WO1989009065A1

WO1989009065A1 - Large-scale production of bovine leukocyte interferon

Info

Publication number: WO1989009065A1
Application number: PCT/US1989/001159
Authority: WO
Inventors: Karen L. Jacobsen
Original assignee: University Of Georgia Research Foundation, Inc.
Priority date: 1988-03-23
Filing date: 1989-03-21
Publication date: 1989-10-05
Also published as: AU3365989A

Abstract

A large scale method of preparation of natural bovine leukocyte interferon, a potent anti-viral agent effective, having a molecular weight by SDS gel electrophoresis under reducing conditions of approximately 19,000 daltons and anti-viral activity that is stable to pH 2.0. The purified bovine interferon has immunologic activity and antiviral activity against pathogenic bovine viruses. Calves receiving this bovine interferon had few of the side effects usually seen in human patients undergoing interferon therapy, with less diarrhea and more weight gain than controls. Monoclonal antibodies to the bovine interferon can be used for large-scale purification of natural bovine interferon, as well as for characterizing the antiviral and immunologic activities of bovine interferon in the laboratory, as well as for therapy of auto-immune disease.

Description

LARGE-SCALE PRODUCTION OF BOVINE LEUKOCYTE INTERFERON

Background of the Invention

The present invention relates to a method for large scale production of natural bovine leukocyte interferon. The phenomenon of viral interference, first described in 1935, is the ability of one virus to interfere with the replication of another (challenge) virus. Thus, the quest was underway for the mediator of viral interference for over 20 years before Isaacs and Lindenmann assigned the name interferon (IFN) to it in 1957. Their discovery of a soluble antiviral factor released from chick chorioallantoic membranes after exposure to a heat-inactivated influenza virus was the beginning of interferon research. Interferons are now recognized to be low molecular weight proteins and glycoproteins that affect a variety of functions in animal cells, including virus replication, cell growth, and the immune response.

Alpha, beta, and gamma (also known as leukocyte, fibroblast, and immune, respectively) are the three known species of interferons. Alpha and beta interferons are collectively called Type I interferons, and can be produced by virtually all nucleated cells. In in vitro interferon production, alpha interferon is the major species released from stimulated leukocyte or lymphoblastoid cultures, whereas beta interferon is usually produced by fibroblasts or epithelial cells. Viruses, synthetic polynucleotides, bacteria, bacterial products, foreign nucleic acids, and certain polymeric chemicals can be used to stimulate production of alpha and beta interferons. Gamma interferon, also called Type II interferon, is a true lymphokine because it is released from T-lymphocytes after stimulation with mitogens, antigens, or interleukin-2. Alpha, beta, and gamma interferons differ in their antigeniσ, biologic, and physiochemical properties. As a general rule, alpha and beta interferons are acid stable, whereas gamma interferons are acid labile. However, there are some acid labile alpha interferons. Interferons may be either proteins or glycoproteins, depending on their species of origin and whether they are produced naturally or recombinantly. Human alpha interferons are most likely proteins (vs. glycoproteins) , since no carbohydrate was detected on analysis of ten homogeneous natural human alpha interferons. However, O-glycosylation cannot be excluded. It appears, however, that mouse, rat, cow, and rabbit alpha interferons are glycoproteins. Interferons produced bacterially are proteins and Jack carbohydrate moieties.

The human genome contains at least 15 to 17 different alpha interferon genes. These genes code for the production of structurally distinct polypeptides, and are the basis for the diversity of subspecies of interferons. However, whether all of the different genes are actually expressed (i.e., transcribed and translated) during physiologic encounters between viruses and cells has not yet been determined. Among interferons from domestic animal species, the bovine interferons constitute the groups about which the most is known presently. Bovine alpha interferon genes have been grouped into the following two homologous, but distinct classes: (1) class 1 (containing 10 to 12 members) and (2) class 2 (containing 15 to 20 members) . Four of the bovine alpha genes have been cloned and expressed in Escherichia coli. Differences in the biological activity of subspecies of interferons for veterinary applications have not been investigated. Human alpha interferon subspecies can differ quantitatively in their antiviral activities up to 200-fold, and in the target cells of their cross-species activities.

Alpha, beta, and gamma interferons significantly reduce the rate of division of normal and tumor cells. It has been suggested that rapidly growing cells are affected by interferon to a greater extent, thus forming the basis for exploration of interferons as antineoplastic agents. In some studies, recombinant interferons did not inhibit cell growth to the same extent as natural interferons. Individual species and subspecies of interferon can have quantitatively different antiproliferative activities. In contrast to the antiviral effects of interferon, the antiproliferative effects are refractory and are maintained only by constant exposure to interferon. Although originally thought to be species- specific substances, interferons are now known to have defined host ranges of cross-species activities. All three species of interferon (alpha, beta, and gamma) have been shown to have cross-species activities, although the host range can vary for the different interferon species. The degree of cross-reactivity observed also may vary with the type of cells (i.e., epithelial versus fibroblastic feline cells) and challenge virus used. The phylogenetic relationship has little bearing on cross-species activity, as many interferons are more active on cells of distantly related animals than on those of closely related animals (i.e., human leukocyte interferon is more active on bovine and feline cells than on monkey cells) . In vitro cross-species antiviral activity has been demonstrated for various bovine interferons on human, monkey, rabbit, pig, sheep, horse, and dog cells. Cross-species activities have been documented in vivo and have also been observed for cell growth inhibitory, priming, and immunomodulatory activities of interferons.

