US3925544A

US3925544A - Bovine vaccines and methods of making and using same

Info

Publication number: US3925544A
Application number: US428658A
Authority: US
Inventors: Isaac L Shechmeister; Jr Joseph R Kolar; Jr William G Kammlade
Original assignee: Southern Illinois University Carbondale
Current assignee: Southern Illinois University Carbondale; Southern Illinois University Foundation
Priority date: 1971-07-12
Filing date: 1973-12-27
Publication date: 1975-12-09
Anticipated expiration: 1992-12-09

Abstract

A polyvalent vaccine effective in immunization of bovines against infectious bovine rhinotracheitis virus (IBR), bovine viral diarrhea virus (BVD) and parainfluenza-3 virus (PI-3) is composed of a suspension, in a vehicle such as an aqueous solution of formaldehyde suitable for parenteral injection of the three viruses in killed form. The three viruses are separately propagated and subsequently suspended in aqueous solutions containing 0.4 percent by volume formalin, and the respective suspensions are maintained at a temperature of about 4*C. for 1 week. Portions, for example equal portions, of the three suspensions are combined to produce a polyvalent vaccine. An equal volume of an aqueous solution containing from 1 to 5 percent weight/volume of a soluble alginate, such as sodium alginate, having certain specified properties may be added to the vaccine as an adjuvant to enhance its effectiveness. Such polyvalent vaccines are adapted for parenteral administration in the vaccination of bovines.

Description

United States Patent 1191 Shechmeister et al.

1 BOVINE VACCINES AND METHODS OF MAKING AND USING SAME [75] inventors: Isaac L. Shechmeister; Joseph R.

Kolar, Jr.; William G. Kammlade. Jr., all of Carbondale, 111.

173] Assignee: Southern Illinois University Foundation, Carbondale. 111.

22 Filed: Dec.27, 1973 [21] Appl. No.: 428,658

Related US. Application Data [62] Division of Set. No. 161.701, July 12. 1971. abandoned. which is a division of Scr. No. 8.631. Feb. 4. 1970. Pat. No. 3.629.413.

[52] US. Cl. 424/89 [51] Int. Cl. A61K 27712 [58] Field of Search 424/89 [56] References Cited UNITED STATES PATENTS 3.075.883 H1963 Scherr et al. 424/89 3.629.413 12/1971 Shechmeister et al 424/89 3.838.004 9/1974 Mebus et a1. 424/89 OTHER PUBLICATIONS Hermodsson et al., Nature 194(4831): 893-894, June 2, 1962, Properties of Bovine Virus Diarrhea Virus. Gillette, Diss. Abtr. 278: 363-364 (1966), Characterization Studies of Two Strains of Bovine Viral Diarrhea Virus."

Kolar et al., Am J. Vet. Res. 33(7): 1415-1420, July 1972 Use in Cattle of Formalin-killed Polyvalent Vaccine with Adjuvant against infectious Bovine Rhinotracheitis, Bovine Viral Diarrhea and Parainfluenza 3 Viruses."

Shechmeister et al., Vet. Bull. 38, No. 1381 (1968). Abst. of Am. .1. Vet. Res. 28: 1373l378 (1967),"

1 Dec. 9, 1975 Use of Sodium Alginate Adjuvant in Immunization against Equine influenza."

Femelius et al.. Am. J. Vet. Res. 32(12): 1963-1979. Dec. 1971. Evaluation of Soluble Antigen Vaccine Prepared from Bovine Viral Diarrhea Mucosal Disease Virus-infected Cell Cultures."

Femelius et al., Am .1. Vet. Res. 33(7): 142-1431 July 1972 Evaluation of B-Propiolactone-lnactivated and Chloroform Treated Virus Vaccines Against Bovine Viral Diarrhea Mucosal Disease."

Primary Examiner-Shep K. Rose Attorney. Agent. or Firm-Koenig. Senninger. Powers and Leavitt 57 ABSTRACT A polyvalent vaccine efiective in immunization of bovines against infectious bovine rhinotracheitis virus (1BR), bovine viral diarrhea virus (BVD) and parain fluenza-3 virus (Pl-3) is composed of a suspension. in a vehicle such as an aqueous solution of formaldehyde suitable for parenteral injection of the three viruses in killed form. The three viruses are separately propagated and subsequently suspended in aqueous solutions containing 0.4 percent by volume formalin. and the respective suspensions are maintained at a temperature of about 4C. for 1 week. Portions, for example equal portions, of the three suspensions are combined to produce a polyvalent vaccine. An equal volume of an aqueous solution containing from 1 to 5 percent weight/volume of a soluble alginate, such as sodium alginate, having certain specified properties may be added to the vaccine as an adjuvant to enhance its effectiveness. Such polyvalent vaccines are adapted for parenteral administration in the vaccination of bovines.

3 Claims, 9 Drawing Figures US. Patent Dec. 9, 1975 Sheet 3 of9 3,925,544

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:3! m-=02: 2:8 *0 omzoamoh 3252-5 @0293 mm m afiuaae go maoadpu QUE CROSS-REFERENCE TO RELATED APPLICATION Feb. 4, 1970, now U.S. Pat. No. 3,629,413 dated Dec. 21, 1971.

BACKGROUND OF THE INVENTION The present invention relates to vaccines and more particularly to a polyvalent vaccine useful in the treatment of infectious diseases in bovines.

Three viruses which commonly infect bovines such as beef cattle are infectious bovine rhinotracheitis (IRB), bovine viral diarrhea (BVD) and parainfluenza-3 (PI-3) also known as the shipping fever" virus. Because they effect beef cattle, these viruses have given rise to a commercially significant problem. In the past, there have been available monovalent vaccines for the treatment of these respective viruses and a bivalent vaccine for the treatment of two of these three viruses. However, these vaccines have been prepared from live viruses. The use of such vaccines containing live viruses has been contraindicated in the case of pregnant cows, and the effect of such vaccines on other animals has not been fully evaluated. Also, there has not been available for treatment of the aforementioned viruses a vaccine containing an adjuvant to enhance its effectiveness.

In summary, there has not heretofore been available a single polyvalent vaccine which is effective against the three aforementioned viruses, which can be recommeded and used in the treatment of all bovines and which requires only a single administration for effective protection of such bovine animals instead of multiple administrations of several different vaccines.

