CA1163556A - Vaccine for infectious bovine rhinotracheitis - Google Patents

Abstract

VACCINE FOR INFECTIOUS BOVINE RHINOTRACHEITIS

ABSTRACT

A vaccine for infectious bovine rhinotracheitis (IBR) is prepared by nonionic detergent extraction of IBR virus infected cells. The extracted viral envelope protein can be prepared in injectable dose form by combination with an oil-type adjuvant. The vaccine produces a high level of antibody response, and incapable of eliminating virus shed for animals infected after immunization.

Description

~ ~ 63~56 BACKGROUI~ AND PRIOR ART

I~fectious bovine rhinotracheitis (IBR) was first recognized as a disti~ct disease of cattle during the 19;0's.
Consistent with the characteristics o~ o~her heipes~iruses, I3R ~irus (IBRV) replicates i~ a wide range of cell types - a~d produces diverse dise se manifestations which include respiratory tract disease, conjuncti~itis, vulvovaginitis, abor~ion, balanoposthitis, meninsoencephalitis, aiimentary ~rac~ disease and fa~-al systemic infec~ion. ~owever, IBR
is known mainly as a respiratory tract disease cnaracterized by tracheitis, rhinitis, and fever. IBR infection is the most commonly diagnosed cause o abortion in pregnan. cattle.
See Rir~bride e~ al, Am. Vet. Med. Assoc., 162, 5~6-560 (1973)o The ~irus is readily transmitted and has worldwi~e distribution. So~e cattle develop a latent infection, which can be reactivated.

Control of IBR is based l~rgely on vaccination, and a number or different kinds of IBR vaccines have bee~
developed. See Kahrs, J. Am. Vet. Med. Assoc., 171, 105;-1060 tl977). The~e include parenteral and intranacal vaccines, both of which use live attenuated IBRV. The parenteral va~cines, which are usually administered in~ramuscularly, may cause abortio~ i~ pregnant cattle, and are therefore con~raindica~ed for pregnznt cattle. ~ur~her, vaccination of suckli~s calves which are nursing pregna~t cat~le may cause abortion due to the shedding of virus by the ~accinated calves. In~ranaszl vaccines are safer in 'hese respects, a~d are ca?able OL produci~g humoral ~r.ti~odies at .iters com?arable with those o intramuscula- va-cines. Howeve_, ~ 1 63556 there are problems associated with administration of intra-nasal vaccines. Head restraint is required, and care mus.
be taken to administex the vaccine deeply into both nostrils. There is a tendency for vaccinated animals to blow the ~acclne out when they snort a~ter vaccination.

- The degree of efficacy of prior art vaccines is variable, and the duration of protection is limited. In general, it is believed that in~ramuscular ~accination gi~es protection of longer dura~ion than intranasal vaccination.
lo With intranasal ~accination, co~servative practice dictates annual revaccination, while at least occasional revaccination is recommended with the intramuscular vaccines. Inacti~ated IBR vaccines ha~e also been developed, ~ut there is controversy about their effectiveness. Annual revaccination is recommended.
Further disadvantages of inactivated vaccines include concern for f~tal hypersensitivity reaction (anaphylaxis) and nonfatal urticaria.

Heretofore no subunit IBR vaccine has been reported~
However, a subunit approach to the preparation of some other virus vaccines has been explored. See, for example, Rubin et al, Progr. Med. Virol., 21, 144-157 ~1975). Cappel has reported experi~ents with a subunit vaccine of herpes sLmplex ~_ ~ virus in rabbits. Arch. Vlrol. 52, 29-35 (1976). The subunit vaccine was found as effective as immuniza'ion wi h live or U.V inactivated herpes simplex virus. Similarly, Cox et al have reported experiments with a subunit vaccine ~or human rabies. In'ect. and Immun., 16, 753~7S4 (1977). 30th Ca?pel and Cox et al worked with non-ionic detergen. extracts o_ -~he virions, since it is known that non-ionic detergents are ,, ~

~ ~ 6~556 capable of solubilizing envelope glycoproteins of viruses.
See Helenius et al, "Solubilization of Membranes by Detergents", Biochem. et Blrphys. Acta, 415, 23-79 (1975). However, Sokal stated as a general rule that subunit vaccines contain-ing soluble antigens would be epxected to provide a specific immunogenicity or specific protective action which is markedly lower than that of virion vaccines. Sokal, Vaccines of the Future: Immunogenicity of Viral Components, Chapt. 9, pp.
129-135j in Viruses and Immunity, Koproski, 1975, Academic Press, Inc.