Unlike antibodies, interferons are not virus specific and are active against both RNA and DNA viruses. The antiviral activity of interferon is mediated indirectly, through effects on host cells, rather than interacting directly with the virus particle. Interferon induces a series of changes in intracellular enzyme levels, thus creating an "antiviral state" in cells rendering them unable to support virus replication.

Every family of mammalian viruses has its own unique strategy of replication, and different families of viruses appear to be affected by interferon through different mechanisms. One or more aspects of the virus replication process are interpreted by the genetic apparatus of the cell as an activation signal for interferon production, and the cell then elaborates specific mRNA for interferon. The interferon mRNA is translated into an interferon protein at the ribosomes and interferon is then released into the extracellular fluid. Although the exact site of glycosylation of interferon proteins is not known, it has been suggested that the addition of carbohydrate moieties to interferon proteins takes place within membranous structures prior to excretion.

Interferons are produced by cells very early in the course of viral infection. Thus, they are available much earlier than antibodies. Also in contrast to antibodies, interferons have antiviral activity against a wide range of virus families. However, viral families differ in their susceptibility to the antiviral effects of interferon. In addition, different families of viruses may respond differently to a given interferon in the same cell line, and this spectrum of activity differs among different cell lines from a given animal species. The immunomodulating effects of interferon also play a role in its antiviral activity, including stimulation of both natural killer cells and cytotoxic T lymphocytes.

Most early antiviral clinical trials with human interferons were uncontrolled in design and unimpressive in results. In 1974, the first controlled trial was conducted that indisputably showed that topical nasal human alpha interferon (1.4 x 10⁷ U/patient) could inhibit the symptoms and virus shedding of rhinovirus 4 infection in man. The effect of parenteral human alpha interferon (5 x 10⁵ U/kg/day for 7 days) was next demonstrated against human herpes zoster infection. Other human virus infections or conditions that have shown positive responses to interferon therapy in controlled clinical trials include: varicella, cytomegalovirus, herpes simplex type I, papilloma virus, the common cold (rhinovirus 13) , and multiple sclerosis. Most of the above trials in the late 1970's were performed with KSCN- precipitated human leukocyte (alpha) interferon provided by the Finnish Red Cross.

Viral infections in cattle are a major source of economic loss to both the beef and dairy industry. Large numbers of U.S. feedlot cattle succumb to virally-initiated respiratory disease yearly, resulting in monetary losses of such proportions that the U.S.D.A. regards respiratory disease as the number one economic problem in beef cattle. Viral respiratory diseases and viral diarrheas are also among the most economically important infectious diseases in dairy cattle.

Studies measuring the endogenous production of interferon by calves and cows exposed to viruses have documented a role for interferon in the bovine antiviral defense system. Interferons, as potent antiviral agents, represent a hope for prophylaxis and/or therapy of many heretofore untreatable diseases of cattle. Cattle produce significant amounts of endogenous interferon when exposed to either viruses or synthetic ribonucleotide interferon inducers. Such endogenous interferon has also been shown to protect against heterologous virus challenge in cattle. Intranasal vaccination of calves with infectious bovine rhinotracheitis (IBR) virus resulted in high levels of interferon in nasal secretions by 2 days, which peaked at 3 to 4 days, and were maintained through 8 days. A temporal association was observed between nasal secretion interferon and protection against IBR challenge. Intranasal vaccination with IBR vaccine has also been shown to result in reduction of severity of foot and mouth disease (FMD) and decreased nasal excretion of FMD virus. In in vitro testing, human interferons have antiviral activity against IBR, parainfluenza-3 (PI-3) , bovine respiratory syncytial virus (BRSV) , and vesicular stomatitis virus (VSV) , and bovine interferons have antiviral activity against IBR, PI-3, BRSV, VSV, bovine viral diarrhea virus, bovine adenovirus, rotavirus_r FMD virus, goat respiratory synctial virus, and swine pseudorabies virus. Although these studies have been useful in documenting that interferon plays a major role in the defense against bovine viral disease, stimulation of endogenous interferon has obvious practical disadvantages because of limited and highly variable amounts of interferon produced by the body.

Despite the promising results of in vitro studies and studies of the bovine endogenous interferon response, studies of the exogenous administration of interferons to cattle have been only recently accomplished. Recombinantly derived human alpha interferon (10⁶ U/kg/day for 7 days) administered intramuscularly (IM) to calves afforded complete protection against vaccinia virus (a pox virus) in an experimental model. Human leukocyte-A interferon was administered both IM and intranasally (IN) to calves both prior to and after IBR challenge. Viral shedding and the appearance of virus- neutralizing antibodies occurred later and respiratory tract disease was less severe in interferon-treated calves. Natural human leukocyte interferon administered orally at very low dosages (0.05, 0.5, and 5.0 U/lb/day for 3 days) to calves prior to IBR challenge resulted in increased levels of IBR antibodies 14 days after infection. Enhanced PI-3 seroconversion was also reported during a natural shipping fever outbreak and higher IBR and BVD antibody levels after modified-live vaccination. Administration of bacterially-derived bovine alpha interferon (IN) to calves prior to challenge with IBR and Pasteurella hemolytica resulted in significantly lower numbers of days sick and levels of serum fibrinogen and greater functional activity of neutrophils in interferon-treated calves. Only half as much lung tissue was pneumonic in interferon- treated calves, but these calves shed only slightly less virus than controls. It was suggested that bovine alpha interferon may have a greater immunomodulatory than antiviral effect in this model. Large-scale production of interferons can be accomplished both by natural methods and through the use of recombinant DNA technology. Although human interferons from both sources are available in ample supply for human research use and clinical trials, this is not true for most veterinary interferons. Several animal (bovine, porcine, and equine) leukocyte interferons have been produced in small amounts. Techniques have been developed for recombinant production of bovine alpha, beta, and gamma interferons. However, bovine recombinantly derived interferons have had limited availability for veterinary research use, and none are approved for clinical use presently.

