Comp. Immun. Microbiol. infect. Dis. Vol. 15, No. 3, pp. 221-228, 1992 Printed in Great Britain. All rights reserved
0147-9571/92 $5.00+0.00 Copyright © 1992 Pergamon Press Ltd
A TISSUE CULTURE VACCINE WITH LAPINIZED CHINESE (LC) STRAIN OF HOG CHOLERA VIRUS (HCV) M . FERRARI Istituto Zooprofilattico Sperimentale della Lombardia e della Emilia, via A. Bianchi 7, 25100 Brescia, Italy Akstract--The lapinized chinese (LC) strain of hog cholera virus (HCV), was adapted to grow in a cell line from minipig kidney (MPK) where it reached a titer, as determined by immunofluorescence, significantly higher than in rabbits. Inasmuch as the immune serum to HCV neutralized the culture-adapted virus, it was concluded that its antigenicity did not undergo any change after adaptation to MPK cells. The MPK-LC adapted virus (MPK-LC-HCV) showed also a higher immunogenic activity in rabbits, in comparison with the original LC virus. The MPK-LC-HCV protected pigs against challenge infection with virulent HCV. Thus, the vaccinated pigs did not show any clinical signs of disease, nor have they been responsible of virus shedding after they were exposed to the challenge infection 1 month or 6 and 11 months later. All vaccinated pigs seroconverted after vaccination and the antibody titers were on the same range of those reported in pigs vaccinated with the traditional vaccine prepared in rabbits. In the same pigs the antibody concentration underwent a booster effect following challenge infection. It was suggested the MPK-LC-HCV vaccine as an alternative product that might be used to prevent HCV infection. Key words: Lapinized chinese hog cholera virus, minipig cell line, vaccine, pigs.
VACCIN PREPARI~ AVEC LA SOUCHE LAPINISI~E CHINOISE (LC) DU VIRUS DE LA PESTE PORCINE C L A S S I Q U E ( P P C ) A D A P T I V E ,~ L A C U L T U R E C E L L U L A I R E R4sum~-La souche lapinisre dite chinoise (LC) du virus de la peste porcine classique (PPC) a 6t6 adaptre ~i se developper dans une culture cellulaire de rein de mini-porcelet (RPM) o6 elle rejont un titre, determin6 par l'immunofluorescence, qui est d'une manirre significative plus 61ev6 que dans les lapins. Du moment que le serum immune anti PPC a neutralis6 le virus adapt6 ~ la culture cellulaire, on est ainsi arriv6 ~i la conclusion que ses antigrnes ne subissaient aucun changement aprrs l'adaption aux cellules RMP. Le virus RMP-LC multipli6 en culture cellulaire (RMP-LC-PPC) a demontr6 aussi una activit6 immunologique beaucoup plus grande chez les lapins, si on le compare avec le virus LC original. Le RMP-LC-PPC protrgeait les porcins contre l'infection provoqure par le PPC virulent. C'est pourquoi les porcins vaccin6s ne montraient aucun symptome de la maladie, ils n'etaient mrme pas responsables de la elimination du virus bien qu'ils aient &~ exposrs /t l'infection exp~rimentale provoqure et ce pendant 1, 6 ou 11 mois aprrs. Tousle porcelets vaccinrs presentaient une rrponse immunitaire aprrs la vaccination ainsi que les titres des anticorps etaient du mrme niveau que ceux reportrs dans les porcins vaccin6s avec le vaccin traditionnel pr6par6 sur les lapins. Dans les m~mes porcelets, la concentration des anticorps augmentaient suivant l'infection experimentale avec le virus virulent. On a conseill6 la vaccin RMP-LC-PPC en tant que produit alternatif qui peut etre utilis6 pou prevenir l'infection de la PPC. Mots-clefs: Virus lapinisre chinoise peste porcine classique, culture cellulaire de mini-porcelet, vaccin, porcelets.