SUM~RY OF INVENTION

This invention is based in part on the discovery of the means for producing IBR vaccines giving markedly higher antibody responses than any previous experimental or commer-cial IBR vaccines. Further, the vaccines prepared in accordance with the present invention are capable of producing higher anitbody responses than those found in cattle which have recovered from natural ~BR infections.
, The vaccines of the present invention are prepared 20~ from virulent IBRV, such as field isolates of IBRV or substantially unattenuated IBRV. The IBR virus in its natural or virulent form is propagated in a cell-medium mixture capable of promoting the multiplication of the virus.
After propagation, the infected cells are separated from the mixture, and are extracted with an aqueous solution of a non-ionic detergent to obtain an aqueous solution of the antigenic protein. The non-solubilized residue is separated from the supernatant solution, which is then employed as the antigenic ingredient of the ~Taccine. The soluble antigen `\
~ 1 6355~

in aqueous solution is preferably combined with a suitable adjuvant to provide parenteral vaccine in injectable dose form. Oil-ty?e adjuvants are preferred, such as Freund's Incomplete Adjuvant.

At an effective dose level in combination with a suitable adjuvant, a level of antibody response can be obtained which confers immunity to the disease and also prevents the development of an IBR infection. This is indicated by the fact that vaccinated animals when challenged lo with live virulent IBRV do not shed virus. This finding is -most unexpected since virus shed is always found after challenge of calves vaccinated with any existing vaccine, and there are no known literature references suggesting that prevention of IBR virus shed is attainable by vaccination.
The vaccinated cattle do not develop lesions or clinical signs of IBR illness.

The vaccines of the present invention can be used in pregnant animals, in suckling calves, and in calves with maternal antibodies. The vaccines are free from active IBR virus, and therefore cannot cause infection or transfer of IBRV. Further, ~ecause the vaccines are of limited antigenic composition, serologic tests can be devised to distinguish vaccinates from infected animals. This cannot be done with currently available IBR vaccines. More specifically, the vaccine of the present invention could be used in a local or a national eradication program, because control of virus sheddins is essential to controllins virus transmission. Presently available IBR vaccines do not prevent shed of virus or establishment o-^ latent infections.

. : ' ~ 1 ~3556 DETAILED DESCRIPTION

Any virulent strain of infectious bovine rhino-tracheitis virus tIBRV) can be used in practicing the present invention. For example, the Cooper strain can be used, which is supplied as an IBR challenge virus by the Veterinary Services Laboratory, U.S.D.A., Ames, Iowa.
Virulent IBRV strains can be obtained from other sources, such as the American Type Culture Collection. For example, ATCC No. VR-188 is suitable. Alternatively, field isolates lo can be collected from IBRV infected cattle, thereby assuring that such isolates are fully virulent. However, such virulent strains can be subjected to a number of in vitro cell passages while retaining virulence, as indicated by the ability to infect cattle and cause clinical signs of IBR, for example, an average temperature elevation of over 103.5F.

The virulent IBRV is propagated by cell culture.
Preferably, bovine cells are employed which are capable of propagating IBR virus to a high titer, such as bovine ~ lung cells, kidney cells, testicle cells, etc. Established lines of bovine cells adapted for in vitro culture can be used, although good results can also be obtained with freshly collected cells. For purposes of the virus propagation, .
the cells are combined with a suitable nutrient medium, which is adapted to promote the growth of the virus to a high titer.
For example, the basal medium may comprise Eagle's minimum essential medium (MEM). To promote viral multiplication, incluslon of fetal calf serum (FCS) is desirable. For example, 10% FCS can be used for growth of the cells, and a 5% PCS
level for maintenance of the cells after infection while the virus is multiplying. Within 24 hours after inoculation ~ ~ ~35~8 .

with the IBRV, the cells have been killed and the propagation of the virus is completed. The desired viral envelope protein is associated with the cell membranes where it has collec~ted before being formed into IBR viral particles.