Peterhans, et al. , reported in Res.Vet.Sci. 20,99-100 (1976) an unsuccessful attempt at the large scale production of large quantities of natural bovine interferon from calf testicular cells and leukocytes. Luna, et al. , in Experientia 40, 1410-1412 (1983), described using Newcastle's Disease Virus as the inducer of bovine leukocytes to obtain titers of approximately 9,000 units interferon/ml. It is not clear from their report whether they removed or inactivated the virus following induction, or whether their method was used on a large scale. More recently, Cohen, et al., reported in Methods in Enzvmoloσy 119, 436-144 (1986), that bovine leukocytes stimulated with Sendai virus according to the method described by Cantell, et al., used for large scale production of human alpha interferon (Methods in Enzvmoloαv, 78, 29-38 (1981)), resulted in the isolation of both acid stable and acid labile interferons having molecular weights of 16,000 and 25,000 daltons by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS- PAGE) . However, the authors also state that the yield of the bovine interferon were about one-tenth (1000 units/ml) the yield of human leukocytes induced and tested in the same way.

It is therefore an object of the present invention to provide methods for large scale production of natural bovine leukocyte interferon.

It is another object of the present invention to provide a natural bovine leukocyte interferon preparation for use in treating animals including cattle, horses, dogs, cats, sheep, goats, monkeys, rabbits and humans.

It is still another object of the present invention to provide monoclonal antibodies to a natural bovine leukocyte interferon for use in assays, large scale purification procedures, and potentially for therapy of immune-mediated disease in humans or animals.

Summary of the Invention

A method for large-scale preparation of natural bovine leukocyte interferon, a potent anti-viral and anti-proliferative agent, having a molecular weight by SDS gel electrophoresis under reducing conditions of approximately 19,000 daltons and anti-viral activity that is stable at pH 2.0 and partially stable at 56°C for 15 to 120 min. The natural bovine interferon has antiviral activity against pathogenic bovine viruses and has immunologic activity. Normal calves receiving this bovine interferon had few of the side effects and clinicopathological changes usually seen in human patients undergoing interferon therapy, and gained more weight than controls.

Monoclonal antibodies to the bovine interferon can be used for large-scale purification of natural bovine interferon, as well as for assaying for and characterizing the antiviral and immunologic activities of bovine interferon. The antibodies may also be used in the therapy of autoimmune diseases in humans and animals and also for potentiating interferon's antiproliferative activity.

Brief Description of the Drawings

Figure 1 is the elution profile from Sephadex G1Q0 of bovine leukocyte interferon containing no FBS concentrated by precipitation with 0.5 M KSCN at pH 3.5 and desalted. Bovine leukocyte interferon, in 15 ml volumes (90 g) , was applied to a Sephadex G-100 column (2=5 x 100 cm) equilibrated with PBS/NaCl/EG buffer and eluted with buffer at a flow rate of 5 ml/hour at 4°C. Fractions of 2.5 ml each were collected. Fractions 245 through 325 were collected and pooled fractions of 10ml each were concentrated and assayed for bovine leukocyte interferon activity. The column was calibrated with thyroglobulin (670,000 MW) , gamma globulin (158,000 MW) , ovalbumin (44,000 MW) , yoglobin (17,000 MW) and cobalamine) (1,350 MW) . The first large protein peak represents inactivated Sendai inducer virus from the preparation.

Figure 2 shows a comparison of molecular weights and purity of 16.5 g column purified bovine leukocyte interferon (B, C) with 5 βg recombinant bovine alpha_! interferon (D) using 10% SDS-PAGE under reducing conditions. (standards, A, are phosphorylase B: 92,500 daltons; bovine serum albumin: 66,200 daltons;

\ ovalbumin: 45,000 daltons; carbonic anhydrase: 31,000 daltons; soybean trypsin inhibitor: 21,500 daltons; and lysozyme: 14,400 daltons.) Samples were boiled in the presence of 2% SDS and 2-mercaptoethanol. After electrophoresis the gel was stained with Coomassie Blue and silver stain.

Detailed Description of the Invention

The present invention is a method for the large scale production and purification of a natural bovine leukocyte interferon characterized by a molecular weight of approximately 19,000 daltons by SDS-PAGE under reducing conditions, and anti-viral activity that is stable at pH 2.0 and stable after very long term storage 4°C, -20°C and -70°C. The anti-viral activity is destroyed by digestion with trypsin. In contrast to human alpha interferon, it is believed that this bovine leukocyte interferon is glycosylated.

This is the first known preparation of a purified bovine alpha interferon produced by induction of bovine leukocytes having these characteristics, although other bovine alpha interferon preparations are known. Because recombinantly-derived interferons contain only a single interferon subspecies, the biological activities of these interferons may differ from those of naturally-derived interferons, especially since interferon subspecies can vary in their degree and range of antiviral activity. It has been documented that recombinant interferons do not inhibit cell growth to the same extent as natural interferons. Individual subspecies of alpha interferons can have quantitatively different antiproliferative and antiviral activities. It is predicted that the natural bovine leukocyte interferon is useful in a variety of species for prophylaxis of viral infections, therapy of viral infections, modulation of immune responses, therapy of neoplastic diseases and leukemias, therapy or eye disease, and therapy of enteric diseases. The natural bovine leukocyte interferon can be administered intravenously, intramuscularly, subcutaneously, orally, intraperitoneally, by intrauterine (or intravaginally) or intramammary routes, by application to mucous membranes, topically, either in an ointment or by solution. U.S. Patent No. 4,462,985 to Cummins teaches a method for the oral administration of interferons of heterologous species. It has also been documented that various bovine interferons have activity on human, monkey, rabbit, pig, sheep, horse, and dog cells.

