WO1983002394A1

WO1983002394A1 - Bluetongue virus vaccine, method of producing same, and method of immunizing ruminants therewith

Info

Publication number: WO1983002394A1
Application number: PCT/US1983/000052
Authority: WO
Inventors: A&M University System Texas; Stewart; McCONNELL; Georgia A.; CUMMINGS; Jr. Charles W. LIVINGSTON
Original assignee: Texas A & M Univ Sys
Priority date: 1982-01-19
Filing date: 1983-01-14
Publication date: 1983-07-21
Also published as: JPS58502210A; ZA83114B; CA1226544A; NZ203030A; DK422783D0; IL67633A0; DK422783A; EP0098867A1; AU1155583A

Abstract

Ruminants, particularly cattle, sheep, and goats, can be protected against bluetongue virus (BTV) infection, particularly International Serotypes IT 10, IT 11, IT 13, and IT 17, by parenteral inoculation with the vaccine of the present invention. The vaccine is prepared by attenuating the individual BTV serotypes in at least thirty serial passages through a continuous cell line not originating from ruminants, in particular the "Vero" cell culture derived from the African Green Monkey.

Description

BLUETONGUE VIRUS VACCINE, METHOD OF PRODUCING SAME, AND METHOD OF IMMUNIZING RUMINANTS THEREWITH

This invention relates to a vaccine for immuniz¬ ing ruminants against International serotypes of bluetongu virus (BTV) and a method pf m'a ing said vaccine, and a me¬ thod of immunizing ruminants with said vaccine. Bluetongue, an arthropod-borne viral disease, oc curs in cattle, sheep, goats, and wild ruminants. Blue¬ tongue lesions in affected animals resemble infectious bo- vine virus diarrhea, vesicular stomatitis, malignant catar rhal fever, mycotic stomatitis, rinderpest, photosensitiza- tion, and foot and mouth disease. Bluetongue virus (BTV) has been incriminated as a cause of hydranencephaly in cat¬ tle and of infertility, abortion, and birth of defective young in cattle and sheep. Twenty serotypes are reported in the literature as causing problems ranging from inap- parent infection to acute fulminating infection. Chronic, persistent virus shedding cattle have also been recognized. The virus infects the endothelial cells of the blood vas¬ cular system.and the periendothelial cells and the peri- cytes of capillaries, pericapillary arteriodes, and venules Stair, et al., 5 Pathologia Vet 164 (1968). The virus is associated with the reticulo-endothelial system and may occur within the spermatozoa of infected bulls. Luede et al, 3 Proc. 20th World Vet Congress 2039 (1976) .

The direct losses resulting from mortality caused by BTV are sometimes high, but indirect losses are of even greater economic importance. With BTV there is a marked loss of body condition and marketing of slaughter animals may be delayed. In BTV-infected sheep, wool growt may be impaired by the development of wool breaks which produce a defective or low yielding fleece.. The marked debility following BTV infections may result in a lowering .of resistence to secondary bacterial or chlamydial infec¬ tions and other predatory factors. The reproductive effi¬ ciency of infected animals is also adversely affected. Abortions and defective offspring are observed in infected animals, and some animals may be barren for one or more breeding seasons. The most significant damage inflicted by bluetongue infections is economic loss resul^'ting from embargoes and stringent testing requirements imposed on • producers who export cattle, cattle semen, and sheep from bluetongue endemic areas.

Under natural conditions, transmission of the virus occurs via the bites^" of at least four Culicoides species, e.g., sand flies, midges. The biological trans¬ mission^* of BTV between cattle and sheep by the same culi- coid vector has been demonstrated experimentally. uedke et al, 28 AJVR 457 (1967) . Cattle, sheep, and many species of wild ruminants may act as reservoirs of BTV producing a means for the virus to overwinter. A persistent BTV vire- mia which can last as long as three years has been identi- fied in cattle. Hourrigan, 51 Aust Vet J 170 (1975). Once BTV becomes established in a country, the virus is virtual¬ ly impossible to eradicate. Erasmus, 51 Aust Vet J 209 (1975) .

The etiologic agent of bluetongue belongs to the family Reoviridae, genus Orbivirus. This name designation was changed from "reo-like" virus to Orbivirus by the In¬ ternational Committee on viral nomenclature in 1971. The viral etiology of bluetonge was established by Theiler in 1906. Erasmus, 51 Aust Vet J 165 (1975). Since then sev- eral reports have appeared in the literature which (a) con¬ firm the isolation of bluetongue virus from cattle, sheep, goats, and a number of wild ruminants in various parts of the world, (b) describe the transmission, the epizootiolog and the clinical and pathological features of the blue¬ tongue diseases, and (c) describe infections resulting from different BTV serotypes. For example, see DuToit, 19 Onderstepoort J Vet Sci Anim Indus 7 (1944) ; Komarov and Goldsmit, 8 Refuah Vet 96 (1951); Price and Hardy, 124 J Am Med Assn 255 (1954); Shope et al, 111 J Exp Med 155 (1960); Livingston and Hardy, 25 AJVR 1958 (1964); Luedke et al, 30 AJVR 511 (1969); Hourrigan and Klingsporn 51 Aust Vet J 170 (1975) . Immunological control of bluetongue in the

United States was first attempted by McKercher et al, 18 AJVR 310 (1975) , with a BTV International serotype 10 vac¬ cine grown in fertile hen eggs. This product was patterne after that of Alexander, 21 Onderstepoort J Vet Sci Anim Indus 231 (1947) , who first succeeded in propagating the bluetongue virus in chicken embryos. Today, vaccination is routinely carried out in South Africa and Israel using an egg-attenuated polyvalent live virus vaccine containing a number of bluetongue strains. An egg-adapted vaccine produced by Cutter Laboratories and used in the United States has been taken off the market because of severe reactions in vaccinated sheep. Subsequently, Kemeny and Drehle, 22 AJVR 921 (1961), adapted the BTV International type 10 from eggs to bovine kidney cell cultures. This modified live virus vaccine, produced by Colorado Serum Company, is used for sheep in the United States.