- On completion of the propagation, the cells are preferably separated from the liquid media. In one procedure, !
this can be accomplished by centrifugation. For example, the cells will be pelleted out of the cell culture medium by centrifugation at 100,000 x g for 60 minutes. The loseparated cells are then extracted with a non-ionic detergent, which solubilizes the membrane-associated viral envelope protein and inactivates the IBR virus. ~he cell pellets may be resuspended in water containing the non-ionic detergent for the solubilization. Detergent concentrations of O.5 to 5%
can be used. The non-ionic detergents which have been found :
effective for solubilizing viral envelope glycoprotein are advantageous. For example, Triton X-100 50ctyl phenoxy ; ~polyethoxyethanol) can be used. This non-ionic detergent :: ::: , is produced by Rohm & Haas Co., Philadelphia, Pennsylvania, ~20 ~ and is distributed commercially by Sigma Chemical Co., St.
Louis, Missouri. Nonidet P 40 is another non-ionic detergent which has been reported in the literature as desirable for selective dissolving of viral envelope protein. Nonidet P 40 is produced by BDH Chemicals Ltd., Poole, England, and dis-tributed in the United States by Gallard-Schlesinger Chemical Mfg. Corp., Carle Place, New York. Such non-ionic detergents solubilize viral envelope pro~ein without appreciable denatura-tion of the protein.

, ~ 3 ~3556 Contacting oX the aqueous detergent solution with the cellular membranes can be prompted by mechanical disruption or dispersion of the cells. For example, the resuspended cells can be passed through a homogenizer or subjected to sonication, thereby promoting the contacting of the non-ionic detergent with the cellular membranes and the IBR virus.
Very little of the desired viral envelope protein is obtained from the IBR viral particles themselves, but complete inactiva-tion of the IBR virus is desirable. It is desirable to carry ; 10 out the contacting at a neutral or slightly alkaline pH, such as a pH in the range of 6.0 to 9Ø A suitable buffer can be included in the aqueous solution to maintain the desired pH.
The water used for the extraction is preferably sterile deionized water. The extraction can be completed in one to two hours. For example, with homogenization and/or sonication, and continued stirring of the cell suspension in the aqueous detergent, the extraction can be completed in one hour.
; ~ Preferably, the suspension is maintained at a temperature below 10C. during the extraction, such as a temperature of about 4C. On completion of the extraction, the residue of ~;~ ; solid material is separated from the supernatant solution of solubilized protein. For example, the separation can be ~~ accomplished by centrifugation. The cell debris may be pelleted by centrifugation at 100,000 x g for 60 minutes.
::
The extract of subviral protein thus obtained can be used directly to prepare vaccines, where, as preferred, it contains a sufficient concentration of protein to permit it to be mixed with an adjuvant to ?repare injectable doses of vaccine. The amount used per dos2 should be effective tc prèvent IBR disease, either as a single injection or as a ~ 1 63556 sequence of two injections. For example, the extract may contain from 3 to 6 milligrams (mg) of extracted protein solids per milliliter ~ml). The injectable dose form of the vaccine may be prepared by mixing equal parts of a suit-able adjuvant and the subviral protein extract as obtained (without concentratio~). While the vaccine (protein extract with adjuvant) may contain from 0.5 to 10 mgjml of total protein (solids basLs), it will usually contain an amount within the range from l to 6 mg/ml of the extracted protein.
For example, where the dose of the vaccine is to comprise ~

2 milliliters, the total dose will then comprise rom 2 to 12 mg of total protein, such as a total dose of 4 to 8 mg.

If desired, the antigenic protein may be concentrated or recovered prior to preparation of the vaccine, such as by salting out or ultrafiltration. Where the antigenic protein is recovered in solid form, it is preferably resuspended in water at the desired concentration for incorporation in the vaccine. Then, as previously indicated, the solution of vlral envelope proteln is combined with a suitable adjuvant.
Since the antigen is in solution, it is preferred to combine the aqueous solution with an oil-type adjuvant. For example, Freund's Incomplete Adjuvant may be used. This adjuvant may be purchased from a commercial source, such as Difco Labora-tories, Detroit, ~lichigan, or it may be prepared by combining mannide monooleate with paraffin oil in the proportions by volume of 1.5:8.5. The resulting vaccine is a water-in-oil emulsion with the antigen in the dispersed water phase. Whlle the proportior.s of adjuvant to protein extract can var~ considerably, approximately equal proportions are desirable.