Using the method of the present invention, from 0.2 to 24 liters of crude bovine leukocyte interferon have been produced at a time, each liter of interferon requiring an initial four liters of peripheral bovine blood. Leukocytes may be obtained from cattle blood collected by venipuncture from live cows or at slaughter. The method can readily be scaled up to produce larger quantities. The natural bovine leukocyte interferon of the present invention is produced by virus-induced bovine leukocytes as subsequently described. Bovine leukocytes are isolated from multiple cows and pooled cells are maintained as spinner cultures in media containing 0 to 8% fetal bovine serum in a 35-40°C water bath. Using leukocytes from several cows averages out individual differences in interferon-producing ability. Flasks are filled to no more than 2/3 capacity. The priming dose of virus is added to leukocyte suspensions immediately and the inducing dose is added within 2 hours. Leukocytes are gently stirred during incubation of up to 66 hours. Supernatants containing interferon are harvested by pelleting cells by low speed centrifugation (1700 x g, 20 minutes), followed by ultracentrifugation (175,000 x g, 2 hours) or pH 2.0 dialysis to remove or inactivate virus. This bovine leukocyte interferon has been assigned an Investigational New Animal Drug (INAD) number from the FDA to allow use in food- producing animals.

In one embodiment of the production method, media containing 8% bovine serum is used and leukocytes are primed with 60 hemagglutinating units/ml Sendai virus, followed within four hours by induction with 240 hemagglutinating units/ml Sendai virus. When incubation was for 12 hours, mean interferon titers of numerous production lots were over 15,opo units/ml. These titers are significantly higher than any other reported titers for bovine leukocyte interferon. The specific activity was 11,100 units/mg protein for preparations made with 8% fetal bovine serum, and this value is considerably higher for preparations with no fetal bovine serum. Following virus inactivation or removal, the bovine leukocyte interferon is concentrated by precipitation with solid potassium thiocyanate (KSCN) , added to a final concentration of 0.5 M. The pH is adjusted to 3.5 with 2 N HC1 at 4°C. The sample is centrifuged (2300 x g, 40 minutes) and the precipitate is resuspended in a minimum amount of phosphate buffered saline (PBS) buffer at pH 7.2. Adjusting the PBS buffer to contain 25% ethylene glycol and 1 M NaCl maximizes final yield of the purified bovine leukocyte interferon. The resuspended precipitate is stirred overnight and undissolved precipitate is removed by centrifugation (1700 x g, 15 minutes) . Clear supernatant is then dialyzed against cold PBS overnight. In another embodiment of the production method, leukocytes are cultured in medium containing up to 8% fetal bovine serum. This allows co-precipitation of the interferon with the albumin in the fetal bovine serum, such that interferon yields are maximized from the KSCN precipitation step.

In another embodiment of the production method, leukocytes are cultured in medium containing 0% bovine serum such that specific activity of the final purified interferon product is highest. In alternative embodiment of the concentration process, interferon prepared using 0% fetal bovine serum is concentrated by large-scale ultrafiltration using a membrane having a molecular weight cutoff that retains the interferon (generally in the range of 10,000 to 14,000 daltons).

Following concentration, the interferon is purified by application of the interferon to a Sephadex G-100 liquid gel chromatography column. The column is eluted with PBS buffer and fractions are collected, concentrated, and assayed for interferon activity. The molecular weight of interferon- containing fractions is estimated by comparison with protein standards passed through the column. Pooled fractions containing interferon are reduced with 2- mercaptoethanol and analyzed by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE) . Final purification of the bovine leukocyte interferon is performed using SDS-PAGE or binding to an antibody. In another embodiment of the purification method, antibody columns are utilized to isolate pure interferon in a single step. Monoclonal antibodies also enable development of a radioimmunoassay for the rapid detection of bovine interferons. Such an assay has advantages over conventional bioassays for interferon in that many subjective factors of the assay procedure are eliminated. Variability in tissue culture cell lines and in challenge virus potency are common problems encountered with cytopathic effect reduction assays routinely used for interferon assay. Despite the limitations of a radioimmunoassay (biologically inactive interferon molecules in a preparation are also detected) , its speed, consistency, and simplicity make it a useful adjunct to bioassay methods.

Monoclonal anti-interferon antibodies have many further uses in in vitro and in vivo investigations of the antiviral and immunomodulating effects of bovine alpha interferons. They are also potentially useful in therapies of autoimmune diseases. Enhanced antiproliferative action of interferon targeted by bispecific monoclonal antibodies has been described by

Alkan, et al., J. Interferon Res. 8:25-33(1988). Example 1: Large scale production of Bovine leukocyte

Interferon: Type of inducing virus, concentration and time of exposure on interferon titer in Leukocyte Supernatant.

