Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
  • A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/08—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from viruses
  • C07K16/081—DNA viruses
  • C07K16/085—Orthoherpesviridae (F), e.g. pseudorabies virus or Epstein-Barr virus
  • C07K16/087—Herpes simplex virus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• This invention relates to a passive vaccine for pseudorabies virus. More specifically, the invention relates to monoclonal antibodies to pseudorabies virus glycoproteins, useful in alleviating pseudorabies virus infections in swine. BACKGROUND OF THE INVENTION
• PRV Pseudorabies virus
• PRV infections are variously called infectious Bulbar paralysis, Aujeszky's disease, and mad itch. Infections are known in important domestic animals such as swine, cattle, dogs, cats, sheep, rats and mink.
• the host range is very broad and includes most mammals and, experimentally at least, many kinds of birds (for a detailed list of hosts, see D.P. Gustafson, "Pseudorabies", in Diseases of Swine, 5th ed. , A.D. Leman et al., eds., (1981)). For most infected animals the disease is fatal. Adult swine and possibly rats, however, are not killed by the disease and are therefore carriers.
• PRV vaccines have been produced by a variety of techniques and vaccination in endemic areas of Europe has been practiced for more than 15 years. Losses have been reduced by vaccination, but vaccination has maintained the virus in the environment. No vaccine has been produced that will prevent infection. Vaccinated animals that are exposed to virulent virus survive the infection and then shed more virulent virus. Vaccinated animals may therefore harbor a latent infection that can flare up again. (See, D.P. Gustafson, supra) .
• PRV Live attenuated and inactivated vaccines for PRV are available commercially in the United States and have been approved by the USDA (See, C.E. Aronson, ed. , Veterinary Pharmaceuticals & Biologicals, (1983)).
• PRV is a herpesvirus.
• the herpesviruses generally are among the most complex of animal viruses. Their genomes encode at least 50 virus specific proteins and contain upwards of 150,000 nucleotides.
• Among the most immunologically reactive proteins of herpesviruses are the glycoproteins found, among other places, in virion membranes and the membranes of infected cells.
• the literature on PRV glycoproteins refers to at least four viral glycoproteins (T.
• Monoclonal antibodies have also been used to partially characterize several PRV glycoproteins with respect to glycosylation and synthesis within infected cells (Hampl et al., supra.; and N. Lukacs et al., J. Virol., 53, pp. 166-73 (1985)(gl and gII)).
• N.S. Balachandran, et al., Infect. Immun. , 37, pp. 1132-37 (1982) and R.D. Dix, et al., Infect. Immun. , 34, pp. 192-99 (1981) refer to protecting mice from homologous virus challenge using passive immunization with monoclonal antibodies reactive with herpes simplex virus glycoproteins.
• D.P. Gustafson et al. , J. Am. Vet. Med. Assoc, 160, pp. 623-28 (1972) refer to passive administration of PRV hyperimmune serum and gamma globulin preparations and their effectiveness as a short-term prophylaxis against Aujesky's disease in swine.
• R.A. Crandell et al., J. Am. Vet. Med. Assoc, 171, pp. 59-63 (1977) refers to suckling pigs given hyperimmune serum incurring a lower rate of mortality than control pigs after exposure to PRV.
• the short-term prophylactic treatment of the instant invention is useful after exposure of a herd of pigs to PRV. Highly susceptible young pigs must be protected at that time in order to survive. Vaccination programs would then be started after initial protection is achieved to protect the pigs for a longer time.
• the monoclonal antibodies of the instant invention are more advantageous than hyperimmune serum or gamma globulin in a passive immunization program for such protection because they are easier to mass produce and have greater specificity as well as less chance of passing on incidental infection.
• the present invention relates to a method of treating PRV infection in swine by administering an anti-PRV effective amount of a composition comprising a monoclonal antibody against PRV gll, gIII, or gp50 to a swine in need of such treatment. More particulary, the invention relates to a method of treating PRV infection in swine by administering an anti-PRV effective amount of a composition comprising a monoclonal antibody selected from the group consisting of 3A4, 3D1, and 3D11 to a swine in need of such treatment. The present invention also relates to a monoclonal antibody selected from the group consisting of 3D11, 3D1, and 3A4.
• the present invention also relates to a hybridoma selected from the group consisting of hybridomas 3A-4C, 3D-1C, and 3D-11C.
• the present invention also relates to a vaccine for passive immunization of swine against PRV infection comprising a monoclonal antibody against PRV gll, gIll, or gp50.