Heretofore, there has been little information regarding vaccination for BTV in cattle. A successful vaccine has been greatly needed for some time because cat- tie have a major role in the epizootiology of BTV. It has been recognized that an immune cattle population would contribute significantly to the overall control of the disease. Erasmus, 51 Aust Vet J 213 (1975).

Accordingly, the object of the present invention is to provide a vaccine having the aforesaid advantages and in particular to provide a effective vaccine for protecting cattle, sheep, and goats against BTV. In accordance with the present invention, it has been found that bluetongue viruses can be adapted to and propagated in cell cultures derived from tissues that do not originate from cattle, sheep, goats, or wild un- gulates of the family Bovidae and the virulence so modi¬ fied and reduced that no symptoms of the bluetongue virus disease are observed upon parenteral inoculation.

Accordingly,, the present invention provides for a vaccine for immunizing ruminants against International serotypes of bluetongue virus IT 10, IT 11, IT 13, and

IT 17, comprising at least about 10 tissue culture infec¬ tious doses of one or more of bluetongue virus serotype IT 10 , IT 11, IT 13, or IT 17 per ml, wherein the virus serotype is attenuated so that upon parenteral administra- tion to said ruminants the vaccine is capable of stimulat¬ ing production of a host defense mechanism comparable to that produced by natural infections without producing the pathological responses of disease due to the bluetongue virus serotypes. The present invention also provides for a method of preparing a vaccine for parenteral innoculation of ru¬ minants against a bluetongue virus, which comprises culti¬ vating and attenuating the bluetongue virus by 30 or more serial passages through Vero cell cultures in a nutrient fluid.

The present invention also provides for a method of immunizing cattle, sheep, or goats against bluetongue virus which comprises parenterally administering to said cattle, sheep, or goats at least 2 ml of a vaccine compris- ing at least one or more of bluetongue International virus serotype IT 10, IT 11, IT 13, or IT 17, wherein said virus is attenuated by 30 or more serial passages through Vero cell cultures in a nutrient fluid at an incubation tempera¬ ture of from about 30°C to about 36°C, each passage lasting from about 1 day to about 10 days.

The present invention also provides for a method of immunizing cattle, sheep or goats against bluetongue virus which comprises parenterally administering to said

- ϋR OM cattle, sheep, or goats at least 2 ml of a vaccine compri- sing at least about 10 tissue culture infectious doses of bluetongue virus International serotype 13 per ml, wherein said virus is attenuated by at least 3 blind pas- sages in pathogen-free embryonating hen eggs prior to 'said 30 or more serial passages through Vero cell cultures.

The vaccine is safe in that it will not cause overt disease in ruminants that receive it by the paren¬ teral route. Further safety is provided in that the viruses will not pass from vaccinated ruminants to other ruminants in contact with those vaccinated, thereby re¬ ducing the possibility of reversion to virulence by animal passage in these species. This constitutes a significant advance in the control of BTV. The basic method for producing the present vaccine comprises cultivating and attentuating the blue- tongue virus by at least thirty serial passages through "Vero" cell cultures in a nutrient fluid. The Vero cell line is derived from the African Green Monkey as described in more detail below. The serial passages are conducted at incubation temperatures of from about 30 C to about 36 C, and each passage lasts from about 1 day to about 10 days.

The vaccine may be produced in liquid form suit- able for parenteral inoculation into ruminants. Such liquid vaccine contains at least about 10 4 tissue culture infectious doses of each BTV serotype being protected against per ml. A suitable dosage for mmunizing cattle, sheep, or goats is about 2 ml of such liquid vaccine. Live virulent bluetongue viruses can be ob¬ tained from cattle and sheep infected with the viruses according to the methods of isolation and identification described in the literature. For example, see Livingston and Moore, 23 AJVR (1962); Goldsmit and Barzilai, 22 efuah Vet 279 (1965); Luedke et al, 30 AJVR 511 (1969). The serotypes of bluetongue virus for which this invention is most useful are International serotypes 10, 11, 13, and 17 which occur in the United States. It is understood that these specific viruses are by way of illύstratio only and that the present invention can be adapted to other BTV serotypes. These four viruses are serological- ly related as determined by the conventional agar gel precipitin test (e.g., each virus contains the same group antigen) but do not cross-protect as determined by sheep inoculation tests or by the conventional serum neutrali¬ zation tests. These four serotypes are included in a listing of several bluetongue virus serotypes presented by DeVilliers, 3 Intervirology 47 (1974). The BTV IT, 10, IT 11, IT 13 and IT 17 serotypes have been deposited in The American Type Culture Collection (ATCC) Rockville, Maryland, U.S.A., and catologued in the Catalogue of Strains as VR-187, VR-872, VR-873, and VR-875, respect¬ ively. In preparing the vaccine of this invention, it has been found that attenuation and modification of the virulent BTV may be accomplished by cultivating and mul¬ tiplying each BTV serotype in at least 30 continuous serial passages (BTV IT 13 may be first adapted to em- bryonating hen eggs with three blind passages) in a susceptible permanent cell line, such as "Vero," a continuous cell line derived from the-African Green Monkey (Cercopithecus aethiops) subculture levels 128 through 150, at low temperatures, preferably 30-36 C, and most preferably 32 C. After each series of five pas¬ sages within the 30 serial passages, the virus is puri¬ fied by filtration through a membrane with an average pore diameter of 220nm, by chloroform treatment prior to use^* in master pools, and by standard terminal dilution techniques.