Collection and Induction of Leukocytes. For each lot of bovine interferon produced, 3-12 healthy, lactating Holstein cows from the University of Georgia Dairy were used as blood donors. Multiple cows were used for each production lot to minimize variability among cows in interferon-producing ability of leukocytes. One to 4 liters of blood were collected from each cow by jugular venipuncture into chilled 2 liter vacuum bottles containing acid citrate dextrose. Alternatively, blood could have been collected at slaughter. Buff coats from pooled blood samples were harvested by centrifugation and residual red blood cells were lysed by 2 washes in 8 volumes of cold 0.83% NH4CI. Leukocytes were resuspended in Minimum Essential Medium modified with Earle's salts with glutamine and supplemented with sodium bicarbonate (2 g/lit r) , tricine (0.3 g/liter) , neomycin (50 mg/liter) , and heat-inactivated fetal bovine serum (FBS) (0%, 0.4%, or 2% final concentration) . No FBS is used in preparations for purification of the interferon to homogeneity. Cell viability was consistently > 95% as determined by trypan blue exclusion.

Leukocyte suspensions (1.2 x 10⁷ viable cells/ml) were maintained as spinner cultures in 1, 2, or 4 liter Erlenmeyer flasks filled to not more than 1/2 capacity. All flasks were incubated in a single large 37°C water bath specifically designed to permit simultaneous interferon production in up to 12 flasks under tightly controlled conditions of temperature and spin rate. The priming dose of virus was added to leukocyte suspensions immediately and the inducing dose was added within the next 2 hours. Leukocytes were gently stirred throughout incubation. Supernatants containing interferon were harvested by pelleting cells at low speed centrifugation (1000-1700 x g, 20 minutes) after approximately 20 hours. Ultracentrifugation (175,000 x g, 2 hours) was used to remove virus particles. In control cell cultures, media was used in place of priming and inducing virus. Quantity and strain of Inducing Virus.

The Sendai virus (Cantell strain) has been determined to be the optimal inducing virus for production of bovine leukocyte interferon according to the method of the present invention. Sendai virus is propagated in 10-day-old pathogen-free embryonating eggs. Allantoic fluid is harvested, centrifuged (1700 x g) , and frozen at -70"C prior to use as stock virus. Stock sendai virus is titered in hemagglutinating units (HA)/ml. Ultracentrifuged preparations of Sendai virus are tested by interferon assay to establish that inducing virus preparations do not contain measurable amounts of interferon. Interferon assays routinely use vesicular stomatitis virus (VSV) as "challenge virus" (the virus that lyses tissue culture cells in an interferon assay) . Indiana strain VSV is propagated in Maden-Darby Bovine Kidney (MDBK) cells and titered by plaque assay in MDBK cells, resulting in titers in PFU/well.

A cytopathic effect inhibition assay for interferon is performed on MDBK cells grown in monolayer in 96-well microtiter plates, adapted from the method of Familletti, et al., Methods Enzymol. 78,387-394 (1981). All interferon samples are ultracentrifuged at 175,000 x g to remove inducing virus prior to assay. Samples are cultured in trypticase soy broth and thioglycolate to assess sterility and frozen at -70°C prior to assay. Thawed samples, in triplicate, are diluted serially 1:2 in 96-well flat-bottomed microtiter plates (100 μl/well) . MDBK cells (4 x 10⁴ cells/100 μl) are added to all wells and allowed to form monolayers during 24 h of incubation. The interferon is then removed and challenge virus (VSV) added to the wells after 24 hours (8 x 10³ pfu/well) . After another 20-24 hours or when the virus control shows 100% CPE, supernatants are decanted and plates are stained with 0.5% crystal violet in 80% methanol. The interferon titer is calculated as the reciprocal of the dilution which protects 50% of the cells against viral lysis. Since there is not yet an international standard for any bovine interferon, the assay is standardized by including in each assay our own bovine leukocyte interferon standard with a titer of 3200 units/ml. This has been calibrated against the WHO standard for human leukocyte interferon, 69/19.

Kinetics for the large-scale production of natural bovine leukocyte interferon were investigated using 3 viral inducers, the Sendai virus, Newcastle disease virus (NDV) , and infectious bovine rhinotracheitis (IBR) at various doses and incubation times. The differences in mean titers for interferon incubated at different times with Sendai virus and NDV inducers is shown in Table 1. Induction with IBR virus results in very little interferon production. Highest titers of bovine leukocyte interferon (15.314 U/ml) were obtained using Sendai virus (60 HA/ml priming dose and 240 HA/ml inducing dose) and incubating 12 hours. The effect of priming with Sendai virus at various doses and times is demonstrated in Table 2. Up to 24 liters (over 360 million U) of natural bovine leukocyte interferon were produced at one time, quantities sufficient for clinical use in cattle. The effect of FBS concentration and ethylene glycol in the PBS buffer on interferon yield is shown in Table 3.

TABLE 1: Bovine leukocyte IFN production with Sendai virus and NDV inducers: differences in mean titers for IFN incubated at different times.