221
222
M. FERRARI INTRODUCTION
It is known that vaccination is the most reliable method to reduce the spread of the infectious diseases, at least, it seems to be so at the very beginning of a program aimed to stop the propagation of the disease. One of the most common infections, where the application of such a principle led to a significant improvement of the control of the disease, is the hog cholera. Several vaccines have been used to protect pigs against hog cholera virus (HCV) infection. At the early stage of vaccination programs, inactivated preparations, obtained by exposure of the viral antigen to the activity of some chemical products, such as formalin [1] or crystal violet [2], have been used. However, when it was clear that the inactivated vaccines were only able to prevent the clinical manifestations of the disease but not to avoid the possibility by the vaccinated pigs to become infected, they progressively were replaced by the modified-live (ML) vaccines. One of the most popular ML vaccine is the lapinized chinese (LC) strain of HCV, consisting in a suspension of spleen from infected rabbits. However, several successful attempts have been made in order to develop vaccines through the adaptation of the virus in tissue culture [3-5]. Accordingly, a study was planned with the purpose: (a) to test the ability by the LC virus to grow in two cell lines, one of which originated from rabbit kidney (RK-13) and the other obtained from minipig kidney (MPK); (b) in the case of a successful result, to determine whether the tissue culture adapted virus protect pigs against challenge infection with virulent HCV. This paper gives the findings made [6-8].
EXPERIMENTAL PROCEDURES--RESULTS
(1) Attempts to cultivate LC strain of HCV in RK-13 and MPK cell lines [7] Virus. The virus was obtained from the spleen of a rabbit inoculated intravenously (i.v.) with 800 median rabbit infectious doses (RIDs0) of LC strain of HCV in a volume of 2 ml. The rabbit was killed when its body temperature reached about 41°C. The infected spleen was removed and used to prepare a lymphocyte cell suspension according to the procedure described in the next paragraph. Infection of the cell lines. The inner portion of the spleen from the infected rabbit was washed in minimum essential medium (MEM), minced and homogenized in a wire-netting. Cells were then washed three times in MEM by centrifugation at 800 r.p.m, and the final pellet was treated with 0.83% NH4C1 cold solution for 1 rain to destroy the red blood cells. 2 ml of this cell suspension were slowly layered over 8 ml of fetal calf serum, then centrifuged at 800 r.p.m, for 10 min at 4°C and the pellet was diluted to a concentration of 108 cells in MEM containing 10% (v/v) of fetal calf serum. This lymphocyte cell suspension was co-cultured [9] with RK-13 and M P K cell suspensions in MEM, each suspension containing 5 × 1 0 6 cells/ml. The mixtures, previously resuspended in 100 ml of the appropriate medium (MEM enriched with 10% fetal calf serum, or MEM containing 2 x aminoacids-vitamins solution, for RK-13 or MPK, respectively) were inoculated into 150 cm 2 plastic flasks, and incubated at 37°C in 5% CO2 atmosphere. Subpassages were carried out after a 5 day incubation by culturing trypsinized cell suspensions of infected cells with an equal amount of uninfected fresh harvested RK-13
Lapinized chinese strain of hog cholera virus grown in minipig kidney cell line
223
or M P K cells. The virus antigen in the infected cells was detected at each serial passage by immunofluorescence (IF) according to the methodology described elsewhere [10]. According to the findings made by the IF tests, the virus was detected in RK-13 cells only in the first two serial passages. Therefore, after two additional passages, it was decided to discontinue the study on the RK-13 cell line which apparently it was not susceptible to LC virus replication. However, the M P K cells appeared to be fully susceptible to the virus inasmuch as the viral antigen was regularly detected in the cells from the second passage onwards. So, after a total of 20 subpassages have been made, the evaluation of the susceptibility of M P K cells to the virus was made in rabbits. Thus, experimental infection, serum neutralization and protection tests were carried out in New Zealand white rabbits. For the experimental infection, two tests were made. In the first test, six