To begin the preparation of the vaccine of the present invention, whole blood collected from infected sheep, such as that supplied by the USDA, Arthropodborne Viral Diseases Laboratory, Denver, Colorado, may be used. The lowest available sheep passage levels of BTV strains are utilized. The serotypes BTV IT 10, 11, and 17, are

OMPI processed as disrupted washed red blood cells, inocula¬ ted onto monolayer cultures of Vero cells, grown accord¬ ing to the techniques of Dulbecco and Vogt, 99 J. Exptl Med 167 (1954) and Younger, 85 Proc Soc Exp Biol and Med 202 (1954), and serially subjected to blind passage until suitable cytopathic effect develops. Each is then plague-purified three times using a terminal dilution scheme, and the plague-purified viral prpgeny is then subjected to attenuation. Ovine-virulent BTV IT 13 (strain 67-41B) is. first adapted to the chicken embryo by inoculating intra- veneously into twelve-day-old embryos 0.1 ml of a disrup¬ ted suspension of washed sheep red cells. These embryos are collected at selected intervals after the initial inoculation, and heart tissues from the collected embryos are serially passed three times as 20% suspensions in chicken embryos using a similar inoculum size, each such passage lasting from three to seven days. Subsequently, this specific serotype BTV IT 13 is adapted to Vero cells, and plaque-purified by the standard Dulbecco technique, 38 Proc Nat Acad Sci 747 (1952) .

The passage time intervals should be such as to allow for maximum virus replication between passages, typically

-8-

from one to ten days and preferably from two to seven days. The optimum passage time can be determined by εtaκ3ard cell culture procedures, for example, by cyto¬ pathic observations wherein the virus is allowed to 5 replicate during each passage to the point where gross cytopathic effect (CPE) can be observed and 75-100% of t cell monolayer is destroyed.

In accordance with the invention, therefore, a meth 0 is provided for attenuating virulent strains of BTV, whi when used alone or in optimal proportions and parenteral inoculated, preferably intramuscularly, into sheep, goat or cattle, will immunize against homologous BTV. The method comprises introducing an inoculum of virulent BTV 5 into a nutrient cell culture medium at a temperature of from about 30'C to about 36'C, preferably 32^*C, for a period of from.one to ten days, and thereafter serially passing the viruses through additional such Vero cell cultures for a total of at least 30 consecutive passages 0

The viral preparations produced by this invention m be diluted to adjust potency and may have added to them stabilizers, such -as lactose, dextrose, or other non-tox substances. The viral preparations may also be desiccat -**^* e.g., by freeze-dryin , for storage purposes or for sub¬ sequent formulation into liquid vaccines. Stabilizers useful in freeze drying of viruses are described in Rightsel et al, 3 Cryobiology 423 (1967) and Greiff et a Advances in Freeze Drying, 103-122 (1966).

The present invention is described in more detail i the following specific examples. These examples describ the BTV vaccine of present invention, as well as the - 4-

ethod of preparing the vaccine and the method of immuniz¬ ing ruminants with the vaccine, both of which are also part of the present invention.

Example IA

A sample of live virulent bluetongue virus, BTV Texas Station Strain, an IT 11 serotype, (Sheep No. 2989, day after infection 7, whole blood in OPG), was obtained from whole blood stored at 4^*C. A 3.0 cc sample of blood was centrifuged at 1000 rpm for 10 minutes. The supernatant fluid was discarded, and the packed red blood cells were resuspended in 1.0 cc of a balanced salt solution fphos- . phate buffered saline ( PBS)J, The cells were disrupted in - a Ten Broeck tissue grinder, and the disrupted cells were ^' brought to a final volume of 3.0 cc with PBS. A quantity of 0.5 cc lysed red blood cell suspension was added to a decanted monolayer of Vero cells and allowed to adsorb for 1 hour at 30-34^*C in standard tubes (16 x 125 mm) prepared as described below.

. The Vero cell line used is the cell line initiated by Yasamura and Kawakita at the Chiba^* University in Chiba, Japan. 21 Nippon Rinsho 1201 (1963). This cell line was brought from the Chiba University to the Laboratory of Tropical Virology, National Institute of Allergy and Infectious Diseases, National Institutes of Health,' in the 93rd passage. Subsequently, the cell line was deposited with the American Type Culture Collection, at passage level 113 and distributed by the ATCC as CCL81 Vero passage No. 122. Each cell line tube containing 2 ml of growth medium consisting of Eagle's Minimum Essential Medium in Earle's balanced salt solution (EMEM) supple¬ mented with non-essential amino acids (NEAA) 1%, fetal bovine serum 10%, sodium pyruvate 1%, and Kanamycin 200 mcg/ml, was seeded with 1 cc of suspended Vero cells (ca. 250,000 cells/ml). The pH was adjusted with sodium bicarbonate to maintain a pH of about 7.0 to 7.2. The cells were allowed to grow at 34.5^*C for about three days until a confluent monolayer of cells was achieved.