Sendai virus dose (HA/ml)

Incubation

Time (hours) 100 200 300

6 2,440^abC 7,720^a 3,500^abC

12 4,800^a ll,028^a 15,314^a

18 3,000^ab l,850^SbC 8,400^ab

24 3,200^ab l,867^ab 4,000^abC

30 l,500^abC 900^abC 3,200^abC

36 l,533^abq l,514^abC l,600^{bcd "}

42 l,950^abC 850^abcd l,500^bGd

48 l,060^abC 400^bCd 920^Cd

54 700^c 400^Cd 900^Cd

60 2,160^abC 280^Cd 480^d

66 l,200^bC 200^d 575^d

NDV Dose (ELD-. /mi; 1

Incubation Time

(hours) 3 x 10^A 3 X 10⁶ 3 X 10⁷ 3 X 10⁸ 3 X lθ' ab

100 100' 640^a ab

400

12 80ab 250^s 1 , 175^a 1,200^£ ¹8 100ab 200^E 1 ,280^a 800^£

66 34^c 200 abed Means of log- titers with the same symbols were not significantly different at P<.05. Comparisons were made by ANOVA and the least significant difference test for samples within each virus dose. TABLE 2: Production of Bovine Leukocyte IFN With

Sendai Virus*: Effects of Varied Priming Doses and Priming Times

Varied Dose Varied Time

Priming dose+ Priming time* (hours) IFN IFN (% of total titer prior to titer virus dose) (U/ml) induction) (U/ml)

0 3,200 0 3,200

10 6,400 0.5 12,800

20 6,400 1.0 6,400

30 3,200 2.0 3,200

40 3,200 3.0 3,200

50 _.800 -0_o 3,200

*Sendai virus inducer (300 HA/ml) was used. All samples were incubated for 24 hours after induction.

+Priming time was 2 hours prior to induction. #Priming dose was 20% of total virus dose.

TABLE 3: Purification of BoL-IFN: Effect of FBS concentration in crude IFN and use of ethylene glycol in buffer on recovery of BoL-IFN

EXP. la Exp. lb Exp. 2 EXP. 3

% FBS in crude IFN 2% 2% 0.4% 0%

Crude 750,000 750,000 1,219,000 1,440,000

IFN (U)

IFN (U) recovered from precipi¬ tate⁸ N.D.^b 360,000 204,800 7,200

IFN (%) recovered from precipi¬ tate N.D. 48% 16.8% 0.5%

Ethylene glycol in buffer⁰ No Yes No Yes

IFN (U) recovered from column^d 26,250 259,350 51,674 6,747

IFN (%) recovered from column N.D. 72% 25.2% 93.7%

Total IFN recovered from crude 3.5% 34.6% 4.2% 0.5%

Specific activity (U/mg) of final IFN N.D. 5.5 X 10⁴ 4.0 X 10⁴ 6.0 X 10⁴

IFN was concentrated by precipitation with KSCN at pH 3.5 and pellet was redissolved in buffer (below).

N.D. = not determined. ^c Buffer was either PBS or PBS with 25% ethylene glycol.

Redissolved IFN was applied to a Sephadex G-100 column and eluted with buffer (above) . Example 2: Purification and characterization of bovine leukocyte interferon.

Following removal of inducer virus from leukocyte culture supernatants by ultracentrifugation, the interferon was concentrated by precipitation with

KSCN. Solid KSCN was added slowly to cold cell supernatant to a concentration of 0.5 M and the pH adjusted to 3.5 with 2 N HC1. After standing overnight at 4°C, the interferon-containing precipitate was collected by centrifugation at 2300 x g for 40 min at 4°C and dissolved in approximately 1/lOOth of the cell supernatant volume of PBS pH 7.2, with or without 1 M NaCl and 25% ethylene glycol. The PBS/NaCl/EG was used in all steps when purifying the interferon to homogeneity. Ethylene glycol increases the overall recovery of the purified interferon by 10_.. fold, from 3.5% to 34.6%, and the column recovery from 25% up to between 72% and 93.7%. This was stirred overnight at 4°C to homogenize the pellet and centrifuged at 12,000 x g for 30 min.

The supernatant was applied to a Sephadex G-100 column (2.5 x 100 cm) for purification. Interferon in 15 ml volumes was applied to the column and 2.5 ml fractions were eluted with buffer at a flow rate of 5 ml/hr at 4^βC. Pooled fractions (10 ml) collected from the columns were concentrated by ultrafiltration, sterilized using a 0.2 μ filter, and assayed for interferon activity.

The omission of fetal calf serum from the production media results in interferon titers comparable to those produced with calf serum in the crude preparations, but lower amounts of a more pure interferon recovered after KSCN precipitation, presumably because interferon binds to albumin in the calf serum, and these proteins are co-precipitated during the precipitation. This problem can be overcome by using 0% FBS and concentrating with a large scale ultrafiltration device, thus maximizing both yield and purity. Fractions containing interferon were assayed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in order to determine the purity and precise molecular weight of the interferon. Samples were boiled one minute in buffer containing 2-mercaptoethanol (2-ME) and the gel run for 4 hr at 15 mA constant current.

The interferon activity of the bands was determined by slicing the gel into 2 mm pieces and eluting the interferon from the gel into 0.5 ml PBS at room temperature for 18 hrs. Only slices with molecular weights of approximately 18,000 - 30,000 daltons contained interferon. Multiple bands were present in the samples prepared from leukocytes cultured with FBS. The interferon activity eluted from Sephadex G100 is shown in Figure 1. Figure 2 is the SDS-PAGE comparing the molecular weight and activity of purified natural interferon and recombinant interferon to molecular weight standards. The natural interferon has a molecular weight of 19,000 while the recombinant interferon is approximately 18,400. The antiviral substance produced by these methods is an interferon, as tested for the following standard criteria to classify a substance as interferon: 1) deactivation by trypsin (one ml of 0.05%EDTA-trypsin was added to one ml of bovine interferon and incubated at 37°C for 60 min; trypsin was inactivated by addition of 100 μl of cold FBS and the sample assayed for interferon activity) ; 2) stability at 37°C and 56°C for 60 minutes, 3) lack of toxicity to cells in tissue culture, 4) retention of antiviral activity following washing of the cells, 5) no production of antiviral effect in uninduced leukocyte cultures, and 6) stability at pH 2.0 (10 ml of bovine interferon was dialyzed at 4°C in membrane tubing with a w cutoff of 12,000 daltons, Spectrum Medical Industries, Los Angeles, CA, for 24 h against pH 2.0 phosphate buffer, and then for 24 h against pH 7.2 phosphate buffer. Like human alpha interferon, this bovine interferon was resistant to reduction by 2 ME and boiling 2 minutes in SDS. The antiviral activity was stable at pH 2.0 partially stable at 56°C (50% of the initial activity remaining after incubation for 15 min, 25% after 30, 60, 120 min) . The antiviral activity was partially stable at 37°C (100% of the initial activity remaining for 96 h, 50% after 7 days, and 87% after 30 days) . Activity was stable for 6 to 12 months at 4^βC, -20'C, and -70°C.