SUMMARY OF THE INVENTION Among the objects of the invention may be noted the provision of a novel polyvalent vaccine which is effective in immunizing bovines against the three aforementioned viruses; the provision of such a vaccine which can safely and reliably be used in the vaccination of all bovines; the provision of a polyvalent vaccine of this type which contains the respective viruses in killed form; the provision of a vaccine of this character which produces an immune response in bovines which lasts for an extended period of time; and the provision of methods for preparing and using such polyvalent vaccines in a convenient and economical manner. Other objects and features will be in part apparent and in part pointed out hereinafter.

In accordance with the present invention, there is provided a polyvalent vaccine comprising a suspension, in a vehicle suitable for parenteral injection, of killed infectious bovine rhinotracheitis virus, bovine viral diarrhea virus and parainfluenza-3 virus. The invention is also directed to the method of preparing such a polyvalent vaccine by first separately propagating the three viruses, suspending the respective viruses in separate aqueous solutions containing a final concentration of about 0.4 percent by volume formalin to kill the viruses, maintaining each suspension at a temperature of about 4C. for about 1 week, and combining portions of each suspension to produce a polyvalent vaccine containing the three viruses in killed form. The invention further includes the method of treating bovine infections by parenterally administering the novel polyvalent vaccines to bovines.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. I is a graph showing the average antibody response of cattle to infectious bovine rhinotracheitis virus when inoculated with either infectious bovine rhinotracheitis monovalent vaccine or the polyvalent vaccine of the present invention;

FIG. 2 is a graph showing the average antibody response of cattle to bovine viral diarrhea virus when inoculated with either bovine viral diarrhea monovalent vaccine or a polyvalent vaccine according to the present invention; a

FIG. 3 is a graph showing the average antibody of cattle to parainfluenza-3 virus when inoculated with either parainfluenza-3 monovalent vaccine or a polyvalent vaccine according to the present invention;

FIG. 4 is a graph showing the average antibody of cattle to parainfluenza-3 virus when inoculated with either parainfluenza-3 monovalent vaccine or a polyvalent vaccine in accordance with the present invention expressed on a different basis than in FIG. 3;

FIG. 5 is a graph showing the average antibody response of cattle to parainfluenza-3 virus when inoculated with either pairainfluenza-3 monovalent vaccine or a polyvalent vaccine according to the present invention expressed on still another basis;

FIG. 6 is a graph showing the pooled antibody response of cattle to infectious bovine rhinotracheitis virus when inoculated with a polyvalent vaccine of the present invention;

FIG. 7 is a graph showing the pooled antibody response of cattle to bovine viral diarrhea virus when inoculated with a polyvalent vaccine of the present invention FIG. 8 is a graph showing the average antibody response of cattle to parainfluenza-3 virus when inoculated with a polyvalent vaccine of the present invention; and

FIG. 9 is a graph showing the pooled antibody response of cattle to parainfluenza-3 virus when inoculated with a polyvalent vaccine of the present invention expressed on a different basis than in FIG. 8.

DESCRIPTION OF THE PREFERRED EMBODIMENTS It has now been found, in accordance with the present invention, that a polyvalent vaccine containing antigens of infectious bovine rhinotracheitis virus, bovine viral diarrhea virus and parainfluenza-3 virus, all in killed form, is effective in the immunization of bovines against these viruses. Animal studies, described in detail hereinafter, have demonstrated the effectiveness of such a polyvalent vaccine against the infectious diseases caused by the viruses whose antigens are present in one vaccine.

The polyvalent vaccines of the ivention are readily prepared by first separately propagating the three aforementioned viruses as described below. The three viruses are then separately suspended in an aqueous inactivating medium to kill the viruses. Preferably, the inactivating medium is an aqueous solution containing a final concentration of about 0.4 percent by volume formalin. This final concentration may range between about 0.3 percent and 0.5 percent by volume formalin. Formalin is a 37 percent by weight formaldehyde solution. The separate suspensions are maintained at a temperature of about 4C. for approximately I week. Portions of the three suspensions are then combined to produce the complete polyvalent vaccine. Preferably, equal proportions of the three separate suspensions are combined to yield an effective vaccine. However, these proportions may be varied somewhat without substantially affecting the results obtained.

Preferably, the final vaccine contains a concentration of between about 10 and about organisms or units of each of the three killed viruses per ml. of vaccine l0"-l0 TClD /mlJ. The dosages of vaccine employed in carrying out the invention may be varied widely, but preferably ranges between l and 10 ml., a dosage of 1 ml. being preferred when the concentration of each of the killed viruses is about 10 and a dosage of 10 ml. being preferred when the concentration of each of the killed viruses is about l0". It will be understood that the concentrations and dosages employed may both be varied within the above-noted ranges.

In order to enhance the effectiveness of such a polyvalent vaccine, a soluble alginate may be incorporated into the vaccine as an adjuvant. Such soluble alginates and their properties are fully disclosed in Scherr et al. US. Pat. No. 3,075,883, dated Jan. 29, I963, incorporated herein by reference. As disclosed therein, such soluble alginate may be utilized inthe form of an aqueous solution containing from I to 5 percent weightvolume of a soluble alginate, such as sodium alginate, having the following characteristics:

a. in 5 percent weight/volume aqueous solution,

readily passes a 24-gauge needle;

b. in 4 percent weight/volume aqueous solution, has

a viscosity less than 50 centistokes; c. a chemical equivalent below 250; and d. a milliosmolarity less than 150 per kg. of water on a Fiske osmometer. Additionally, such an alginate solution may advantageously contain alginate insolubilizing ions in the form, for example, of a physiologically acceptable gluconate such as calcium gluconate. The vaccine should not, however, contain calcium or other insolubilizing ions of a concentration sufficient to precipitate as the insoluble alginate get before injection of the vaccine into animals is made.

In the practice of the present invention, we have found it satisfactory to use a commercially available aqueous alginate solution of the above type which contains 4 percent weight/volume sodium alginate and 0.67 percent by weight calcium gluconate. The calcium gluconate content may range between about 0.4 and 0.8 percent by weight. An equal volume, for example, of such an aqueous solution may be added to the vaccine, prepared as described above, to further enhance its effectiveness and to produce a higher level of antibodies.