rabbits were inoculated i.v. in groups of two, with culture fluid fraction (fraction A), cell suspension (fraction B) or disrupted cell suspension (fraction C), respectively [7]. Two additional rabbits were each inoculated i.v. with 5 ml of the original (vaccine) LC strain of H C V and served as controls. The rectal temperature from all inoculated animals was recorded twice daily and when it reached the value of 41 °C, the rabbits were killed and their spleens taken for virus detection in M P K cells by IF tests. The second test consisted in the titration of the infectivity of culture virus under study. The tests were carried out in 27 rabbits according to the methods already described [7] and, with the same procedures other rabbits were given the LC strain of HCV, the latter rabbits to be considered as controls. The results were evaluated on the basis of the body temperatures of the rabbits. A reaction was considered to be positive when temperature reached the value of 40°C or more within 5 days. Titers were calculated according to Reed and Muench [11]. The serum neutralization tests were carried out by mixing each of the two-fold dilutions, from 1:40 to 1 : 10,240, of fraction A from M P K infected cells with 1 : 10 dilution of either HCV-antiserum prepared in rabbits or rabbit normal serum. After 60 min incubation at 37°C, 2 ml of each mixture were inoculated in triplicate i.v. in rabbits. Rectal temperatures of rabbits were taken daily for 5 days after exposure. Final readings were made on the 5th day and the virus was considered to be neutralized by the immune serum in those mixtures which, in contrast with analogous mixtures containing normal serum, failed to evoke a febrile response in the rabbits. Finally, for the protection tests, fraction A from infected M P K cell cultures was serially diluted as above and each dilution was inoculated i.v. in a dose of 2 ml to three rabbits. A similar procedure was used for the inoculation of the LC strain of HCV. Challenge of the immunity of the rabbits was made 30 days after the initial infection by injecting i.v. 2 ml of 1 : 10 of the vaccine concentration of LC strain of HCV. Two additional rabbits which were initially given M E M , were exposed to the challenge virus as above and served as challenge virus controls. Rectal temperatures were taken, as usual, for 5 days after primary infection or challenge exposure. The results of the experimental infection of rabbits with the different fractions of the 20th passage infected cultures, are depicted in Table 1, where it can be seen that all rabbits reacted with fever, as did those inoculated with the vaccine. The virus was detected by IF in the spleens of all inoculated animals. The titer of the virus at its 20th passage level on M P K cells, as determined by experimental infection in rabbits, was 1:7240/2 ml, i.e. about 10 times higher than the vaccinal virus whose titer was of 1:756/2 ml.
224
M. FERRARI Table I. Clinical and virological response of rabbits to intravenous inoculation with different preparations of LC strain of HCV at its 20th passage level in MPK cell cultures Fever 40°C or higher
Inoculum
No. of rabbits
Affected No. of rabbits
Onset/duration (hr)
Virus detection in the spleens of the affected rabbits*
2
2
36/18
Positive
2
2
36/24
Positive
2
2
36/18
Positive
2
2
36/18
Positive
Virus culture
Culture fluid (fraction A) Cell suspension (fraction B) Disrupted cell suspension (fraction C) Control LC strain of HCV (vaccine)
*Virus was detected by direct immunofluorescence tests in MPK cell cultures inoculated with the infected spleens.
The antigenicity of the MPK cultured virus did not appear to undergo any modification insofar as the rabbits inoculated with the mixtures containing the immune serum to HCV remained healthy throughout the observation period (Table 2). The MPK adapted virus and LC strain of HCV appeared to be mutually protective in rabbits. However, the former virus was still immunogenic at the dilution of 1:2560, i.e. a value 8 times higher than the maximum protective dilution (1:320) reported for the LC strain of HCV. (2) Vaccination of pigs with LC strain of H C V grown in M P K cells