Several studies have reported that bovine interferons are labile or partially labile at pH 2.0.

Example 3: Effects of Crude Bovine Interferon on Immune Function In Vitro.

The effect of bovine leukocyte interferon on mitogen-induced lymphocyte blastogenic response and one-way mixed lymphocyte response (MLR) was investigated. Ten healthy female 3-6 month-old Holstein calves served as blood donors for each experiment. Lymphocytes were exposed simultaneously to interferon and either mitogens (PHA, PWM, or ConA) or allogeneic lymphocytes. Decreased responses were observed to all 3 mitogens (p < .05 for PHA) when 800 U IFN/ml was used. Decreased responses were also observed in one-way MLR when 200, 400, or 800 U IFN/ml was used. These results indicate that the effects of interferon on lymphocyte function are similar to those reported for human leukocyte interferon. Exa ple 4: Antiviral Effects of Crude Bovine Interferon In Vitro. The antiviral effects of crude bovine interferon were documented against two unrelated viruses. Protection against cytopathic effect induced by VSV (a rhabdovirus) was established by routine interferon assays. In addition, bovine interferon was investigated for .in vitro prophylactic and therapeutic effects against bovine leukemia virus (BLV) , a retrovirus. BLV was assayed by syncytial induction assay in F-81 cells. Bovine interferon (240 U/ml or 480 U/ml) afforded complete protection against BLV, and lower concentrations afforded partial protection in a dose-related manner. For assessment of therapeutic effect, a cell line chronically infected with BLV was treated with serial dilutions of bovine interferon and BLV was measured in cell supernatants. When administered therapeutically, bovine interferon reduced, but did not completely eliminate, BLV infection in a dose- related manner.

Example 5: Inhibition of Cell Growth bv Bovine Interferon. Samples were collected after incubation for 6 h. Petri dishes (60 mm²) were seeded with 5 ml of MDBK cells (1 x 10⁵ cells/ml) in serum-free media, which, after 24 h was replaced by media containing 10% FBS. After 24 h, 5 groups of 33 plates each were treated for one h either with 100, 500 or 1,000 units of bovine interferon, or with control. An additional 4 ml of media was added to each plate, and the plates reincubated. At 0, 6, 12, 18, 24, 30, 36, 48, 54, 96, and 108 h, three plates were harvested from each group and the average number of variable cells for each set of three was determined by Trypan Blue exclusion. The plating efficiency of untreated MDBK cells was 99% at 180 min and the doubling times was 38 h.

Growth curves of untreated MDBK cells and control cells and cells treated with 100 units of interferon were similar. Cultures treated with 500 or 1,000 units of bovine interferon grew similarly but had 43% and 48% fewer cells, respectively, than untreated cultures at 96 h after cell growth initiation.

Example 6: Effects of Crude Bovine Interferon on Clinical and Immunological Parameters In

Vivo.

Six healthy, two-month-old Holstein steers were injected intravenously with bovine leukocyte interferon 1 x 10³ units per kg body weight per day for 5 days. The interferon was prepared by priming and induction of isolated bovine leukocytes, as described above, followed by centrifugation to remove the cellular debris. Physiological saline was similarly administered to six control calves with identical histories. Daily physical examinations of all calves by employed veterinary students having no knowledge of the treatment protocol revealed no overt side effects aside from slight pyrogenicity in treated calves 5 hours after injection. Although human patients undergoing interferon therapy often become fatigued and anorectic, calves in treatment groups gained more weight than members of the respective control groups. This may have been due, in part, to a lesser incidence of a coccidial diarrhea, diagnosed in all calves after the first day of the experiment, in treatment calves on all experimental days except one.

Circulating interferon levels following treatments were <200 units/ml in all calves. Complete blood counts, including total and differentials, white blood cell counts, platelet and red blood cells counts, packed cell volume, fibrinogen and hemoglobin determinations, and plasma protein determination revealed no abnormalities. Thus many of the side effects commonly associated with interferon therapy in man (leukopenia, lymphopenia, and thrombocytopenia) were not observed. Determination of serum urea nitrogen, total protein, albumin, creatinine, aspartate amino transferase (AST) and gamma- gluta yltransferase (GGT) levels detected no abnormalities of kidney or liver function during the treatment period. No differences in three parameters of the immune response, the mitogen-induced lymphoproliferative response, the one-way MLR, and the serum IgM levels, measured by single radial immunodiffusion, were observed during interferon treatment. Example 7: Monoclonal Antibody Production.

ELISAs and Neutralization Assays: ELISA was developed for testing mouse sera and monoclonal supernatants for antibody activity against natural bovine leukocyte interferon and recombinant bovine alpha interferon from CIBA-GEIGY, Greensboro, N.C. Neutralization assays were also developed to determine if positive mouse sera or monoclonal Abs could neutralize antiviral activity of natural and recombinant bovine alpha interferons.