_ g _ ~ 1 B3~56 , The vaccines of this invention are partlcularly designed for use with cattle, and may be administered to calves and pregnant cattle. The vaccines can also be used with other bovine species, such as oxen or water buffalo.
The volume of the injectable dose can vary, such as a volume - of 1 to 4 ml. However, a 2 ml dose is convenient. In administering the vaccine, a single injection may be given but it is preferred to glve two sequential injections per animal. For example, a 2 ml dose containing from 4 to 8 mg lo of total protein can be administered twice at 30 day intervals. The disease is prevented and the transfer of -IBR virus by viral shed is also prevented, thereby more effectively controlling IBR infection.

In developing and testing the vaccines prepared in accordance with the present invention, the following materials and procedures were used.

Virus - The virulent Cooper strain of IBRV was received at the eighth passage level, from the National Veterinary Services Laboratory, Ames, Iowa. The Cooper .
strain is conventionally used as a challenge strain. It was passed two times in bovine lung cells (BLU) and a stock pool containing 1.0 x 108 plaque forming units (PFU) per ml was frozen at -70C.

Cell culture procedure - Cell cultures utilized in this study were grown in Eagle's minimum essential medium (MEM) supplemented with 10% irradiated fetal calf serum (FCS) (heat inactivated at 56C for 30 minutes), 0.5~ lactalbumin hvdrolysate and antibiotics (100 IV penicillin, 100 ~g kanamycin sulfate and 100 ~g streptomycin sulfate per ml).
rrhe medium was buffered ~ith 0.15~ sodium bicarbonate and . .
.

~7~7 63556 8 .~l N-2-hydroxyethylpiperaæine-N'-2-ethanesulfonic acid (HEPES). Cultures were incubated at 37C in a 5~ CO2 atmosphere.

Virus isolation - I~7asal secretions were collected for virus isolation by insertion of a 15 cm cotton-tipped swab to its full length in the left ventral nasal meatus and manipulation of the swab in a rotary motion until saturated.
Swab samples were immersed in 1.0 ml MEM containing antibiotics (200 IU penicillin, 200 ~g kanamycin sulfate, 200 ~g lo streptomycin sulfate and 15 ~g amphoteracin B per ml) and held at 4C for 30 minutes. Swabs were then removed from the medium and the medium was frozen at -70C until cultured.

Plaque forming units of virus in specimens wexe determined by inoculation of duplicate cultures of BLU
~monolayers with serial 10-fold dilutions (in MEM) of each specimen. The inocula were adsorbed for 60 minutes, mono-; layers were washed with MEM and overlaid with 1% agarose ;~ ~ containing M~M, 5% FCS and antibiotics. Cultures were incubated at 37~C for 72 hours in a 5~ CO2 atmosphere.
The cultures were fixed with 10~ buffered formalin, the agarose layer was removed and the cell sheet was stained with crystal violet. The virus titer was determined by enuneration of PF7Js.

Plaque reduction-neutralization tests - Anti-IBR

serum neturalization titers were determined by plaque-reduction neutralization tests. Two-_old serun dilution of heat inactivated serum (56C for 30 minutes) 7~7ere mixed with equal volunes of M~M containlnc 1000 P~7~ IBR~7 per ml -~ an~ incubated for 60 minu_es at ~7C. Monlayers o~ BLU
cells in 35-mm 6-well plastic tissue culture plates were ~ .

il 1 63556 .
inoculated wi~h 0.2 ml of serum-virus mixture. After adsorp-tion of the serum-virus mixture for 60 minutes at 37C, the cultures were washed with MEM, and overlaid with 1~ agarose containing MEM, 5% FCS and antibiotics. Cultures were incubated at 37C for 72 hours and then fixed with 10%

., buffered formalin. The agarose layer was removed and the cell sheet was stained wi~h crystal violetO The serum neutralization titer was the reciprocal of the highest serum dilution that reduced the plaque count by at least 50%.

lo Protein determination - Protein content was assayed by the method of Lowry et al, J. Biol. Chem., 193, 265-275 (1951) with bovine serum albumin as the standard. Protein content of samples containing non-ionic detergent was deter-mined using a modified Lowry procedure, as described in Anal._Biochem., 86, 346-356 (1977).