The growth medium was poured off, the cell monolayer washed with prewar ed Hanks balanced salt solution (HBSS), and 0.5 cc of virus-infected disrupted red blood cells added. Adsorption was allowed to proceed at 30^*-34'C for one hour. Two cc of a maintenance medium consisting of Basal Medium Eagle (BME) in KBSS, supplemented with 3% horse serum, 2% tryptose phosphate broth, 100 units of penicillin, and 100 meg streptomycin was added. The tubes were then incubated at 30-34^*C in a roller drum incubator. About 3-5 such tubes were utilized per viral passage. In each passage the CPE was allowed to develop until 75% or more of the cell monolayer was destroyed, and the samples were then rapidly frozen at -85^*C.

The contents of the tubes were harvested and sub¬ jected to identical serial passages for 30 to 50 addi¬ tional passages. The 29th, 39th, and 49th passages were subjected to an additional passage for the purpose of in¬ creasing volume. Prior to passage for increasing volume, • the virus suspension was treated with chloroform according to the procedure of Feldman and Wang, 106 Proc. Soc. Exptl. Biol. Med. 736.(1961), and inoculated onto con- fluent monolayers of Vero cells grown in an antibiotic free medium. The maintenance medium in this case was BME(H) supplemented with 10% lamb serum and 10% tryp¬ tose broth. At maximum CPE, the 30th, 40th, and 50th passage pool were harvested, identified, and titrated by conventional methods. The pool thus prepared constituted a bulk vaccine which could he diluted according to the titer or could have added thereto stabilizers or other nontoxic sub- stances. For use as a vaccine, it is combinable with the other serotypes of BTV prepared in a similar manner and desiccated, or it may be prepared in liquid form as a ultivalent vaccine.

In propagating or attenuating the viruses, any nontoxic nutrient fluid tissue culture medium could have been utilized-. In addition to the supplemented EMEM and BME mediums described above, it will be understood that other nontoxic nutrient fluid tissue culture mediums could also have been used.

EXAMPLE IB

The work of Example IA was repeated, substituting a sample of^* live virulent bluetongue virus BTV BT262, an IT 7 serotype, (62-45S, Sheep No. 2790, day after infection 7, whole blood in OPG) for BTV Texas Station Strain. This sample, BTV BT262, was processed in the identical manner specified for the BTV Texas Station Strain in ^"Example IA.

EXAMPLE IC

The work of Example IA was repeated, substituting a sample of live virulent bluetongue virus BTV BT8, an IT 10 serotype, (Sheep No. 2617, day after infection 7, whole blood in OPG) for BTV Texas Station Strain. This sample, BTV BT8 (2617), was processed in the identical manner specified for BTV Texas Station Strain in Example IA.

OMPI . EXAMPLE ID

The work of Example IA was repeated, substituting a sample of virulent bluetongue virus BTV 6741-B, an IT 13

5 ' serotype (BT Ox 190, Sheep No. 2731, day after infection

7, whole blood in OPG) for BTV Texas Station Strain. This sample, BTV 6741-B, was processed in the identical manner specified for BTV Texas Station Strain in Example IA except that, just prior to inoculation onto Vero cell 0 cultures, this sample was adapted to the chicken embryo by inoculating intravenously into twelve-day-old embryos 0.1 ml of a disrupted suspension of washed sheep red cells. These embryos were collected at selected ^"intervals after the initial inoculation, and heart tissues from the 5^' . collected embryos were serially passed three times as 20% suspension in chicken embryos using a similar inoculum size, each such passage lasting from three to seven days. After this adaptation to the chicken embryo, this sample was adapted to Vero cells in the same manner described in o Example IA.

EXAMPLE II

Two ml of a vaccine prepared according to Example IA 5 (IT 11) serially passed 20 times (subsequent to plaque purification) and titrated to contain a virus titer at 30^*-34^*C of 10⁵-0 TCID50/-T1I as determined by CPE was administered intramuscularly to 5 susceptible yearling sheep. (TCID50 = tissue culture infective dose giving a 0 50% CPE at the highest dilution). Five other yearling sheep were maintained as unvaccinated controls. All 10 sheep were previously determined to be sero-negative to BTV. Evidence of severe BTV disease was generally ob¬ served starting with^'pyrexia on the 5th to 8th day, and 5

OIAΕI O other clinical signs were seen from about the 6th to 8th day onward after normal exposure or challenge with viru¬ lent BTV.

The antibody titer of the 5 inoculated yearling sheep was negative by the agar gel precipitin (AGP) test, and less than 1:5 by the serum neutralization (SN) test. One month later the antibody titer was positive by the AGP test for five of the sheep, greater than 1:20 by the SN

.10 test for three of the sheep, and remained undetectable by both tests for none of the sheep.. All 10 yearling sheep 'were challenged parenterally with 2 ml of homologous virulent virus containing 500-1000 Sheep Intective Doses (SID)/ml (determined by sheep challenge). The yearling

15. sheep were observed for 30 days for evidence of disease. All of the 5 vaccinated sheep remained normal with no clinical disease cr symptoms in contrast to 5 unvaccinated sheep which become ill with BTV disease exhibiting typical signs such as febrile response, lack of appetite and

20 general malaise, coronitis, and ulceration of the dental pa .

EXAMPLE IIIA

" Live virulent BTV, cultured and isolated by standard tissue culture techniques and designated as BTV IT 11 were serially passed 10 consecutive times (subsequent to plaque- purification) in continuous Vero cell onolayers at 3-7 day intervals at about 32"C. The 10th passage was tested

30 in sheep and found to have a residual virulence as evi¬ denced by the febrile response elicited and the appearance of mild clinical signs of BTV infection. Whole blood collected at peak of infection was subpassaged into additional susceptible sheep and clinical signs of disease

-**^■■ were observed (virus still virulent).

