than (CH3)2NH2 + less than (CH3)3NH+ less than (CH3)4N+. Interferon can also be recovered using an electrolyte and ethylene glycol as cosolvents. Apparently, both hydrophobic and electrostatic forces are involved in the adsorption of HuIFN-alpha to CPG.

Recombinant mouse interferon-beta (rMuIFN-beta) produced in Escherichia coli was purified to homogeneity and characterized. The purified protein exhibited a single band of Mr 19,900 on SDS-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions, and also exhibited a single band on native polyacrylamide gel electrophoresis at pH 4.3. The observed molecular weight corresponded to that of the polypeptide moiety of natural MuIFN-beta of Mr 19,700. The amino acid composition and the amino-terminal sequence of the purified rMuIFN-beta were identical to those predicted from cDNA sequence. These results indicate that the purified protein is a nonglycosylated MuIFN-beta, which forms no disulfide-linked dimer and probably exists as a monomeric form.

The pharmacokinetic behavior of recombinant (r) HuIFN-beta was compared with that of fibroblast-derived HuIFN-beta in rabbits using a pharmacokinetic model analysis with a computer curve-fitting. The observed data were in good agreement with a two-compartment open model in intravenous experiment, and with a one-compartment open model in intramuscular and continuous infusion experiments. Although the parameters of elimination phase (beta, Kel or K10) were almost the same in both IFNs, there were significant differences for the parameters (alpha, K12, K21, V1, V2, Vdss) of distribution phase in intravenous experiment and for the parameter (Vd) of the steady-state level in continuous infusion experiment. These results indicate that rHuIFN-beta has a larger distribution volume and a higher distribution rate from the central compartment to the peripheral. The serum levels in intramuscular experiment represented so-called flip-flop type of kinetics. Although the absorption rate from the injection site to the blood circulation was similar in both IFNs, the availability of rHuIFN-beta was higher than that of fibroblast-derived HuIFN-beta. These results suggest that the deficiency of carbohydrate moiety conjugated with IFN molecule may influence the distribution property in vivo.

A procedure for isolating HuIFN-beta without the use of SDS or other denaturants has been developed. The procedure involves the chromatography of IFN on an antibody affinity column followed by reversed-phase HPLC. The specific activity of the resulting IFN-beta is 4 X 10(8) u/mg protein, and overall recovery is about 76%. The procedure provides a method for isolating pure HuIFN-beta in its native form with an efficiency much higher than any other method reported to date.

A new type of interferon (IFN)-alpha cDNA (IFN-alpha I') was identified in a cDNA library constructed from Namalva cells infected with Sendai virus. The nucleotide sequence of this cDNA showed homology, with the exception of two nucleotides in the coding region, with the previously identified IFN-alpha I gene (Lawn et al., 1981). An expression plasmid which directs the synthesis of the mature IFN-alpha I' peptide was constructed using vectors carrying the lpp/lac promoter and "runaway" replicon. Furthermore, hybrid genes were constructed by in vitro recombination of IFN-alpha I' and IFN-alpha A at a common restriction endonuclease site located at amino acid positions 121-122. While the specific antiviral and anticellular activities of IFN alpha A and IFN-alpha I' on human cells were comparable, the antiviral activity of IFN-alpha I' on mouse cells was 125-fold higher than that of IFN-alpha A. The specific antiviral activities of the hybrid IFNs on human and bovine cells were similar to that of the amino-terminal parental IFN peptide, while the anticellular activities on human cells of the alpha A/alpha I' hybrid were higher and that of the alpha I'/alpha A hybrid were lower than the parental IFN-alpha A and IFN-alpha I'.