Production of vaccines in accordance with the present invention and results obtained in the testing of the vaccines are illustrated by the following examples.

ExAMæLE I

Production of Vaccines The bovine lung cells (BLU) were propagated in 490 cm2 roller bottles until monolayers were formed. The monolayers were infected by removing the medium and adding 5 ml of stock IBRV, prepared as described above. After 60 minutes adsorption the inoculum was removed, the monolayer was washed with MEM and 30 ml of maintenance medium (MEM
containing 5% FCS) W2S added. The cells were incubated at

3,'C with rolling (1 rpm) until cytopathic effect (CPE) was ~ 12 -~ 1 63556 complete (approximately 24 hours). The infected BLU cells were scraped from the roller bottle surface with a rubber policeman. The cell-medium mixture was centrifuged at 100,000 x g for 1 hour. The resultant pellet was solublized using a modification of the procedure of Vestergaard et al, J. Virol., 24, 87-90 (I977). The pellet was suspended in 0.010 M glycine-0.038 M tris (hydroxymethyl~ aminome~hane (Tris) (pH 9.0) containing 0.5~ Triton X-100 (1 ml per roller bottle). The mixture was homogenized with a homogenizer lo and sonically treated for two-20 second cycles with a sonicator at a setting of 904 efficiency. The mixture was allowed to react for 1 hour at 4C with constant mixing by a magnetic stirrer. The mixture was centrifuged at 100,000 x g for 1 hour and the supernatant harvested as the vaccine. The vaccine was stored at -70C until used. The vaccine was mixed with an equal volume of Freund's Complete or Freund's Incomplete Adjuvant and emulsified with 200 strokes in an adjuvant mixing apparatus. A second subunit vaccine was produced using a modification of the above procedure. The extracting solution was 2.5~ Nonidet P-40 (NP-40) in distilled water prepared as described by Martin et al in Infect. Immun., 5, 248-254 (1972).

Determination of Vaccine Antigenicity Triton X-100 solubilized IBR subunit vaccine and cell control preparations were administered to 10 IBR sero-negative cattle in two intramuscular doses at 20 dav intervals as detailed in Table A. Animals were observed daily throughout the study for clinical signs of disease. Rectal temperatures ~ 1 63556 .
were recorded for 3 days prior to vaccination and for 10 days after vaccination. Nasal secretions were collected for virus isolation prior to vaccination and on days 2, 3 and 7 post-vaccination. Serum samples were collected at weekly intervals.

Experiment 1-. Determination of subunit vaccine antigeni i~

All 10 animals in this experiment remained clinically normal throughout the study. Rectal temperatures following vaccination were within +1C of temperatures recorded prior lo to vaccination. Virus was not isolated from nasal secretions prior to or following vaccination. Serum neutralization titers are set out below in Table B.

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, ExAMæLE III

Twenty IBR seronegative calves, 3 ~o 6 months of age, were ra~domly di~ided into 5 groups (I in each group) .- and housed in separate, enclosed, isolatio~ rooms throughout th~ study. Groups of calves, designaked 1 to S, were vaccinated intramuscularly as detailed in ~able C. Thirty days after vaccination,calves were administered a standard challenge virus inoculum (1 x 106 PFU Coo~er strain IBR, lo lQth passage). A gas-powered a~omizer was used to deliver : 2 ml o inoculum into each nostril.