The novel vaccines of the invention are prepared utilizing a vehicle, preferably an aqueous solution, suitable for parenteral injection as by intramuscular, intradermal or intracutaneous injection into bovine animals.

As demonstrated by the animal studies described below, the vaccines of the invention produce a high level of antibodies in cattle inoculated therewith. Moreover, our vaccines, particularly those containing the abovedescribed adjuvant, produce an immune response which is long-lasting, i.e., for at least a period of 120 days.

Two studies were carries out to demonstrate the practice and effectiveness of the vaccines and methods of the present invention.

In the first study, five vaccines were used. Two of these were complex polyvalent vaccines. One contained a mixture of formalin-killed infectious bovine rhinotracheitis (lBR), bovine viral diarrhea (BVD) and parainfiuenza-3 (Pl-3) viruses plus sodium alginate as an adjuvant. The other vaccine contained a mixture of the same three killed viruses without the adjuvant. Three monovalent vaccines were also prepared, these containing sodium alginate as an adjuvantv and the killed lBR, BVD and Pl-3 viruses respectively. Each of the vaccines was suspended in a sterile aqueous medium as a vehicle suitable for parenteral administration.

All viruses used in preparing the above vaccines were grown in bovine embryonic trachea (BEmT) cell cultures. The cells were passed in minimum essential medium (MEM, of. Eagle, H., Amino Acid Metabolism in Mammalian Cell Cultures," Science, Vol. 130, p. 432-437, I959) with a fetal bovine serum concentration of l0 percent The cells for stocks and viruses propagation were serially passed in 16 02. glass prescription bottles. A saline-trypsin-versene (STV) buffer solution was used to remove the cells from the surface of the glass. After centrifugation, the cells were resuspended in MEM plus serum, distributed in 16 02. bottles at a cencentration of6 X 10 cells per ml. (30 ml. per 16 oz. bottle), and incubated at 37C. A confluent cell monolayer formed in 4 days. The bovine trachea cell line was used in the serum assay procedures and to propagate the viruses.

All viruses employed were propagated by inoculating 0.3 ml. of i0 dilution stock virus into 16 oz. glass prescription bottles of washed, prepared bovine trachea cell monolayers and were incubated at 37C. until a desired titer was achieved (e.g. 4 days). After harvesting of the virus, the tissue culture fluid containing the virus was tested for bacterial contamination by inoculation into thioglycollate medium and nutrient broth. The absence of bacterial growth after 3 days incubation at 37C. gave evidence that the virus cultures were free from bacteria. The viruses in the tissue culture fluid were then assayed for hamagglutinating activity and neutralization studies using the microtiter technique.

Each of the viruses was suspended in an aqueous solution and inactivated with 0.4 percent formalin (final concentration) and maintained for 1 week at 4C. The two polyvalent vaccines were prepared by combining equal proportions of the three killed viral components, i.e. the lBR, BVD and Pl-3 killed viruses. The vaccines with adjuvants were prepared by mixing the respective viral suspension or suspensions with a equal volume of an aqueous solution containing 4.0 percent weightlvolume sodium alginate and 0.67 percent by weight calcium gluconate. This soluble alginate had the characteristics previously set out. The vaccines were then tested for bacterial contamination in nutrient and thioglycollate broth cultures and for residual viral activity in bovine embryonic trachea (BEmT) tissue culture.

The parainfluenza-3 (Pl-3), strain SF-4, of virus used for vaccine production was serially passed in BEmT cells and a hemagglutination (HA) titer of 1:512 was obtained. Infectious bovine rhinotracheitis (lBR) was serially passed in the BEmT cells and a tissue culture inefective dose (TClD titer of 10 per ml. was obtained. The bovine viral diarrhea virus (BVD) was passed 15 times in BEmT cells and a TCID titer of IO -per ml. was achieved. The TCID titers were obtained by titerating the virus in a suspended cell system using a microtiter technique and the determinations were made using the method of Reed, L. J. and Muench, H., A Simple Method of Estimating Fifty Percent Endpoints," Am. J. Hyg. 27, (1938): p.493-497.

Forty-five head of beef cattle, 23 heifers and 22 steers, of mixed Angus and Hereford breeds, each weighing 300-400 pounds, were employed as test animals in the first study. The animals ranged in age from 5-8 months. They had no history of vaccination against any of PI-3, or IBR, or BVD viruses. All animals were in good health at the onset of vaccination and there had been no history of clinical cases of shipping fever (PI-3) IBR, or BVD in the herd.

The 45 head of catter were divided into groups. Groups II and III consisted of 3 and 2 animals, respectively, and the remaining eight groups had five animals per group. Twenty-five ml. of blood was obtained from each animal immediately prior to initial inoculation with vaccine. All serum samples were tested for antibodies against PI-3, IBR, and BVD using the HI test or the neutralization test described infra. The vaccines were parenterally administered by inoculating intramuscularly in the biceps femoris with 10.0 ml. of vaccine or control mixture as indicated in Table 1. Cattle in Groups IV, V, VI, IX, X were reinoculated with an additional 10.0 ml. of an identical preparation 38 days after the first injection. Blood samples of 25.0 ml. were obtained from all cattle on the following days subsequent to the initial injection: 3, 7, l0, I4, 17, 23, 31, 35, 38,45, 52,66, 87, and 120. Table I shows the composition of the vaccines used (prepared as described above) and the injection protocol employed for the cattle in Groups l-X.

TABLE I FOR CA'I'I'LE IN GROUPS I-X erythrocytes are used. Guinea pig erythrocytes were used in the HA determinations.

The following dilutions of the PI-3-MEM -MEM suspension were used in the HA test: 1:10, 1:15, 1:20. 1:30, 1:40, 1:60, 1:80, 1:120, 1:160, 1:240, lz320. 1:480, 1:640, and 1:960. One ml. of the initial 1:10 and 1:15 dilutions ofthe virus were prepared in Kahn tubes. and all succeeding HA and HI tests were performed using the above referred to microtiter procedure.