• the invention relates to a vaccine for passive immunization of swine against PRV infection comprising a monoclonal antibody selected from the group consisting of 3A4, 3D1, and 3D11. DETAILED DESCRIPTION OF THE INVENTION
• EXAMPLE 1 Cell Cultures, Media and Virus Sp2/0-Ag14 cells (M. Shulman et al., Nature, 276, pp. 269-70 (1978); American Type Culture Collection, ATCC CRL 1581)) were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, penicillin (100 units/ml), streptomycin (100 ⁇ g/ml) and 8-azaguanine (10 ⁇ g/ml). The Sp2/0-Agl4 cells were then maintained in the absence of 8-azaguanine for four to six days prior to fusion with spleen cells.
• DMEM Dulbecco's Modified Eagle Medium
• penicillin 100 units/ml
• streptomycin 100 ⁇ g/ml
• 8-azaguanine 10 ⁇ g/ml
• Selection medium used after fusion of spleen cells and Sp2/0-Ag14 cells comprised DMEM supplemented with 20% Zeta Serum (AMF Biological and Diagnostic Products Co., Seguin, TX) , hypoxanthine (13.6 ⁇ g/ml), aminopterin (0.18 ⁇ g/ml), and thymidine (3.9 ⁇ g/ml).
• Hybrid cells shown to secrete neutralizing antibody were cloned in medium comprised of DMEM supplemented with 20% fetal bovine serum, hypoxanthine (13.6 ⁇ g/ml), and 20% conditioned medium obtained from SP2/0-Ag14 cell cultures (cloning medium) .
• Vero African Green Monkey Kidney cells were grown in Eagle Minimum Essential Medium supplemented with 10% fetal bovine serum, penicillin (100 units/ml), streptomycin (100 ⁇ g/ml), and fungizone (0.25 ⁇ g/ml).
• PRV Root strain was propagated in primary rabbit kidney cells as described by Rea et al., J. Virol., 54, pp. 21-29 (1985).
• a female Balb/c mouse (Charles River Breeding Laboratories, Inc., Wilmington, MA), 5-6 weeks old, was injected subcutaneously with 0.2 ml of a soluble, inactivated PRV- infected cell extract (PR-VAC; Norden Laboratories, Lincoln, NE) on Day 0.
• PR-VAC soluble, inactivated PRV- infected cell extract
• the mouse was given booster injections subcutaneously with the same preparation on Day 14 and challenged with PRV (Rice Strain) intraperitoneally on Day 21 with a dose approximately 20 times the 50% lethal dose.
• PRV Raster Strain
• Spleen cells obtained from the immune mouse were fused with Sp2/0-Ag14 cells essentially using the procedure described by R. Kennett, Monoclonal Antibodies, Plenum Press, (1980). Spleen cells and Sp2/0-Ag14 cells were mixed at a ratio of 2:1 and washed twice with DMEM. After the final centrifugation, the cells were dispersed and the centrifuge tube containing the cell mixture was placed in a heating block at 40oC. One ml of 40% (w/v) polyethylene glycol 1450 (J.T. Baker Chemical Co., Phillipsburg, NJ) was added at a rate of 0.25 ml/15 sec.
• 40% (w/v) polyethylene glycol 1450 J.T. Baker Chemical Co., Phillipsburg, NJ
• the cell mixture was then incubated at 40oC for 1 min followed by addition of 1 ml of DMEM at a rate of 0.25 ml/15 sec. An additional 10 ml of DMEM was immediately added at a rate of 0.5 ml/15 sec. During all additions of polyethylene glycol and DMEM the cell mixture was periodically agitated to insure mixing. The cell mixture was subsequently centrifuged for 7 min at 1000 RPM. The cells were then dispersed and DMEM supplemented with 20% Zeta Serum was added. Cells were transferred by pipette into 96-well tissue culture plates so that each well received the equivalent of 2 x 10 5 spleen cells and 1 x 10 5 Sp2/0-Ag14 cells.
• the plates were incubated at 37°C in a humidified incubator with 5% CO 2 .
• One drop of selection medium was placed in each well on each of three consecutive days after fusion. Subsequently, the wells were fed weekly by replacing 75% of the medium with fresh selection medium.