Virus [6, 8]. Pigs were vaccinated with the MPK-LC-HCV, at its 25th passage level or with the LC-HCV prepared in rabbits. The titers, as determined in rabbits, were of 1:1280 or 1 : 756, RID50/2 ml, respectively. The vaccinated pigs were subjected to challenge infection with virulent Brescia strain of HCV, the titer of which, as determined by IF in MPK infected cells, was of 105.74 median tissue culture infectious doses (TCID50) per 0.025 ml. Experimental design: inoculation of pigs. Sixteen pigs, about 60 days of age, were used. Twelve pigs, allotted into two groups of 6, were subjected to inoculation with MPK-LCHCV (Group 1) or LC-HCV (Group 2), respectively, each pig receiving intramuscularly (i.m.) 2.0 ml of the appropriate vaccine. Subsequently, at each 180 or 330 days following vaccination three pigs in Group 1, three in Group 2 and two additional unvaccinated pigs (controls) were separated from the others and subjected to challenge exposure with 2.0 ml of the virulent Brescia strain of HCV which was given also i.m. Temperatures of the pigs Table 2. Neutralization tests in rabbits of LC strain of HCV at its 20th passage level on MPK cells in presence of antiserum to HCV* Serum 1:10 Reference antiserum to HCV Rabbit normal serum
Virus titer
0147-9571/92 $5.00+0.00 Copyright © 1992 Pergamon Press Ltd
A TISSUE CULTURE VACCINE WITH LAPINIZED CHINESE (LC) STRAIN OF HOG CHOLERA VIRUS (HCV) M . FERRARI Istituto Zooprofilattico Sperimentale della Lombardia e della Emilia, via A. Bianchi 7, 25100 Brescia, Italy Akstract--The lapinized chinese (LC) strain of hog cholera virus (HCV), was adapted to grow in a cell line from minipig kidney (MPK) where it reached a titer, as determined by immunofluorescence, significantly higher than in rabbits. Inasmuch as the immune serum to HCV neutralized the culture-adapted virus, it was concluded that its antigenicity did not undergo any change after adaptation to MPK cells. The MPK-LC adapted virus (MPK-LC-HCV) showed also a higher immunogenic activity in rabbits, in comparison with the original LC virus. The MPK-LC-HCV protected pigs against challenge infection with virulent HCV. Thus, the vaccinated pigs did not show any clinical signs of disease, nor have they been responsible of virus shedding after they were exposed to the challenge infection 1 month or 6 and 11 months later. All vaccinated pigs seroconverted after vaccination and the antibody titers were on the same range of those reported in pigs vaccinated with the traditional vaccine prepared in rabbits. In the same pigs the antibody concentration underwent a booster effect following challenge infection. It was suggested the MPK-LC-HCV vaccine as an alternative product that might be used to prevent HCV infection. Key words: Lapinized chinese hog cholera virus, minipig cell line, vaccine, pigs.
VACCIN PREPARI~ AVEC LA SOUCHE LAPINISI~E CHINOISE (LC) DU VIRUS DE LA PESTE PORCINE C L A S S I Q U E ( P P C ) A D A P T I V E ,~ L A C U L T U R E C E L L U L A I R E R4sum~-La souche lapinisre dite chinoise (LC) du virus de la peste porcine classique (PPC) a 6t6 adaptre ~i se developper dans une culture cellulaire de rein de mini-porcelet (RPM) o6 elle rejont un titre, determin6 par l'immunofluorescence, qui est d'une manirre significative plus 61ev6 que dans les lapins. Du moment que le serum immune anti PPC a neutralis6 le virus adapt6 ~ la culture cellulaire, on est ainsi arriv6 ~i la conclusion que ses antigrnes ne subissaient aucun changement aprrs l'adaption aux cellules RMP. Le virus RMP-LC multipli6 en culture cellulaire (RMP-LC-PPC) a demontr6 aussi una activit6 immunologique beaucoup plus grande chez les lapins, si on le compare avec le virus LC original. Le RMP-LC-PPC protrgeait les porcins contre l'infection provoqure par le PPC virulent. C'est pourquoi les porcins vaccin6s ne montraient aucun symptome de la maladie, ils n'etaient mrme pas responsables de la elimination du virus bien qu'ils aient &~ exposrs /t l'infection exp~rimentale provoqure et ce pendant 1, 6 ou 11 mois aprrs. Tousle porcelets vaccinrs presentaient une rrponse immunitaire aprrs la vaccination ainsi que les titres des anticorps etaient du mrme niveau que ceux reportrs dans les porcins vaccin6s avec le vaccin traditionnel pr6par6 sur les lapins. Dans les m~mes porcelets, la concentration des anticorps augmentaient suivant l'infection experimentale avec le virus virulent. On a conseill6 la vaccin RMP-LC-PPC en tant que produit alternatif qui peut etre utilis6 pou prevenir l'infection de la PPC. Mots-clefs: Virus lapinisre chinoise peste porcine classique, culture cellulaire de mini-porcelet, vaccin, porcelets.