One of the main features of an antigenantibody reaction is that the combination is a firm and specific one. Therefore, if the antigen is particulate, its corresponding homologous antibodies may easily be removed from a mixture of antibodies. Since the reaction is specific, this facilitates determining the specific antigens or antibodies present in the system, separating the antibodies if two or more kinds are present in the same serum, and concentrating the antibodies in the serum.

Tests were conducted to determine whether there was any immunological relationship between the three viruses used in preparing the vaccines described above in order to be able to carry out quantitative studies with the immune sera. It was necessary to adsorb out of the sera of the viral antibody components before the third component could be analyzed. The procedure was carried out in 13 X I00 mm.dilution tubes using immune sera against PI-3, IBR and BVD which had been prepared in rabbits. To a 1.0 ml. aliquot of PI-3 immune serum diluted l:l0, was added 1.0 ml. containing a 10 TCID/ml. suspension of IBR virus. To this aliquot of PI-3 immune serum diluted 1:10 was then added 1.0 ml. of 10 TCID/ml. suspension of BVD virus. The tubes were incubated at C. for 4% hours and were titrated. Each of the other two remaining immune sera IBR and BVD, were treated in a similar manner by adsorbing out heterologous antibodies in the sera.

Comparative tests were made of adsorbed anc CATTLE COMPOSITION NUMBER OF AMOUNT IN- GROUP NO. OF VACCINES INJECTIONS JECTED, ML.

1 l-S NOT INJECTED 0 0 ll 6-8 ADJUVANT ONLY I 10 III 9-10 MINIMUM ESSENTIAL MEDIUM (MEM) I I0 ONLY IV 11-15 ADJUVANT IBR 2 10 V 16-20 ADJUVANT BVD 2 10 VI 21-25 ADJUVANT PI-3 2 10 VII 26-30 ADJUVANT IBR BVD PI-Il 10 VIII 31-36 IBR BVD PI-J W/O ADIUVANT I 10 IX 37-40 ADJUVANT IBR BVD PI-J 2 10 X 41-45 IBR BVD PI-3 W/O ADJUVANT 2 10 A microtiter technique developed by Sever and described in Application of a Microtechnique to Viral Serological Investigations," J. Immunol., 88 (1962), pages 320-329, was used for the hemagglutinationactivity (HA) and hemagglutination-inhibition (HI) tests described hereinafter.

As to HA procedure, the myxovirus PI-3 (Strain Sf-4) is capable of agglutinating the bovine, guinea pig, porcine and human type 0 erythrocytes. The highest HA titer is reached when either guinea pig or porcine ml. saline solution). After thorough mixing, the samples were left at room temperature for 20 minutes and then centrifuged at 1500 rpm for minutes. The supernatant fluid was saved. The serum samples were then incubated at 56C. for 30 minutes to remove the complement. This treatment resulted in a serum dilution of 1:10. A 1:15 dilution of each serum was obtained by adding 0.1 ml. of the 1:10 dilution to 0.5 ml. phosphate buffered saline the 1:10 and 1:15 diluted samples were used to make the following dilutions in the HI test: 1:10, 1:15, 1:20, 1:30, 1:40, 1:60, 1:80, 1:120, 1:160, 1:240, 1:320, 1:480, 1:640, and 1:960.

An HA test was performed on each stock virus suspension in MEM to determine 4 HA units per 0.025 ml. After the dilution of the virus was determined, a standard amount of4 HA units per sample was employed in the H1 test.

0.025 ml. samples of a 1:10 dilution of serum and a 1:15 serum dilution were prepared. 0.025 ml. of phosphate buffered saline solution was added to all samples. 0.025 ml. of the viral suspension (4 HA units per 0.025 ml.) was then added to all samples and the samples were sealed. After incubation at room temperature (25C.) for 30 minutes, 0.05 ml. of 0.5 percent guinea pig RBC was added to all samples. The samples were rescaled, incubated at room temperature for 30 minutes, and the results recorded. Known negative and positive antisera were included in each determination as controls. The H1 titer was established as the last dilution in each group of samples to show complete. inhibition of HA.

A hermadsorption technique, using the bovine trachea cell line supplemented the H1 test. When appropriate erythrocytes are added to cell cultures infected with influenza virus, the erythrocytes will adsorb to the infected cell surface. The phenomenon of hemadsorption is dependent upon selective attachment of erythrocytes onto the monolayer surface of tissue culture cells. It is demonstrated by the addition of erythrocytes to a tissue culture system in which propogation of hemagglutinin-producing virus has occured.

Serum specimens were treated similarly to those in the H1 test. The sera were incubated at 56C. for 30 minutes to remove the complement and were then diluted to 1:10 with phosphate buffered saline dilution tubes. A 1:15 dilution of each serum was obtained by adding 0.1 ml. of the 1:10 dilution to 0.05 ml. of phosphate buffered saline. The 1:10 and 1:15 diluted samples were used to make the following dilutions used in the hemadsorption test: 1:10, 1:15, 1:120, 1:30, 1:40, 1:60, 1:80, 1:120, 1:160, 1:240, 1:320 1:480, 1:640, and 1:960.

An HA test was performed on each stock virus suspension in MEM to determine 4 HA units per 0.1 ml. After determining the dilution of virus that contained 4 HA units, the dilution that contained 4 HA units could be calculated and used as the standard amount of virus for each bottle in the hemadsorption test.

The following procedure was used in carrying out the hemadsorption tests. Bovine trachea cell monolayers were prepared in 1 02. bottles with MEM plus fetal calf serum growth medium. The growth medium was poured from the monolayers and the cell sheet was washed twice with phosphate buffered saline. Onetenth ml. of the appropriate virus-serum mixture was added to each bottle, and the bottle was then rotated to assure complete coverage of the monolayer by the mixture. The monolayers with the virus-serum mixtures were allowed to adsorb for 1 hour at room temperature (25C.). After the 1 hour adsorption period, 2.5 ml. of MEM maintenance medium was added to each bottle, and the bottles were sealed and incubated at 37C. for 72 hours. The bottles were removed from the incubator and 0.4 ml. of a 0.5 percent suspension of washed guinea pig erythrocytes was added to each bottle, and the bottles were incubated at 4C. for 30 minutes. Hemadsorption was observed in bottles where the unneutralized virus has infected the cells. Hemadsorption was not observed in bottles where neutralized virus had failed to infect the cells. The hemadsorption titer was established as the highest dilution to show a positive reaction.