• the isotype of monoclonal antibody secreted by each hybrid cell line was determined by an enzyme-linked immunosorbent assay using a commercially available isotyping kit (Hyclone Laboratories, Logan, UT) . This assay was conducted according to the manufacturer's specifications. Of the four finally selected hybridomas , two secreted IgG 2a , one secreted IgG 2b , and one secreted IgA (Table 1). Monoclonal antibodies 3A4, 3D1, 3D11, and 2A2 neutralized PRV only in the presence of complement.
• Hybridomas 3A-4C (UC HB-7), 3D-1C (UC HB-13) , and 3D-11C (UC HB-14) which produce monoclonal antibodies 3A4, 3D1, and 3D11 respectively and exemplify the invention, were deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 on December 18, 1985, and have been assigned accession numbers HB8980, HB8981, and HB 8982 respectively (Note: UC is a registered trademark of The Upjohn Co.).
• EXAMPLE 2 In this example we demonstrate that administration of the monoclonal antibodies of Example 1 protected mice from footpad challenge with PRV.
• microneutralization assay used to determine the neutralization titers of ascites fluids is described in Example 1. 3. Passive Transfer Studies
• mice Female CF-1 mice, 6-7 weeks old, were each administered 1 ml of the respective antibody or control preparations by the intraperitoneal route. Three hours after administration of the respective preparations mice were challenged by the footpad route with a volume of 0.05 ml containing PRV at 20 times the 50% lethal dose (LD 50 ). Mice were observed for mortality for a period of 21 days .
• the most protective monoclonal antibodies were those specific for PRV glycoproteins gp50 or gIII .
• monoclonal antibody 3D1 which is specific for PRV glycoprotein gll, was not protective despite those antibodies having a significant neutralization titer (Tables 2 and 3).
• monoclonal antibody 3A4 was the most protective, even though monoclonal antibody 3D11 had higher neutralization titers. Monoclonal antibody 3D11 neutralized in the absence of complement (neutralization titer - 40) at a very low level. Neutralization by the other monoclonal antibodies was dependent on the presence of complement.
• a challenge dose of 20 LD 50 was used in each of the two passive transfer studies .
• the mean day of death (MDD) observed for control mice that were administered Sp2/0 and hybridoma control ascites fluids in Study 1 were each 4.0 days, whereas in Study 2 the MDD for mice that were administered hybridoma control ascites fluid and saline were 5.8 and 7.1 days, respectively. Since the LD 50 determination is only an approximation, the actual challenge doses in Studies 1 and 2 were probably different, therefore accounting for the slight variation in MDD for control mice between studies. This explanation may also account for the differences in the survival of mice given monoclonal antibodies 3A4 and 3D11 when comparing Studies 1 and 2. These passive transfer studies indicated that monoclonal antibodies directed against gill and gp50 are protective, whereas the monoclonal antibodies directed against gll do not protect mice against PRV challenge.
• Study 1 Thirty-six crossbred pigs, 5-6 weeks old and of mixed sex, randomly allotted into six groups (six pigs/group), were used.
• Study 2 Thirty crossbred pigs, 5-6 weeks old and of mixed sex, randomly allotted into five groups, were used. Sixteen pigs of the same age were also used to determine the LD50.
• the monoclonal antibodies were prepared by culturing hybridoma cell lines in 16-liter-suspension culture vessels operated as 10 liters. Each 10-liter harvest was concentrated to 1 liter with a Pellicon Ultrafiltration unit using 10,000 MW cut-off membranes. The concentrate was brought to 4oC and 1 liter of saturated ammonium sulfate at 4°C was added slowly under vigorous stirring, allowed to stand for 1 hour, and centrifuged at 3000 xg. The precipitate was placed in Spectra/Por 6 dialysis tubing (2.7 mm dia.) and dialyzed for 24 hours at a 1:100 dialysate:buffer volume ratio.
• Dialysis buffer was 0.15 M NaCl in Milli-Q quality water. Each dialysate was then sterile filtered through 0.45 ⁇ filters and later divided into 6 doses in 50 ml sterile serum bottles.
• the LD50 was determined by the method of L. J. Reed and H. Muench, Am. J. Hygiene, 27, pp. 493-97 (1938).