Calves were examined daily ~or cli~.ical signs of ~ . disease Lrom 7 days prisr to vaccination ~ntil t~e study : ~was terminatedO Rectal tempexatures were r~corded for 3 : . days prior to vaccination, for 10 days 'ollowing each vaccination and 'or 14 days following chal'enge. ~asal ecretions were collected ~or virus isolation thr2~ times : p~ior to vacclnation and daily:for 7 davs 'ollowing the initial vaccination in each group. FQ110Wi n5 challenge, ~: nasal secretions were collected at the following times:
S minutes post-challenge; 15 minute intervals thru 1 hour ; post-challenge; hourly intervals thIu l; hours post-challenge;
at 18, 20, 22, 24 hours post-challenge; anc a~ 24 hour nter~als thru 14 days post-challenge. Se-1m samples we_e col}ected three times prior to vaccinaticn ~nd 2 t wee~ly :~ intervals following vaccination. .
: :
Following vaccination, 4 o. 4 control calves and 5 of 12 vaccinates developed indur2ted swelLlngs at the site of vaccine inoculation and febrile responses (40 to 41.1 C) : ~:
, .

that pers~sted for 1 to 3 days. . Deep intramuscular inocul~-tion and avoidance or `'inoculum drag" prevented reaction to the second ~accination.

Table D su~mnarizes the serum neutralization titexs ~ollowing ~ra~c~a~ion and challenge.

Following in~ranasa ~hallenge with IBRV, all cal~es L ~-~o in groups 2, 3, 4 and ~ remai~ed clinically no~mal except calYes 5, 10 and 11 which developed mild respiratory ~rac~
i~fection. Clinical signs of disease included elevated te~perature, in~ranasal herpetic pustules a~d fibrino-necrotic ~asal exudate. The clinical course o disease observed in calves 10 and 11 was characterized by an elevated temp~rature which persisted beyond resolution o~ intranasal lesions and nasal exudation. The temperature returned to ~ase-line ~alues 24 ho~rs after antibiotic ~herapy was initiated on post-challenge day 9. Calf 20 died from causes unrela~ed to experimentation o~ post vaccination day 3~. Post mortem examination did ~ot reveal e~idence of disease or any lesion at the site of vaccination. Challenge Pxposure of control calves produced a severe respiratory disezse characterized by ele~ated temperature (to 41. 7C) and ormation of extensive fibrino-ne~rotic slaques on the nasal mucosa.

Yirus was not recoYered from nasal secret~o~s prior to or following vaccinatio~. In sroups 2 and 3 challenge inocul~m ~irus was isolated at 5 ~in and 1; min post challenge, but c~uld not be isolated ~rom nasal secretions of calves at 30 ~inutes after ch llenge ex?osure. Challen5e inoculum ~irus could not be isolated from nasal secretions o~ calves in groups 4 and 5 at; r~nut~s a~er challenge inoculum. ~ollow-ing the initiaL clearance of ch~llenge virus inoculu~, virus ~ 3 ~3556 was isoiated from nasal secretions 18 hours after challense : in groups 2 and 3 and at 22 hours in groups 4 and S. Table E
details shed of virus in nasal secretions for days 1 to 14 : post challenge. Virus was not isolated from calves 13, 14, : 17, 18, or 19 ~ollowing intranasal challenge exposure.
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As used herein, reference to "prevention of IBR
disease" means that the vaccine protects bovines against development of clinical signs of the disease when challenged with virulent IBR virus. In cattle a principal clinical sign of the disease is a temperature elevation to 103.5F
~ or higher ~or two or more days. Vaccinated cattle after challenge, although not evidencing any clinical signs of the disease, may shed vi~ulent I~R virus, which can transfer infection to other non-vaccinated cattle. Therefore, when lo reference is made herein to "prevention of IBR infection", this means that the vaccine also prevents virus shed, as weIl as preventing the disease. This result is new and surprising.

.

~ .

Claims (2)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. The method of preparing antigenic protein for vaccine use in preventing infectious bovine rhinotracheitis (IBR), comprising propagating virulent IBR virus in a cell-medium mixture capable of promoting the multiplication of said virus, separating the infected cells from the mixture, contacting the cellular membranes of the separated cells with an aqueous solution of a non-ionic detergent to obtain an aqueous solution of antigenic viral envelope protein, and separating said aqueous solution from the non-solubilized residue to obtain the antigenic protein in said solution.

2. The antigenic protein prepared by the method of claim 1.