Antibody with specificity for certain antigens of virus particles can neutralize the infectivity of the virus by combining with the viral antigen and preventing viral multiplication. Neutralizing antibody can be detected by the inoculation of prepared virus antiserum mixtures into susceptible animals or onto the surface of mammalian tissue cultures. The neutralization best demonstrates the appearance or rise in titer of antibody during the course of illness or vaccination.

Tissue culture methods employing bovine trachea cells were utilized to determine the neutralizing capacity of the produced antibody. Serial ten-fold stock virus dilutions of l0" through l0"were prepared. The antiserum was incubated at 56C. for 30 minutes to inactivate the complement and was substantially diluted to 1:10, 1:100 and 121000. Undiluted serum was also used. Non-immune serum was treated in the same manner. One ml. of each virus dilution was combined with an equal amount of each antiserum dilution and incubated at room temperature for 1 hour. A 0.025 ml. quantity of each mixture was added to a suspended system of bovine trachea cells. The bovine trachea cell concentration was 2.5 X 10* cells per sample. Virusphosphate buffered saline mixtures and virus-normal serum were used as controlsfThe virus-serum-cell system was incubated for 6 days at 37C. at which time the cells were observed for cytopathological effects. The 1D, titers were determined by the Reed and Muench technique mentioned supra. and the neutralization indices (N1) were calculated bu subtracting the log of the titer of the virus mixed with immune serum from the titer of the control virus.

In discussing the results of the above first study, the following terminology will be used for convenience to designates the vaccines employed. The three monovalent vaccines are designated 1. Of these, 1A is 1BR, 1B is BVD, and 1C is Pl-3. Vaccine ll refers to the polyvalent vaccine of the invention without the adjuvant and Vaccine 111 to the polyvalent vaccine with adjuvant.

Cattle in Groups 11 through X, as shown in Table l, were injected with a vaccine composed of either adjuvant only, MEM only, a single virus plus adjuvant (Vaccine l), triple virus without the adjuvant (Vaccine 11), or triple virus plus adjuvant (Vaccine 111), and did not shown any evidence of hypersensitivity or any other outward reactions as a result of experimental inoculations.

TABLE ll-continued AVERAGE ANTIBODY RESPONSE OF CATrLE INOCULATED The means values of the neutralizing activity of the antisera against IBR virus are presented in Table II and FIG. I.

EITHER IBR VACCINE OR POLYVALENT VACCINE COMPOSED TABLE II OF PI-3, IBR, AND BVD VIRUSES, EXPRESSED AS NEUTRALIZATION INDICES AGAINST IBR VIRUS RECIPROCAL AVERAGE ANTIBODY RESPONSE OF CATTLE INOCULATED POST- OF ANTI- VACCINATION SERUM DIL.

VII VIII EITHER IBR VACCINE OR POLYVALENT VACCINE AGAINST IBR VIRUS VII VIII RECIPROCAL POST OF ANTI- VACCINATION SERUM DIL.

ANIMALS IN GROUPS IV, IX. AND X WERE GIVEN A SECOND INJECTION OF ANTIGEN 38 DAYS AFTER THE INTTIAL INJECTION. l5 ANTIBODIES NOT DETECTED IN GROUPS I, II, AND III AT ANY TIME.

In FIG. I the neutralization index is plotted against the number of days post vaccination. The neutralizing antibodies to IBR virus were first observed in a l0 antiserum dilution on the 14th day after vaccination. Titrations of sera from earlier periods after vaccination antibodies. Consequently, these values are omitted from the tables. In animals from Group IV, injected twice with the Vaccine IA, neutralizing titers reached the maximum value by the 28th day after the second 35 injection. Similarly, in Group IX, the highest antibody response to two injections of Vaccine III also occurred at the same time. The neutralization indices of the two groups, IV and IX, began decreasing at the 87th day afte the initial vaccination. Sera from animals in Group VII, injected once with Vaccine III, reached maximum antibody activity on the 66th day following initial vaccination. Groups VIII and X which received one and two injections, respectively, of Vaccine II also reached a peak of antibody activity at the 66th day after vacci- 66 nation. By the 87th day after vaccination, the neutralizing titers of the serum began decreasing. Injection of vaccines without adjuvant did not produce as high an antibody response as did the vaccines that were prepared with the adjuvant.

TABLE III AVERAGE ANTIBODY RESPONSE OF CATTLE INOCULATED WITH EITHER BVD VACCINE OR POLYVALENT VACCINE COMPOSED or PI-3. IBR, AND BVD VIRUSES EXPRESSED AS NEUTRALIZATION INDICES AGAINST BVD VIRUS SERUM OBTAINED RECIPROCAL ON INDICATED OF ANTI- DAY POST-VACCINATIOISERUM DIL. V

VIII

VII

a8 Asbeoas i hssj 0 02 02 03 0032 02 002 002 O I0 I00 I000 I0 I00 I000 TABLE Ill-continued SERUM OBTAINED RECIPROCAL ON INDICATED OF ANTI- DAY POST-VACCINATIOISERUM DIL. V

ANIMALS IN GROUPS V. IX. AND X WERE GIVEN A SECOND INJECTION OF ANTIGEN ON 38 DAYS AFTER THE INITIAL INJECTION. NO ANTIBODIES DETECTED IN GROUPS I. II. AND III AT ANY TIME.

As in the case of IBR, antibodies to BVD were first 20 observed in the l0 antiserum dilution at the l4th day vaccination. Two injections of vaccine IB caused an antibody response that reached a maximum level on the 28th day following injection. By the 87th day fol- 2 lowing the initial vaccination, the neutralizing activity of the immune serum began to decrease. The animals in Group VII, which received one injection of Vaccine III. produced a maximum response on the 66th day after injection, but the antibody activity of the sera decreased by the 87th day. The cattle in Group IX, receiving two injections of Vaccine III, reache a maximum level of antibody production at the l4th day subsequent to the second injection and began decreasing by the 49th day after the second inoculation. Animals 3 in Group VIII, having received a single injection of Vaccine ll, reached their peak of neutralizing activity against BVD at the 45th day after vaccination. and the antibody ltiter of the immune serum began decreasing by the 52nd day post vaccination. Cattle in Group X. injected twice with Vaccine II, reached a peak of neutralizing activity at the 28th day after the second injection, and the antibody titer began decreasing by the 49th day subsequent to the second injection.