M An additional 9 serial passages were made in Vero monolayer cell cultures at 7-10 day intervals. The 19th virus passage was expanded to build up volume. The 20th viral passage was titrated for viral content, identified by serum neutralization, and used as a trial vaccine.

Vaccination of sheep with the 20th passage material pro¬ duced sigπifiant amount of protective antibodies enabling the sheep to resist virulent BTV. However, this 20th passage material was found to have a residual virulence effect as determined by febrile response. No other signs or symptoms o.f clinical disease were noted.

EXAMPLE IIIB

« Live virulent BTV, cultured and isolated by standard tissue culture techniques and designated as BTV IT 10 was serially passed in the same manner as described in Example IIIA for BTV IT 11. The same serial passages were tested in sheep in the same manner and with the same results as tor BTV ΪT 11 in Example IIIA.

EXAMPLE IIIC

Live virulent BTV, cultured, and isolated by standard tissue culture techniques and designated as BTV IT 13 was serially passed in the same manner as described in Example IIIA for BTV IT 11. The same serial passages were tested in sheep in the same manner and with the same results as for BTV IT 11 in Example IIIA.

EXAMPLE IVA

The 20th passage virus material from Example IIIA was serially passed in tubes containing a confluent monolayer of Vero cells at 30-34^*C for a variable number of passages, At the 29th and 39th passage, the viral progeny were further expanded to make vaccine pools of the 30th and 40th passage level materials. Each pool was produced in antibiotic free medium, titrated to contain about 10⁴-^*0 TCIDso/ml (determined by CPE) and frozen at -^'85'C. Prior to inoculation of Vero cell monolayers, the virus was treated with chloroform to inactivate contaminants and enhance viral infectivity.

The 30th and 40th passage pools were used as vaccines by the procedure described in Example II. Briefly, five susceptible yearling sheep were vaccinated with 2.0 ml of the 30th passage material administered parenterally. Five other yearling sheep were.maintained as unvaccinated controls. All 10 sheep are previously determined to be sero-negative to BTV. Each sheep was monitored daily for pyrexia and for evidence of clinical disease. After 28 days, all 10 sheep are challenged parenterally with virulent BTV by administering a total dose of 2.0 ml containing approximately 500-1000 SID/ml. The sheep were observed for 4 weeks for evidence of clinical disease. All five vaccinated sheep remained normal with no evidence of clinical disease or symptoms, in contrast to the five unvaccinated control sheep which became ill with BTV disease exhibiting typical symptoms for the challenge level, such as febrile response, lack of appetite, and general malaise. The 40th passage material was tested in a similar manner, and the results were comparable to those observed for the 30th passage material.

EXAMPLE IVB

The 20th passage virus material from example IIIB was serially passed in the same manner as in Example IVA to produce 30th and 40th passage pools of BTV IT 10. These 30th and 40th passage pools were tested in the same manne and with the same results as for BTV IT 11 as in Example IVA.

EXAMPLE IVC

The 20th passage virus material from example IIIC wa serially passed in the same manner as in Example IVA to 0 produce 30th and 40th passage .pools of BTV IT 10. These 30th and 40th passage" pools were tested in the same manne and with the same results as for BTV IT 11 as in Example IVA.

5 EXAMPLE IVD

The 20th passage virus material from example II was serially passed in the same manner as in Example IVA to produce 30th and 40th passage pools of BTV IT 10. These o 30th and 40th passage pools were tested in the same manne and with the same results as for BTV IT 11 as in Example IVA.

EXAMPLE V

5 A virus material containing a combination of two serotypes, IT 10 and IT 11, IT 11 and IT 17, and IT 10 and IT 17 was prepared according to the procedure detailed in Example IA-IC. A 20-ml amount of this material containing each of the listed combinations of serotypes obtained at 0 the fiftieth passage level was dispensed into standard vaccine vials and frozen at -90^*C to -80^*C for inoculation purposes. The frozen vials were thawed rapidly, and each of six sheep (2 per dual vaccine) were given 2.0 ml parentally with a titer of about 10^4*5 TCID_5Q/ml. The 5 sheep were maintained in separate isolation facilities a monitored daily for disease. Blood was collected in OPG on the seventh and the fourteenth day after vaccination for virus isolation studies. All sheep were bled for pirevaccination antibody titer.

One month later, the vaccinated sheep were given a virulent virus challenge (one of each of the pair with a different single virus) dose consisting of 500-100 SID/m of the serotypes represented. For four weeks after challenge, the sheep were observed daily with no clinica signs of disease observed. In contrast, unvaccinated sheep^' exhibited typical- manifestations of BTV disease. •

Two months after vaccination, the sheep were given virulent, virus challenge ^'(with the alternate virus not used .previously) dose consisting of 500-100 SID/ml. For four weeks after challenge the sheep were observed daily with no clinical signs of disease observed. In contrast unvaccinated control sheep exhibited typical manifesta¬ tions of.BTV disease. Antibody determinations before an after vaccination showed all sheep to be sero-negative b the agar gel precipitin test before vaccination and four of six to be sero-positive after vaccination. Blood samples collected on the^' seventh and fourteenth day after vaccination were assayed for circulating vaccine virus an found to be negative. The vaccinated sheep were* fully protected from disease as compared to the unvaccinated controls.