B-cell human lymphoblastoid cell line, BALL-1, produced interferon-alpha (IFN-alpha) and cytotoxic factors by the stimulation of hemagglutinating virus of Japan (HVJ). These cytotoxic factors were identified to be tumor necrosis factor-alpha (TNF-alpha) and tumor necrosis factor-beta (TNF-beta) on the basis of molecular level analysis. In the kinetic study, it was found that the maximum production of IFN-alpha was reached at 16-20 hr and that of TNF-alpha was at 10-16 hr after the stimulation by HVJ, whereas the maximum production of TNF-beta was at 40-50 hr, suggesting that the regulation of TNF-alpha and TNF-beta genes are different. These cytokines were purified by monoclonal antibody linked column chromatography, respectively. IFN-alpha was further separated into 9 subspecies on the basis of the difference of isoelectric point by FPLC chromatofocusing method. From the N-terminal amino acid sequence analyses of the 7 species of 9 subspecies, these subspecies were classified into 3 subtypes according to the Weissmann's notation. On the other hand, the amino acid sequence of the tryptic peptides of purified TNF-alpha and TNF-beta coincided well with the parts of the complete amino acid sequences which had been deduced from complementary DNA of TNF-alpha and TNF-beta of BALL-1 cell.

Mouse interferon (MuIFN) preparations containing both the alpha and beta species were treated with chemical modifying reagents specific for lysine, cysteine and tryptophan residues. The purpose was to ascertain whether reagents which modify specific amino acid residues distinguished MuIFN-alpha from MuIFN-beta, and if biologic properties could be correlated with specific amino acid residues. MuIFN species were characterized by their activity on homologous and heterologous cells and by antibody neutralization. Modification of lysine residues with increasing concentrations of fluorescamine resulted in a concomitant loss of biologic and antigenic activities of both MuIFN species. Modification of cysteine residues with 5,5'-dithio-bis (2-nitrobenzoic acid) showed an enhancement of antiviral activity of MuIFN-alpha but a gradual decrease in all activities of MuIFN-beta. Modification of tryptophan residues with 2-methoxy-5-nitrobenzyl bromide enhanced somewhat MuIFN-alpha activity, but resulted in the loss of MuIFN-beta activity. These results show that amino-modifying reagents rendered both major species of MuIFN inactive; however, they can be distinguished by reagents which affect cysteine and tryptophan residues. The fact that biologic activity is accompanied by the loss of antigenicity implies that the biologic and antigenic sites may be the same or closely associated. Chemical modification of interferons may provide a useful approach for relating the properties of interferons to their primary structure.

Seven stable mouse hybridomas secreting monoclonal antibodies to human fibroblast beta interferon (IFN-beta) were isolated, all seven of which belonged to IgGl subclass and kappa type. While neutralizing the antiviral activity of human fibroblast IFN-beta, they failed to neutralized both that of human IFN-alpha and human IFN-gamma. These monoclonal antibodies neutralized the antiviral activity of human fibroblast IFN-beta but not that of human IFN-alpha and IFN-gamma. Two of the seven monoclonal antibodies, YSB-1 and YSB-2, showed particularly high neutralization titers in the ascitic fluid. Monoclonal antibodies were purified from cultures of hybridoma grown in a serum-free medium. The purified monoclonal antibodies, YSB-1(5 micrograms/ml) and YSB-2(1 microgram/ml), neutralized IFN-beta from 10EU/ml to 1EU/ml. Human fibroblast IFN-beta was purified to 3.5 X 10(7) IU/mg protein (1.0 X 10(8) IU/mg protein in the peak fraction) by the monoclonal antibody (YSB-2) affinity column chromatography, whereas human recombinant IFN-beta obtained from E.coli was also purified to 6.5 X 10(7) IU/mg protein (1.1 X 10(8) IU/mg protein in the peak fraction) by the monoclonal antibody (YSB-1) affinity column chromatography.