The HI activity of the immune sera against PI-3 virus HI antibodies toward PI-3 were first observed on the 14th day after vaccination. Maximum HI activity of the sera from cattle in Group VI, having received two injections of the Vaccine IC, was observed at the 28th day following the second injection, and the titer began decreasing by the 49th day after the second inoculation. Sera from Group VII, which recieved one injection of Vaccine III, reached a maximum HI titer at 66 days after vaccination. By the 87th day following vaccination, the HI activity of the immune sera began to decrease. Cattle in Group IX, which received two injections of Vaccine III, also produced a maximum antibody responses to PI-3 at the 66th day following the initial injection. The antibody level of the sera decreased by the 87th day after the initial vaccination. Animals in Group X, inoculated twice with the Vaccine II, produced a maximum response to PI-3 28 days following the second vaccination, and by the 49th day subsequent to the second injection the antibody level began to decrease.

Antibody activity of the sera toward PI-3 was also determined by the procedure of hemadsorption inhibition (HAdI). The mean values of the HAdI activity are recorded in Table V and FIG. '4.

TABLE V is presented in Table IV and FIG. .3.

TABLE IV AVERAGE ANTIBODY RESPONSE OF CATTLE INOCULATED EITHER PI-3 VACCIIEIIE OIZJEIbYVALENT VACCINE OF PI-3. IBR. AND BVD VIRUSES EXPRESSED AS RECIPROCAL OF HI ACTIVITY AGAINST Pl-3 SERUM OBTAINED GROUP ON INDICATED DAY POST-VACCINATION AVERAGE ANTIBODY RESPONSE OF CATTLE INOCULATED WITH EITHER PI-3 VACCINE OR POLYVALENT VACCINE COMPOSED OF Pl-3. IBR, AND BVD VIRUSES EXPRESSED AS RECIPROCAL OF HEMADSORPTION ACTIVITY AGAINST PI-3 SERUM OBTAINED ON INDICATED DAY VI VII VIII IX X POST-VACCINATION VI VII VIII IX X 3 10 I0 I0 I0 10 3 10 I0 10 I0 10 I0 10 I0 10 I0 I0 I0 I0 I0 I0 I0 l0 I4 22 I0 I6 10 IS I4 26 I2 I8 I0 I7 I7 38 20 30 I8 26 I7 42 I8 30 I9 28 23 72 30 34 34 34 23 76 34 36 38 38 3| I00 64 64 52 48 31 I04 72 68 60 56 35 220 I04 72 I08 35 25 30 44 30 38 U. '0 i fit it B 45 335 I92 I04 272 I20 45 352 203 04 233 20 52 448 288 I28 384 I76 52 448 288 I36 4I6 I92 66 480 352 I44 416 208 66 SIZ 384 I44 480 208 87 384 304 I28 368 I I2 87 416 336 I20 384 I I2 I20 288 256 92 272 84 6 I20 288 272 84 272 88 ALL TITERS OF I0 ARE I0. THE INDICATED HI TITERS ARE BASED ON A RISE ABOVE A BASE LINE OF I0. GROUPS I. II. AND III SHOWED NO RISE IN ANTIBODY TITER ABOVE A BASE LINE OF I0.

" SECOND INJECTION.

-NOT DONE.

ALL TITERS OF I0 ARE l0. THE INDICATED HEMADSORPTION TITERS ARE BASED ON A RISE ABOVE A BASE LINE OF I0. GROUPS I. II, III SHOWED NO RISE IN ANTIBODY TITER ABOVE A BASE LINE OF I0.

" SECOND INJECTION.

-NOT DONE.

13 The Mill titers parallel the III titers. but the llAdl test is more sensitive than the HI test. An analysis of the HAdl activity of the immune sera toward Pl-3 would be repetitious of the HI activity analysis. An inspection of the HAdI titers in Table V and FIG. 4 reveals the similarity in antibody activity to HI titers found in Table IV and FIG. 3.

The average neutralizing activity of the immune sera toward PI-3 is recorded in Table VI and FIG. 5

TABLE VI I4 tion. and by the 49th day alter the second inoculation AVERAGE ANTIBODY RESPONSE OF CATTLE INOCULATED WITH EITHER Pl-3 VACCINE OR POLYVALENT VACCINE COMPOSED OF PI-3, IBR, AND BVD VIRUSES EXPRESSED AS NEUTRALIZATION INDICES AGAINST PI-J VIRUS DAY RECIPROCAL POST- OF ANTI- VACCINATION SERUM DIL.

I0 I00 I000 0 I0 I00 I000 0 I0 I00 I000 0 I0 I00 I000 0 seq this ANIMALS IN GROUPS VII IX. AND X WERE GIVEN A SECOND INJECTION OF ANTIGEN IIII DAYS AFTER THE INITIAL INJECTION. ANTIBODIES NOT DETECTED IN GROUPS I. II, AND III AT ANY TIME.

Antibodies toward Pl-3 were first demonstrated in a higher level of antibodies than did the complex vaccin l0 dilution of antiserum 14 days after initial vaccination. Maximum neutralizing activity of the serum in the cattle of Group VI, which received two injections of the Vaccine IC, was recorded on the 28th day after the second injection. The antibody level began decreasing by the 49th day subsequent to the second inoculation. Animals in Group VII, injected once with Vaccine III, developed a maximum response to PI-3 66 days following the initial vaccination. By the 87th day post vaccination, the NI of neutralization index began to decrease. Maximum neutralizing activity of sera from Group VIII, having received one injection of Vaccine II, was reached at the 66th day after the initial injection and began decreasing by the 87th day post vaccination. Animals in Group IX. which received two injections of Vaccine III, developed maximum neutralization titers in the sera at the 28th day following the second injecwithout the adjuvant (Vaccine II). Antibodies to th three viruses persisted for over 4 months in the sera c animals injected twice with the adjuvant-containin vaccine. The test results also indicate that the thre killed viruses of the polyvalent vaccines of the inver tion do not interfere with each other and that the v ruses may be administered in a single polyvalent var cine.