This example shows that the attenuated bluetongue virus contained in the vaccine is fully protective and does not circulate in blood in high enough titers to be detected by conventional laboratory methods. EXAMPLE VI

Vaccines of fiftieth passage level material were pre pared for each of the four serotypes as in Examples IA-ID. Each vaccine was undiluted and was dispensed into standar vaccine vials and frozen at -90"C to -80^*C for inoculatio purposes. The frozen vaccines were thawed rapidly, and each of .20 sheep were given^' 2.0 ml parenterally. All sheep were bled for prevaccination antibody titer and wer monitored daily for evidence of pyrexia or other untoward response to the vaccine. One month later each of five sheep were challenged with a different serotype of virule BT virus, i.e., five with BTV IT 10, five with IT 11, fiv with IT 13 and -five with IT 17. Similar challenges were given to unvaccinated controls.

One serotype, IT 10, failed to protect against virulent virus challenge. The other three strains were protective. All serum samples assayed for antibody were negative. A retrospective titration of the vaccine residue show the vaccine titer of IT 10 to contain less

2 than 10 TCID__n/ml of virus. This example shows that the vaccine failed because of processing difficulties.

Where sufficient virus was present, adequate protection was elicited in the vaccinated sheep.

EXAMPLE VIIA

A vaccine was prepared as in Example IA but with the fiftieth passage level material. The vaccine was diluted to contain either 10⁴ TCID₅₀/ml, 10³ TCID₅₀/ml, or 10² TCIDc_Q/ml and was dispensed into standard vaccine vials and frozen at -90'C to -80^*C for inoculation purposes. The frozen vials were thawed rapidly, and each of 72 sheep

OMP 4 were given 2.0 ml parenterally, 24 with a titer of 10

TCID₅₀/ml, 24 with a titer of 10³ TCID₅₀/ml, and 24 with titer of 10 TCID_5Q/ml. All sheep were bled for prevac- cination antibody titer and marked for future study.

One month later, all vaccinated sheep were bled for post-vac-cinal antibody titers and were given a virulent virus challenge dose consisting of 500-1000 SID/ml of a homologous virus. For four weeks after challenging, the sheep were observed daily with no overt clinical signs of disease observed. There was, however, a body temperature rise measured in each of the groups. But, the percentage

' ' 4 of febrile responses was lower in the 10 TCID_j._f./ml and unvaccinated control group. Antibody determinations before and after vaccination showed the vaccinated sheep to be sero-negative to the vaccine by the agar gel pre- cipitin test and the serum neutralization test (serum dilution 1:5). However the vaccinated sheep were fully protected from disease. This example shows the vaccine t be protective but it has lost its antigenic marker as determined by the serological tests used.

EXAMPLE VIIB

The work of Example VIIA was repeated, substituting sample of fiftieth passage level BTV IT 10 for BTV IT 11. This sample (IT 10) was processed in. a similar manner except that dilutions of 10 TCID-g/ml, 10² TCID_5Q/ml, an

10 TCID__n/ml were used. Similar results were obtained f 10 3 TCID_5Q/ml dose, but the other two dose levels were unacceptable. EXAMPLE VI IC

The work of Example VIIA was repeated substituting a sample of fiftieth passage level BTV IT 17 for BTV IT 11. This sample (IT 17) was processed in a similar manner except that dilutions of 10⁵ TCIDso/ml, 10⁴ TCID₅₀/ml, and 10³ TCID5o/ml were used. Similar results to those found in Example VIIA were obtained.

EXAMPLE VIII

A virus material containing the four serotypes, BTV IT 10, IT 11, IT 13, and IT 17, is prepared according to the procedure detailed in Example IA-ID. A 2000-ml amoun of this virus material containing all four serotypes ob¬ tained at the fiftieth passage level was dispensed into standard vaccine vials and frozen at -90^*C to -80^*C for inoculation purposes. The froze.n vials were thawed rapidl and each of 1000 sheep were given 2.0 ml parenterally, with a titer of about lO⁵- τciD5o/ l. The sheep were ' maintained in separate flocks," 500 in "A" flock, 250 in "B" flock, and the -rest in flocks "C" and "D". A random selection of 50 sheep from flocks A and B were bled for prevaccination antibody titer and marked for future study. Ten percent of flocks C and D were also pre-bled and similarly marked.

One month later, the tagged vaccinated sheep were given a virulent virus challenge dose consisting of 500-1000 SID/ml of each of the four serotypes. For four weeks after the challenge, the sheep were observed daily with no clinical signs of disease observed. In contrast, unvaccinated sheep exhibited typical manifestations of BTV disease. Antibody determinations before and after vac- cination showed the sheep to be sero-negative by the agar

OMPI gel precipitin test and the serum neutralization test (serum dilution 1:5). However, the vaccinated sheep were fully protected from disease as compared to the unvacci¬ nated controls.

This example shows that the attenuated bluetongue virus contained in the vaccine is fully protective but has lost the antigenic marker as determined by the serological tests used. The work recited in this example was repeated two additional times at yearly intervals with similar results.