221
222
M. FERRARI INTRODUCTION
It is known that vaccination is the most reliable method to reduce the spread of the infectious diseases, at least, it seems to be so at the very beginning of a program aimed to stop the propagation of the disease. One of the most common infections, where the application of such a principle led to a significant improvement of the control of the disease, is the hog cholera. Several vaccines have been used to protect pigs against hog cholera virus (HCV) infection. At the early stage of vaccination programs, inactivated preparations, obtained by exposure of the viral antigen to the activity of some chemical products, such as formalin [1] or crystal violet [2], have been used. However, when it was clear that the inactivated vaccines were only able to prevent the clinical manifestations of the disease but not to avoid the possibility by the vaccinated pigs to become infected, they progressively were replaced by the modified-live (ML) vaccines. One of the most popular ML vaccine is the lapinized chinese (LC) strain of HCV, consisting in a suspension of spleen from infected rabbits. However, several successful attempts have been made in order to develop vaccines through the adaptation of the virus in tissue culture [3-5]. Accordingly, a study was planned with the purpose: (a) to test the ability by the LC virus to grow in two cell lines, one of which originated from rabbit kidney (RK-13) and the other obtained from minipig kidney (MPK); (b) in the case of a successful result, to determine whether the tissue culture adapted virus protect pigs against challenge infection with virulent HCV. This paper gives the findings made [6-8].
EXPERIMENTAL PROCEDURES--RESULTS
(1) Attempts to cultivate LC strain of HCV in RK-13 and MPK cell lines [7] Virus. The virus was obtained from the spleen of a rabbit inoculated intravenously (i.v.) with 800 median rabbit infectious doses (RIDs0) of LC strain of HCV in a volume of 2 ml. The rabbit was killed when its body temperature reached about 41°C. The infected spleen was removed and used to prepare a lymphocyte cell suspension according to the procedure described in the next paragraph. Infection of the cell lines. The inner portion of the spleen from the infected rabbit was washed in minimum essential medium (MEM), minced and homogenized in a wire-netting. Cells were then washed three times in MEM by centrifugation at 800 r.p.m, and the final pellet was treated with 0.83% NH4C1 cold solution for 1 rain to destroy the red blood cells. 2 ml of this cell suspension were slowly layered over 8 ml of fetal calf serum, then centrifuged at 800 r.p.m, for 10 min at 4°C and the pellet was diluted to a concentration of 108 cells in MEM containing 10% (v/v) of fetal calf serum. This lymphocyte cell suspension was co-cultured [9] with RK-13 and M P K cell suspensions in MEM, each suspension containing 5 × 1 0 6 cells/ml. The mixtures, previously resuspended in 100 ml of the appropriate medium (MEM enriched with 10% fetal calf serum, or MEM containing 2 x aminoacids-vitamins solution, for RK-13 or MPK, respectively) were inoculated into 150 cm 2 plastic flasks, and incubated at 37°C in 5% CO2 atmosphere. Subpassages were carried out after a 5 day incubation by culturing trypsinized cell suspensions of infected cells with an equal amount of uninfected fresh harvested RK-13
Lapinized chinese strain of hog cholera virus grown in minipig kidney cell line
223
or M P K cells. The virus antigen in the infected cells was detected at each serial passage by immunofluorescence (IF) according to the methodology described elsewhere [10]. According to the findings made by the IF tests, the virus was detected in RK-13 cells only in the first two serial passages. Therefore, after two additional passages, it was decided to discontinue the study on the RK-13 cell line which apparently it was not susceptible to LC virus replication. However, the M P K cells appeared to be fully susceptible to the virus inasmuch as the viral antigen was regularly detected in the cells from the second passage onwards. So, after a total of 20 subpassages have been made, the evaluation of the susceptibility of M P K cells to the virus was made in rabbits. Thus, experimental infection, serum neutralization and protection tests were carried out in New Zealand white rabbits. For the experimental infection, two tests were made. In the first test, six rabbits were inoculated i.v. in groups of two, with culture fluid fraction (fraction A), cell suspension (fraction B) or disrupted cell suspension (fraction C), respectively [7]. Two additional rabbits were each inoculated i.v. with 5 ml of the original (vaccine) LC strain of H C V and served as controls. The rectal temperature from all inoculated animals was recorded twice daily and when it reached the value of 41 °C, the rabbits were killed and their spleens taken for virus detection in M P K cells by