Mouse Immunization and Fusions: Mice were immunized and boosted with column-purified natural bovine interferon. Sera were antibody positive to natural bovine interferon at 1:10,000 dilutions.

Several hybridomas secreting monoclonal antibodies that bound to natural bovine leukocyte interferon were obtained. Non-neutralizing antibodies are useful for purification and development of more efficient interferon assays (RIAs, ELISAs) , while neutralizing antibodies can be used to perform blocking experiments in vitro and iα vivo to clarify bovine interferon'≤ role in the antiviral immune response of cattle. Modifications and variations of the present invention, a method for large scale purification of bovine leukocyte interferon and product thereof, will be obvious to those skilled in the art from the foregoing detailed description. Such modifications and variations are intended to come within the scope of the appended claims.

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Claims

I Claim: "29~

1. A natural bovine leukocyte interferon preparation comprising a single polypeptide of approximately 19,000 Daltons by SDS gel electrophoresis under reducing conditions having anti¬ viral activity which is stable at pH 2.0, wherein said interferon is produced by virus-induced bovine leukocytes.

2. A preparation for the treatment of animals comprising a natural bovine leukocyte interferon of approximately 19,000 Daltons by SDS gel electrophoresis under reducing conditions having anti¬ viral activity which is stable at pH 2.0 when said interferon is produced by virus-induced bovine leukocytes.

3. A monoclonal antibody to a natural bovine leukocyte interferon of approximately 19,000 Daltons by SDS gel electrophoresis under reducing conditions having anti-viral activity which is stable at pH 2.0, which is produced by virus-induced bovine leukocytes.

4. A method for isolating a natural bovine leukocyte interferon of approximately 19,000 Daltons by SDS gel electrophoresis under reducing conditions having anti-viral activity which is stable at pH 2.0 which is produced by virus-induced bovine leukocytes comprising reacting a monoclonal antibody to the interferon with an interferon-containing solution.

5. An assay for natural bovine leukocyte interferon comprising a monoclonal antibody to a natural bovine leukocyte interferon of approximately 19,000 Daltons by SDS gel electrophoresis under reducing conditions having anti-viral activity which is stable at pH 2.0 which is produced by virus-induced bovine leukocytes.

6. A method for large scale production of natural bovine leukocyte interferon comprising: priming isolated bovine leukocytes to produce interferon by exposure to a virus; inducing the primed bovine leukocytes to produce interferon with a second exposure to a larger number of virus particles; and removing or inactivating the virus.

7. The method of claim 6 wherein the virus is selected from the group consisting of Sendai virus, Newcastle's disease virus, and infectious bovine rhinotraσheitis virus.

8. The method of claim 7 wherein the leukocytes are induced with Sendai virus between 0 and approximately 120 minutes of priming.

9. The method of claim 7 wherein the leukocytes are primed with between 0 and 50% of the total virus dose used to induce interferon production.

10. The method of claim 6 further comprising removing the virus by ultracentrifugation until virus particles are removed.

-Si¬ ll. The method of claim 6 further comprising concentrating the interferon.

12. The method of claim 11 wherein the interferon is concentrated by ultrafiltration with a membrane having a molecular weight cutoff of between approximately 10,000 and 14,000 molecular weight.

13. The method of claim 11 wherein the interferon is concentrated by precipitation with KSCN.

14. The method of claim 11 further comprising removing from the concentrate the contaminants having molecular weights less than about 18,000 daltons and greater than about 30,000 daltons.

15. The method of claim 14 wherein the contaminants are removed by chromatography on a molecular weight sieve.

16. The method of claim 15 wherein the interferon is chromatographed in a buffer containing ethylene glycol.

17. The method of claim 14 further comprising purifying the interferon by polyacrylamide gel electrophoresis.

18. A method for treating animals comprising administering a natural bovine leukocyte interferon

• ^• preparation wherein said interferon is a single polypeptide of approximately 19,000 Daltons by SDS gel electrophoresis under reducing conditions having anti¬ viral activity which is stable at pH 2.0 when said interferon is produced by virus-induced bovine leukocytes.

19. The method of claim 18 wherein the animal is selected from the group consisting of cattle, horse, humans, dogs, sheep, pigs, goats, monkeys, rabbits and cats.

20. The method of claim 18 wherein the interferon preparation is in a form selected from the group consisting of solutions for injection, solutions for topical administration, ointments for topical administration, intra-respiratory tract sprays, compositions for application to mucous membranes, compositions for intra-rectal administration, compositions for intra-uterine administration, compositions for intra-mammary administration, and compositions for oral administration.

21. The method of claim 18 wherein the interferon is administered in a dose and form which acts to modulate components of the immune system.

22. The method of claim 18 wherein the interferon is administered in a dose and form which is effective in the treatment of neoplasms and leukemias.

23. The method of claim 18 wherein the . interferon is administered as a prophylactic.

24. The method of claim 18 wherein the interferon is administered in a dose and form resulting in weight gain.

25. The method of claim 18 wherein the interferon is administered in a dose and form to treat enteric infections.

26. A method for using antibodies to a natural bovine leukocyte interferon preparation comprising a single polypeptide of approximately 19,000 Daltons by SDS gel electrophoresis under reducing conditions having anti-viral activity which is stable at pH 2.0, wherein said interferon is produced by virus-induced bovine leukocytes, as an immunomodulator or to potentiate cell proliferation.