EXAMPLE IX

.. _ A vaccine was prepared as in Example VIII but with fortieth passage level material. A 2-ml sample of such - vaccine with a virus titer of about 1θ5^«5 TCID5o/ml was administered parenterally to susceptible cattle (5 each) and goats (10 each) which had been previously determined to be sero-negative to BTV. Other cattle and goats are maintained as unvaccinated controls^'. The antibody status of all animals prior to vaccination was less than 1:5 for goats and 1:10 for cattle by the serum neutralization test and negative by the agar gel pre- • cipitin test. One month later just prior to challenge, the antibody titer varied from negative to positive by the agar gel precipitin test and from 1:10 to 1:40 on serum neutralization assay. All aminmals were challenged parenterally with virulent BTV of all four serotypes, total dose of 2.0 ml containing 500-1000 SID/ml of each of the four serotypes of BTV. The cattle and goats were observed for 30 to 90 days for evidence of clinical disease. All the vaccinated animals remained normal with no clinical disease or symptoms noted.

OMPI EXAMPLE X

A 200-ml sample of each of the fortieth passage viru materials obtained as described in Example IVA-IVD was used for inoculation purposes. Prior to vaccination, all experimental yearling sheep were found to have an antibod titer of less than 1:5 by the serum neutralization test and to be agar gel precipitin test negative. A 2-ml amount of a polyvalent vaccine containing equal titer equivalents of BTV IT 10, 11, 13 and 17 prepared by diluting mother pools of stock virus of each serotype to contain about 1θ5-0 TCIDso l was administered paren¬ terally to each of 20 susceptible yearling sheep with other unvaccinated sheep maintained as unvaccinated controls. Each sheep was monitored daily for evidence of pyrexia or other untoward response to the vaccine. One month later, the 20 vaccinated yearling sheep were divide into four groups of five each, and each group was chal¬ lenged with 500-1000 SID/ml of virulent BTV, i.e., each group of five sheep received BTV IT 10, 11, 13 and 17, respectively. Appropriate unvaccinated control sheep were included for each group.

Serum samples collected just prior to virulent virus challenge and tested by the agar gel precipitin test showed that greater than 60% of the vaccinates had group specific BTV antibody in their serum, whereas no antibody was found in the unvaccinated controls. All twenty of the vaccinated sheep were re-challenged at 30 day inter- vals with each remaining BTV serotype and again monitored daily for signs of clinical disease. In each challenge experiment, the scheme of the challenges being shown in Table ϊ,. the vaccinated sheep remain normal with no clinical disease or symptoms in contrast to the unvac¬ cinated controls which became ill with BTV disease exhibiting typical symptoms such as febrile response, edema, lameness, lack of appetite, and general malaise.

Table 1: Scheme for Rotating Virulent Virus Challenge

Sheep

(5/group) IT 10 IT 11 IT 13 IT 17

10

A Day 30* Day 60 Day 90 Day 120

B Day 120 Day 30 Day 60 Day 90

C Day 90 Day 120 Day 30 Day • 60

D Jay 60 Day 90 Day 120 Day 30

15

*Each group was challenged with serotype given on day specified. Appropriate controls were included for each test day.

20 . EXAMPLE XI

•A 200-ml sample of vaccine prepared as in Example X was used for inoculation purposes. Prior to vaccination, 80% of the experimental cattle were found to be agar gel -c precipitin test negative. A 2-ml dose of a polyvalent _^- vaccine containing equal titer equivalents of BTV IT 10, 11, 13 and 17 prepared by diluting mother pools of stock virus of each serotype to contain about 10⁴ TCIDC._Q/IΠ1 was administered parenterally to each of 69 pregnant (termina 0 aspect of gestation period) cows with other cows main¬ tained as unvaccinated contact controls. Fifty days later, the cows were bled for serum precipitin antibody assay and for testing for presence of virus in the blood.

5 In addition, similar samples were collected for assay 96 days after vaccination from the cows and 58 nursing calve born to vaccinated cows.

All blood samples assayed for virus were negative.

All vaccinated animals remained normal with no clinical disease or signs noted after vaccination. The product was safe, non-abortogenic or teratogenic as evidenced by clinical evaluation of newborn calves. In addition, 41% of the nursing calves were agar gel precipitin test antibody positive, showing that maternal antibody was obtained via colostrum intake.

EXAMPLE XII

A 500-ml sample of vaccine prepared as in^* Example X was used for inoculation purposes. Prior to vaccination, all cattle were pregnancy palpated, and 58% cows tested were found to be pregnant. A 2-ml dos.e of a polyvalent vaccine containing equal titer equivalents of BTV IT 10, 11, 13 and 17 prepared by diluting mother pools of stock virus of each serotype to contain about 10⁴ TCIDso/ml was administered to each of 180 cows in various stages of pregnancy from 45 days to 8 months and to the 4 herd sires. The cattle were monitored daily for evidence of clinical disease and abortion. Sixty days later, the cow were pregnancy palpated, and 72% of the cows were found t be pregnant. This study shows that the vaccine is non- abortogenic and does not cause transient or long term infertility.

EXAMPLE XIII

A 50-ml sample of vaccine prepared as in Example X was used for inoculation purposes. Prior to vaccination.

O all experimental white-tail deer fawn were found to be free from circulating BTV.. Eight of the 12 fawn had aga gel precipitin antibody (maternal antibody) from colostr intake. A 2-ml amount of polyvalent vaccine containing equal titer equivalents of BTV IT 10, 11, 13 and 17 pre¬ pared by diluting mother pools of stock virus of each serotype to contain about 10⁴ TCID50/111I was administered parenterally to- each of 12 white-tail deer fawn. Each fawn was monitored daily for evidence of pyrexia or othe untoward response to the vaccine. Four and 9 days after vaccination, each fawn was bled and tested for evidence vaccine virus. One month later, 2 of the vaccinated dee fawn were challenged with 250-500 SID/ml of virulent BTV i.e., each -deer fawn received BTV IT 10, 11, 13 and 17, respectively. The remaining deer fawn served as contact controls. Eight and 15 days after virulent virus chal¬ lenge, each fawn was bled and tested for evidence of virulent virus.