IF tests. The second test consisted in the titration of the infectivity of culture virus under study. The tests were carried out in 27 rabbits according to the methods already described [7] and, with the same procedures other rabbits were given the LC strain of HCV, the latter rabbits to be considered as controls. The results were evaluated on the basis of the body temperatures of the rabbits. A reaction was considered to be positive when temperature reached the value of 40°C or more within 5 days. Titers were calculated according to Reed and Muench [11]. The serum neutralization tests were carried out by mixing each of the two-fold dilutions, from 1:40 to 1 : 10,240, of fraction A from M P K infected cells with 1 : 10 dilution of either HCV-antiserum prepared in rabbits or rabbit normal serum. After 60 min incubation at 37°C, 2 ml of each mixture were inoculated in triplicate i.v. in rabbits. Rectal temperatures of rabbits were taken daily for 5 days after exposure. Final readings were made on the 5th day and the virus was considered to be neutralized by the immune serum in those mixtures which, in contrast with analogous mixtures containing normal serum, failed to evoke a febrile response in the rabbits. Finally, for the protection tests, fraction A from infected M P K cell cultures was serially diluted as above and each dilution was inoculated i.v. in a dose of 2 ml to three rabbits. A similar procedure was used for the inoculation of the LC strain of HCV. Challenge of the immunity of the rabbits was made 30 days after the initial infection by injecting i.v. 2 ml of 1 : 10 of the vaccine concentration of LC strain of HCV. Two additional rabbits which were initially given M E M , were exposed to the challenge virus as above and served as challenge virus controls. Rectal temperatures were taken, as usual, for 5 days after primary infection or challenge exposure. The results of the experimental infection of rabbits with the different fractions of the 20th passage infected cultures, are depicted in Table 1, where it can be seen that all rabbits reacted with fever, as did those inoculated with the vaccine. The virus was detected by IF in the spleens of all inoculated animals. The titer of the virus at its 20th passage level on M P K cells, as determined by experimental infection in rabbits, was 1:7240/2 ml, i.e. about 10 times higher than the vaccinal virus whose titer was of 1:756/2 ml.
224
M. FERRARI Table I. Clinical and virological response of rabbits to intravenous inoculation with different preparations of LC strain of HCV at its 20th passage level in MPK cell cultures Fever 40°C or higher
Inoculum
No. of rabbits
Affected No. of rabbits
Onset/duration (hr)
Virus detection in the spleens of the affected rabbits*
2
2
36/18
Positive
2
2
36/24
Positive
2
2
36/18
Positive
2
2
36/18
Positive
Virus culture
Culture fluid (fraction A) Cell suspension (fraction B) Disrupted cell suspension (fraction C) Control LC strain of HCV (vaccine)
*Virus was detected by direct immunofluorescence tests in MPK cell cultures inoculated with the infected spleens.
The antigenicity of the MPK cultured virus did not appear to undergo any modification insofar as the rabbits inoculated with the mixtures containing the immune serum to HCV remained healthy throughout the observation period (Table 2). The MPK adapted virus and LC strain of HCV appeared to be mutually protective in rabbits. However, the former virus was still immunogenic at the dilution of 1:2560, i.e. a value 8 times higher than the maximum protective dilution (1:320) reported for the LC strain of HCV. (2) Vaccination of pigs with LC strain of H C V grown in M P K cells
Virus [6, 8]. Pigs were vaccinated with the MPK-LC-HCV, at its 25th passage level or with the LC-HCV prepared in rabbits. The titers, as determined in rabbits, were of 1:1280 or 1 : 756, RID50/2 ml, respectively. The vaccinated pigs were subjected to challenge infection with virulent Brescia strain of HCV, the titer of which, as determined by IF in MPK infected cells, was of 105.74 median tissue culture infectious doses (TCID50) per 0.025 ml. Experimental design: inoculation of pigs. Sixteen pigs, about 60 days of age, were used. Twelve pigs, allotted into two groups of 6, were subjected to inoculation with MPK-LCHCV (Group 1) or LC-HCV (Group 2), respectively, each pig receiving intramuscularly (i.m.) 2.0 ml of the appropriate vaccine. Subsequently, at each 180 or 330 days following vaccination three pigs in Group 1, three in Group 2 and two additional unvaccinated pigs (controls) were separated from the others and subjected to challenge exposure with 2.0 ml of the virulent Brescia strain of HCV which was given also i.m. Temperatures of the pigs Table 2. Neutralization tests in rabbits of LC strain of HCV at its 20th passage level on MPK cells in presence of antiserum to HCV* Serum 1:10 Reference antiserum to HCV Rabbit normal serum
Virus titer