A second study was carried out employing three vac cines. The first vaccine was a polyvalent vaccine (Vac cine I) consisting of equal proportions of bovine virz diarrhea virus, Southern Illinois University strai (BVD-SIU). infectious bovine rhinotracheitis viru: Southern Illinois University strain (lBR-SIU) and p2 rainfluenza-3 (SF-4), Southern Illinois University strai (Pl-3-SIU). The second vaccine (Vaccine II) was polyvalent vaccine consisting of equal proportions c the above three viruses and of Pasteurella multocida, an organism frequently isolated along with PI-3 in cases of shipping fever. The third vaccine (Vaccine III) was a polyvalent vaccine consisting of equal proportions of BVD and [ER viruses (Goff strains obtained from Affiliated Laboratories, Whitehall, Illinois) and the Pl-3- SIU virus. Each of the vaccines was prepared with a sodium alginate adjuvant composed of an aqueous solution containing 4.0 percent weight/volume of sodium alginate and 0.67 percent by weight calcium gluconate. The adjuvant was mixed in a 1:1 ratio with the suspension of viruses for the complete vaccine in each instance.

The Pl-3-SIU virus employed was serially passed 5 times in bovine embryonic trachea (BEmT) cells and a hemagglutination (HA) titer of 1:640 was achieved. The lBR-SIU and lBR-Goff viruses were serially passed 6 times each and tissue culture infective dose (TCID titers of 10" and 10 respectively, were reached. The BVD-SIU and BVD-Goff viruses were serially passed seven times in BEmT cells and TCID titers of 10" and 10, respectively, were achieved. Vacine 111 which incorporated Pasteurella multocida contained 1 X 10 organisms per ml.

All viruses employed were propagated as previously described and each virus was inactivated with 0.4 percent formalin (final concentration) and maintained for 1 week at 4C. All other procedures employed in the first study were followed in the second study.

Thirty-six head of beef cattle, 22 heifers and 14 steers, of Hereford breed, each weighing 750-950 pounds, were employed as test animals in the second study. The animals were 15 months old. They had no history of vaccination and there had been no history of clinical cases of shipping fever, 1BR, or BVD in the herd.

The 36 head of cattle were divided into five groups. The vaccines were parenterally administered by inoculating the animals intramuscularly in the biceps femoris 16 TABLE V111 AVERAGE ANTIBODY RESPONSE OF CATTLE INOCULATED WITH POLYVALENT VACCINE COMPOSED OF P1-3, 1BR. AND

BVD VIRUSES EXPRESSED AS NEUTRALIZATION INDICES AT A 1:100 ANTISERUM DILUTION AGAINST lBR-SIU 2nd lnjectlon of original vacclne Figures bued on 1 H000 lltllltfllm dilution Neutralizing antibody was first discovered in a 1:100 dilution of antiserum in the animals in Groups 11, Ill, and IV, 14 days following the initial vaccination. The titers continued to rise, and when the neutralization index of 1.0 was reached, the second injection of the same vaccine was administered. Groups II and IV received the second inoculation on the 51st day and Group III on the 37th day following the initial injection. The maximum neutralizing titers in Group II were achieved on the 14th day following the second injection or on the 65th day after the initial injection. Maximum neutralizing activity of sera from Group 111 was seen on the 28th day following the second injection.

Antibody activity to lBR-SIU was still present in the sera from Groups 11, Ill, and IV 93 days following the first injection.

Table IX and FIG. 7 show the neutralizing activity of antisera of animals in Groups 11, III, and 1V against BVD-SIU.

TABLE 1X AVERAGE ANTIBODY RESPONSE OF CATTLE INOCULATED WITH POLYVALENT VACCINE COMPOSED OF PI-3. 1BR, AND BVD VIRUSES EXPRESSED AS NEUTRALIZATION INDICES AT A 1:100 ANTISERUM DILUTION AGAINST IBR-SIU muscle using disposable l9-gauge needles and syringes. g GROUPS The Same procedures described above for the first VACCINATION l n m w study were used in carrying out the same tests In the I 00 0 00 00 second study. 3 00 00 0.0 Table V11 shows the composition of the vaccines and Z 8-8 8g 88 8:? the injection protocol employed in the second study. 4 0.7 m

TABLE V11 COMPOSITION OF VACClNES AND INJECTION PROTOCOL FOR CATTLE 1N GROUPS l-V COMPOSrrION NUMBER OF AMOUNT IN- GROUP CATTLE NO. OF VACCINES INJECTIONS .IECTED. ML.

1 7 Not injected 0 0 ll 7 SIU strains of 1BR. BVD, PI-3 2 5 111 7 SIU strains of 1BR. BVD. Pl-3 2 10 1V 7 SIU strains of 1BR. BVD. PI-3 and Past. multocida 2 5 v a lBR-Goff, BVD- Goff, Pl-3-S1U 2 5 The procedures for determining the antibody levels 8 8 I g 3.; in the sera of animals in the second study were slmllar 5 J to that of the first study, except for two minor varIa- 65 on M 33 3.0 tions which did not affect the results. 79 0.0 3.4 3.0 2.21 The means neutralization values of antisera agaInst 93 0.0 3.1 "2.8 2.4

lBR-SIU virus are presented in Table V111 and FIG. 6.

2nd injection of original vaccine "Figures based on a 1:1(100 antiserum dilution As with IBR-SIU, neutralizing antibody was first detected on the 14th day after the initial injection. In Groups II and IV, the maximum titers were observed on the l4th day following the second injection. Maximum neutralization activity in Group III was seen on the 28th day after the second inoculation. Antibody to IBR-SIU persisted in the sera 93 days after the initial injection.

The hemagglutination-inhibiting activity of the sera against PI-3 virus is seen in Table X and FIG. 8.

TABLE X AVERAGE ANTIBODY RESPONSE OF CATTLE INOCULATED WITH POLYVALENT VACCINE COMPOSED OF PI-Il, IBR.

All titers of IO i II). The indicated HI liters are based on a rise above a base line of IO.