Antigen presentation by macrophages is accompanied by the production of interleukin-1 (IL-1) for successful T cell triggering. We have described EBV infected B cell lines that served as antigen presenting cells (APC) and now report the ability of these cells and their supernates, as well as EBV negative B cell lines, to support IL-2 production by a T cell line JM in the presence of the monoclonal antibody that recognizes the CD3 complex. Both EBV-transformed B cell lines and EBV-negative lines, and their supernates, exhibited IL-1-like activity in two different IL-1 dependent assays. These IL-1-like molecules were released constitutively from five of six EBV positive lines and three of five non-infected B cell lines. The activity from both, one virally infected and one non-infected B cell line, eluted from Sephadex G-75 in two peaks at 15-18K and 30-35K and could not be neutralized by antibody specific for macrophage IL-1. Supernates having IL-1-like molecules also contained a higher molecular weight inhibitor of proliferation. These data indicate that: both EBV-positive and EBV-negative B cell lines are capable of elaborating the IL-1-like activity; and the IL-1-like activity from these cells can be serologically distinct from macrophage IL-1. This suggests the presence of a family of molecules with IL-1 activity that derive from different cell types.

A monoclonal antibody against murine interferon-beta (MuIFN-beta) was prepared using standard methods. Antibodies were immobilized by coupling to Sepharose and used for large-scale purification of poly(I) . poly(C)-induced mouse L cell IFN. Antibodies isolated from the serum of one nude mouse which was transplanted with the anti-MuIFN-beta antibodies producing hybridoma were able to bind at least 7 X 10(7)U MuIFN-beta. In one single antibody affinity chromatography step MuIFN-alpha was separated from MuIFN-beta and a 1000-fold purification of MuIFN-beta was obtained. The purified material had a specific activity of 5 X 10(8)U/mg protein. The recovery from the antibody column was 100%. SDS-PAGE analysis of the purified material revealed the presence of one single protein band with a molecular weight of 33 kD, representing MuIFN-beta.

A multitray culture system was devised for cell culture and interferon (IFN) production on a large scale. The system consisted of a cell culture apparatus which has stacked multitrays and a reservoir. All components of the system were made of nontoxic, nonmutagenic, washable, and autoclavable materials, so that the system can be repetitively used in suitable condition for cultivations of anchorage-dependent cells with low running cost. Large quantities of mouse IFN (containing both MuIFN-alpha and MuIFN-beta) from mouse L cells and human beta interferon (HuIFN-beta) from human fibroblast cells, respectively, were obtained very efficiently using this system in comparison with roller bottle, bulk culture vessel, and microcarrier systems.

Rat fibroblast interferon (RfIFN), produced in serum-free media, has been purified 2,000-fold to 88% purity by a simple method that involves only two chromatographic steps: controlled pore glass and phenyl agarose chromatography. Over 70% of the initial activity was recovered. Gel electrophoresis (SDS-PAGE) and silver staining of the purified RfIFN revealed two major bands of RfIFN activity corresponding to Mr of 26,000 and 22,500 daltons, respectively. A very small amount of RfIFN was also recovered from a region of the gel corresponding to a Mr of 18,500. The average specific activity of the purified material was 3.7 X 10(8) U/mg. From these results it can be calculated that the specific activity of homogenous RfIFN is 4.0 X 10(8) U/mg.

A preparation of recombinant human leukocyte interferon A (rIFN-alpha A) obtained from Escherichia coli cells was found by reverse-phase high-performance liquid chromatography (HPLC) to contain three species. The three species, named Mf-1, Mf-2, and Ms in the order of their elution, were separated and characterized to elucidate their structural differences. The amino acid compositions, the amino-terminal amino acid sequences (Cys1-Asp2-Leu3-), and the carboxy-terminal amino acids (Glu) of the three species agreed with those predicted from the cDNA sequence, although the sequence analysis yields of Mf-2 and Ms were extremely low. They were found to have the same disulfide bonds, Cys1-Cys98 and Cys29-Cys138, by amino acid analysis of the tryptic peptides, but the recovery of the peptides linked by a Cys1-Cys98 disulfide bond for Mf-2 and Ms was extremely low. The results demonstrate that although the primary structures of the three species are almost identical, small difference(s) exists among them especially at the amino-terminal portion.