• Nasal swabs collected from pigs were each placed in one ml of Eagles Basal Medium (BME; M.A. Bioproducts) supplemented with 3% fetal bovine serum (FBS) and antibiotics. Swabs were stored at -70o until they were assayed for the presence of virus. For the virus isolation assay, the nasal swabs in BME were thawed and the individual swabs were discarded. Samples (0.1 ml) were inoculated in duplicate onto porcine kidney-15 (PK-15; ATCC CCL33) cell monolayers and incubated for 1 hr at 37o to allow virus adsorption. An overlay of medium-199 (Flow Laboratories) supplemented with 4% FBS, antibiotics, and 1% agar was placed on the cell cultures. After 3 days the cell monolayers were stained with neutral red and the plaques were enumerated.
• BME Eagles Basal Medium
• FBS fetal bovine serum
• Pigs were treated as outlined in Table 5. Pigs in groups 1 through 3 were administered 5 ml ascites fluid containing monoclonal antibodies (3A4, 3D11, or 3D1) by the intraperitoneal (IP) route. The amount of immunoglobulin was not quantitated in this study. Pigs in group 4 served as positive controls and received 5 ml serum obtained from a pig recovered from PRV infection (convalescent pig serum). Pigs in group 5 served as negative controls and received 5 ml ascites fluid containing no monoclonal antibody.
• IP intraperitoneal
• Study 2 Pigs were administered about 150 mg of the respective antibody preparations by the intraperitoneal route (Table 4) .
• Control pigs were given either fetal calf serum proteins prepared in a manner similar to the monoclonal antibody preparations or convalescent pig serum.
• swine were challenged intranasally with a volume of four ml (two ml/nostril) containing PRV (Rice strain) at 20 times the 50% lethal dose (20 LD 50 - 5.0x10 4 plaque forming units/pig). Swine were observed for mortality for a period of 14 days and nasal swabs were obtained daily form each pig.
• swine were weighed prior to challenge (day 0) and at termination (day 14) or death.
• aRelative standard deviation is a combination of relative standard deviations from two or more independent fractions that were mixed to produce the formulated dose. bNote that the calculated concentrations of IgG have large standard deviations. The IgG concentrations were determined several times and in most instances were calculated to be within two standard deviations of 150 mg. 6. Criteria for Efficacy Evaluation
• mAb monoclonal antibody
• FBS fetal bovine serum
• c The neutralization titer is expressed as the reciprocal of the highest dilution of serum that protected greater than 50% of the cells from cpe.
• dFBS control was used as a negative control for protection and contained no PRV-specific antibody.
• Pigs that were given monoclonal antibody preparations did not gain weight as readily as pigs that were given convalescent pig serum. This may have been due to either the effect of convalescent pig serum antibodies in decreasing the onset of morbidity or the adverse effect of contaminants such as ammonium sulfate, phenol red, and bovine serum proteins present in monoclonal antibody preparations .
• Monoclonal antibody 2A2 did not protect in this study at the levels tested. While not wishing to bound by theory, this is possibly because it is an IgA immunoglobulin and not IgG as were those that protected. Also, as set forth supra, we did not quantitate the amount of immunoglobulin in Study 1 and therefore only a small amount of 2A2 may have been administered in the experiment.
• Composition of vehicle(s) e.g., water, saline, buffer suspensions of purified and/or processed monoclonal antibody or unaltered ascites fluid.
• Route of administration - intraperitoneal, intravenous, intramuscular or subcutaneous , oral or intranasal.
• Dosage regimen at first signs of pseudorabies in the herd, treat all susceptible (non-vaccinated) pigs. One dose is generally adequate but a second dose may be required in the event of further pseudorabies infections. No more than two doses should be administered due to the possible adverse immune reactions to mouse protein.
• Duration of protection - for the first dose up to about 10 days; for the second dose, no more than about one week.
• Vaccination with commercial vaccines during treatment administration of vaccines currently available commercially is contraindicated since the antibody of the monoclonal therapeutic agent will react with the vaccine antigen and cause rapid clearance of antigen and monoclonal antibody.
• Commercial vaccine may be administered at about seven days post therapy as long as a second dose of monoclonal antibody therapy is not contemplated.
• Amount of monoclonal antibody About 20 mg to about 300 mg of monoclonal antibody may be administered per pig. The preferable amount is from about 50 to about 150 mg per pig. Preferably about 1 ml to about 10 ml of a composition comprising the monoclonal antibody will be administered. The maximum administered will be about 50 ml. The amount of material administered will be route dependent and animal dependent (e.g., based on age, size, and general health). Less volume can be administered into the muscle or intravenously than can be administered intraperitoneally or subcutaneously.

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  • 1986-12-19 AU AU68339/87A patent/AU597934B2/en not_active Expired - Fee Related
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