Parainfluenza-Zl-SIU is the only virus common to all vaccines of Groups II-V. Sera from Groups II, IV, and

TABLE xn AVERAGE ANTIBODY RESPONSE OF CATI'LE INOCULATED WITH POLYVALENT VACCINE COMPOSED OF PI-3. IBR-GOFF. AND BVD-GOFF VIRUSES EXPRESSED AS NEUTRALIZATION INDICES AT A l:l00 ANTISERUM DILUTION AGAINST BVD-GOFF DAY GROUPS POST-VACCINATION I V I I 0.0 0.0 14 0.0 0.I 2I 0.0 0.9 37 0.0 1.] SI 0.0 2.4 65 0.0 3.5 79 0.0 3.1 93 0.0 2.8

2nd injection of original vaccine Neutralizing antibody was first detected on the 14th day following the initial injection. Maximum titers were achieved on the 65th day after the first injection or on the I4th day following the second injection. The presence of antibody was still detected on the 93rd day after the first vaccination.

The agglutinating activity of the antisera ofGroup IV against Pasteurella mulmcida is shown in Table XIII.

TABLE XIII AVERAGE ANTIBODY RESPONSE OF CATTLE INOCULATED WITH POLYVALENT VACCINE COMPOSED OF I'l-3 IBR. DVD, AND PAST. MULTOCIDA EXPRESSED AS RECIPROCAL OF AGGLUTINATING ACTIVITY AGAINST PAST. MULTOCIDA DAY GROUPS V showed maximum HI titers on the 14th day following POSTNACCINATION l w the second inoculation. The maximum HI titer in I 10 10 Group III was on the 28th day after the second inocula- 3 10 I0 tion. Hemagglutination-inhibition titers persisted in the Z :8 :g sera after the 93rd post-vaccination day. 5 :t :8 Table XI and FIG. 9 show the neutralizing activity of 37 lo 0 .51 10 '10 antisera against IBR Goff VII'LIS. 65 '0 lo 79 I0 10 Table XI 93 10 I0 AVERAGE ANTIBODY RESPONSE OF CATTLE INOCULATED WIT POLYVALENT VACCINE COMPOSED OF PI-3. IBR-GOFF. AND BVD-GOFF VIRUSES EXPRESSED AS NEUTRALIZATION INDICES AT A l:l00 ANTISERUM DILUTION AGAINST lBR-GOFF DAY GROUPS POST-VACCINATION 7 0.0 0.0 l l 0.0 00 I4 0.0 0.4 2i 0.0 L0 37 0.0 1.8 Sl 0.0 '3.I 65 0.0 4.3 79 0.0 4.I 93 0.0 3.7

2nd injection of original vaccine Maximum neutralizing activity was achieved on the 14th day following the second injection. Titers were first detected on the 14th day following the initial injection. Neutralizing activity was still present on the 93rd day following the initial injection.

The mean neutralizing activity of sera to BVD-Goff is shown in Table XII and FIG. 9.

2nd injection of original vaccine All titen of I0 & l0. The indicated agglutinatint titers are based on a rise above 1 base line of IO.

No agglutinating titers were detected in the sera ol animals injected with Vaccine II. The inability of ani' mals to develop detectable antibody toward Past. multocida is unexplainable. Even after a second injection 01 antigen, agglutinating titers were not achieved.

The results of these two studies show that high titer: of antibodies to BVD, IBR, and PI-3 viruses were detected in animals inoculated with the killed polyvalen' vaccine of the invention and that these titers were con siderably higher when the polyvalent vaccine wa: mixed with the alginate adjuvant. In addition, the titer: of antibodies to each virus obtained after injection 0 the polyvalent vaccines were of the same order of mag nitude as those produced by inoculation of single viru preparation.

In view of the above, it will be seen that the severe objects of the invention are achieved and other advan tageous results attained.

As various changes could be made in the above methods and products without departing from the scope of the invention. it is intended that all matter contained in the above description or shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.

What is claimed is:

l. A vaccine comprising a suspension of l to percent weight/volume of a soluble alginate adjuvant vehicle suitable for parenteral injection and an admixed volume of an aqueous solution containing a final concentration of between about 0.3percent and 0.5 percent formaldehyde and formalin-killed bovine viral diarrhea virus in a concentration of lO-l0 TClD /ml. which has been maintained at a temperature of about 4C. for approximately 1 week.

2. A vaccine as set forth in claim 1 wherein said vehicle is constituted by an aqueous 0.4 percent solution of formaldehyde.

3. A vaccine as set forth in claim I wherein said adjuvant is constituted by an equal volume of an aqueous solution containing about I to 5 percent weight/volume of a soluble alginate having the following characteristics:

a. in 5 percent weight/volume aqueous solution.

readily passes a 24-gauge needle;

b. in 4 percent weight/volume aqueous solution, has

a viscosity less than 50 centistokes; c. a chemical equivalence below 250; d. a milliosmolarity less than kg. of water on a Fiske osmometer.

# t i i i

Claims

1. A VACCINE COMPRISING A SUSPENSION OF 1 TO 5 PERCENT WEIGHT/VOLUME OF A SOLUBLE ALGINATE ADJUVANT VEHICLE SUITABLE FOR PARENTERAL INJECTION AND AN ADMIXED VOLUME OF AN AQUEOUS SOLUTION CONTAINING A FINAL CONCENTRATION OF BETWEEN ABOUT 0.3 PERCENT AND 0.5 PERCENT FORMALDEHYDE AND FORMALIN-KILLED BOVINE VIRAL DIARRHA VIRUS IN A CONCENTRATION OF 10*:5-10**8 TCID50/ML. WHICH HAS BEEN MAINTAINED AT A TEMPERATURE OF ABOUT 4*C. FOR APPROXIMATELY 1 WEEK.

3. A vaccine as set forth in claim 1 wherein said adjuvant is constituted by an equal volume of an aqueous solution containing about 1 to 5 percent weight/volume of a soluble alginate having the following characteristics: a. in 5 percent weight/volume aqueous solution, readily passes a 24-gauge needle; b. in 4 percent weight/volume aqueous solution, has a viscosity less than 50 centistokes; c. a chemical equivalence below 250; d. a milliosmolarity less than 150 kg. of water on a Fiske osmometer.