The present study describes the development of a murine monoclonal antibody (LO-22) directed against 12 native species of HuIFN-alpha. Mice were immunized with purified HuIFN-alpha preparations containing all native species of HuIFN-alpha and spleen cells from immunized mice were fused with P3X63MS1 mouse myeloma cells. Positive hybridoma clones secreting antibodies to HuIFN-alpha were identified by means of a new, extremely sensitive biological semisolid binding assay which was able to disclose positive hybridoma clones consisting of less than 30 hybridoma cells. Antibody affinity columns, made by the LO-22 IgG and monospecific, but polyclonal rabbit IgG, showed identical binding abilities as demonstrated by comparative affinity chromatographies in that the same number of IFN species (12 in total) were found in eluates from parallel experiments. Since LO-22 IgG also recognizes porcine leucocyte interferon, it is suggested that the LO-22 is directed against a common epitope which has been very well conserved in the HuIFN-alpha system, per se. It is suggested that the LO-22 IgG can be used for purification of all HuIFN-alpha proteins--be they recombinantly derived or native--but exerting the common determinant. Preliminary experiments have shown that the LO-22 IgG is highly suitable for ELISA tests.

Interferon (IFN) production induced by either Newcastle disease virus (NDV) or Sendai virus was compared in 10 different rat cell lines. Although there was variation in the IFN titers produced, NDV proved to be the best inducer in each cell line with optimum IFN yields occurring with a multiplicity of infection (MOI) of 1.0. A new continuous rat fibroblast cell line (CD) produced high titers of IFN similar to those reported for other high producers; while Ratec cells were shown to be the most sensitive to the antiviral activity of rat IFN. Partial purification and characterization of IFN produced in CD cells was accomplished by column chromatography. Four sorbents with varying modes of action for binding the IFN (Affi-Gel 202, Poly(U)-Sepharose 4B, CM-Sepharose CL-6B, and Phenyl-Sepharose CL-4B) were compared. The Phenyl-Sepharose CL-4B matrix proved to be the most successful for purification of rat IFN; one passage through this column, increased the specific activity more than 100-fold, with a concomitant recovery of 90%-95% biologic activity. The binding characteristics of rat IFN on each of the column matrices, however, demonstrated differences between the physicochemical nature of CD rat IFN and murine and hamster IFNs.

Physician-administered and self-administered interferon (IFN) nasal sprays were compared by measuring residual IFN recovered from the nose at intervals following single doses. Amounts of IFN recovered five minutes after administration and the decay curves of recoverable antiviral and immunoreactive IFN with time were variable between volunteers but similar for both methods. It follows that a self-administered IFN spray would be suitable for future trials of IFN in upper respiratory infections. It was also confirmed that IFN persists in the nose for at least 24 h and is not all recovered by nasal washing, suggesting that it may be bound to, then slowly released from, nasal mucosal cells.

The synthetic polyribonucleotide polyinosinate:polycytidylate [poly(I):poly(C)] was used to induce interferon (IFN) production in a rat leiomyosarcoma cell line. Three separate IFN species were distinguished by antiviral activity on homologous and heterologous cells, interaction with Blue Sepharose CL-6B chromatography matrix, and antibody neutralization studies. Two IFN species, one with a weakly ionic and the other with a hydrophobic interaction with Blue Sepharose, exhibited heterologous antiviral activity, and were acid- and heat-labile. Both had a molecular weight of 30,000 daltons by HPLC-based molecular exclusion chromatography. The weakly ionic species was neutralized only by antiserum to L-cell IFN, whereas the hydrophobic species was neutralized by antiserum to L-cell IFN and antiserum to human leukocyte IFN. The remaining IFN species, with a weakly ionic interaction with Blue Sepharose, was acid- and heat-stable, exhibited no heterologous cell antiviral activity, and was not neutralized by any antisera to IFN. This species had a molecular weight of 39,000 daltons by HPLC-based molecular exclusion chromatography and 34,000 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These rat fibroblast IFNs possess greater heterogeneity than that reported or observed for murine or human fibroblast IFNs. These IFNs could thus be classified as follows: one species of rat IFN-beta and two different species of unique acid-labile rat IFNs.