Blood samples from three of the white-tail deer faw on the ninth day after vaccination contained low levels of BTV IT 10. No other serotypes of vaccine virus were isolated. In addition, blood samples collected from the two challenged deer on the eighth day after challenge contained virulent virus, one BTV IT 10 and one BTV IT 1 None of the deer showed any signs of clinical illness either after vaccination or after challenge. This study shows that attenuated bluetongue virus contained in the vaccine is fully protective but maintains its ability to replicate in very young susceptible reminants.

In accord with the results obtained in the specific working examples described above, each bluetongue virus

OMP will be sufficiently attenuated for use as a vaccine if the following criteria are met:

(_.1) Over seventy percent of the cattle, sheep, and goats parenterally inoculated with about 2 ml of a vaccine containing at

4 least about 10 tissue culture infectious doses of the attenuated virus must survive a challenge infection four weeks later wherein the cattle, sheep, and goats are parenterally infected with 2 ml of a vir¬ ulent virus containing 500-1000 SID/ml; and (2) Pregnant cattle, sheep, and goats at various stages of gestation must not be¬ come diseased and abort after parenteral infection of 2 ml of a vaccine containing between 10³'⁵ and 10⁷:⁵ TCID₅₍₎/ml.

OMPI

Claims

The claims defining the invention are as follows:

1. A vaccine for immunizing ruminants against International serotypes of bluetongue virus IT 10 , IT 11, IT 13 , and IT 17 , comprising at least about 10 tissue culture infectious doses of one or more of bluetongue virus serotypes IT 10 , IT 11 ,. IT 13 , or IT 17 per ml, wherein the virus serotype is attenu¬ ated so that upon parenteral administration to said ruminants the vaccine is capable of stimulating produc- _. tion of a host defense mechanism comparable to that produced by - atural infections without producing the pathological responses of disease due to the bluetongue virus serotypes .

2. The vaccine of claim 1, wherein each . of said one or more bluetongue virus International serotype is attenuated by 30 or more serial passages through Vero cell cultures in a nutrient fluid at an incubation tem¬ perature of from about - 30 C. to about 36 C. , each pas¬ sage lasting from about 1 day to about 10 days .

3. The vaccine of claim 2 , wherein said vac- cine comprises at least about 10 4 tissue culture infectious doses of bluetongue virus International serotype 13 per ml, wherein said virus is attenuated by at least 3 blind passages in pathogen- free embryo¬ nating hen eggs prior to said 30 or more serial passages through Vero cell structures . . The vaccine of claim 2 or 3, wherein said vaccine comprises a dry solid dosage unit form and where¬ in the vaccine is made solid by drying at low temperatures ,

MPI

5. The vaccine of any of claims 2, 3 or 4, wherein each serial passage through Vero cell cultures lasts from about 2 days to about 7 days.

6. The vaccine of any of claims 2 - 5, wherein said ruminants are selected from cattle, sheep, or goats and said vaccine comprises about

10 4 to about 106 tissue culture infectious doses of the bluetongue virus serotype per ml.

7. A method of preparing a vaccine for parenteral innoculation of ruminants against a blue¬ tongue virus, which comprises cultivating and attenuat- the bluetongue virus by 30 or more serial passages through Vero cell cultures in a nutrient fluid.

8. The method of claim 7, wherein said

4 virus is present in an amount of at least 10 tis¬ sue culture infectious doses of one or more of blue- gongue virus International serotype IT 10, IT 11, IT 13, or IT 17 per ml.

9. The method of claim 7 or 8, wherein said virus is bluetongue International serotype 13 (BTV

IT 13) andsaid method comprises:

(1) cultivating and attenuating the

BTV IT 13 virus by at least 3 serial passages in pathogen-free embryonating hen eggs, and

(2) then cultivating and attenuating the BTV IT 13 virus by 30 or more serial passages through Vero cell cultures in a nutrient fluid whereby said vaccine is for parenteral innoculation of ruminants against bluetongue virus International serotype 13 (BTV IT 13) .

10. The method of any of claims 7, 8 or 9 wherein said cultivating and attenuating of the virus by serial passages through Vero cell cultures is accomplished at a temperature of from about 30°C. to about 36°C. and each serial passage lasts from about 1 day to about 10 days.

11. The method of any of claims 7, 8, 9 or 10, wherein each serial passage lasts from about 2 ^to about 7 days.

12. A method of immunizing cattle, sheep, or goats against bluetongue virus which comprises par¬ enterally administering to said cattle, sheep, or goats ^■" at least 2 ml of a vaccine comprising at least one or more of bluetongue International virus serotype IT 10, IT 11, IT 13, or IT 17, wherein said virus is attenuated by 30 or more serial passages through Vero cell cultures in a nutrient fluid at an incubation tem- 5 perature of from about 30°C. to about 36°C. , each pas¬ sage lasting from about 1 day to about 10 days..

13. A method of immunizing cattle, sheep, or goats according to claim 12,. which comprises parenterally administering to said cattle, sheep, or 0 goats at least 2 ml of a vaccine comprising at least

4 about 10 tissue culture infectious doses of bluetongue virus International serotype 13 per ml, wherein said virus is attenuated by at least 3 blind passages in patho¬ gen-free embryonating hen eggs prior to said 30 or more serial passages through Vero cell cultures.