- Email: [email protected]
Attempts to protect rabbits against challenge with virulent, cell-associated, malignant catarrhal fever virus
Veterinary Microbiology, 7 (1982) 419--425 Elsevier Scientific Publishing Company, Amsterdam --Printed in The Netherlands
419
ATTEMPTS TO PROTECT RABBITS AGAINST CHALLENGE WITH VIRULENT, CELL-ASSOCIATED, MALIGNANT CATARRHAL FEVER VIRUS
P.B. ROSSITER
Division of Virology, Veterinary Research Department, Muguga, P.O. Box 32, Kikuyu (Kenya) (Accepted 10 July 1982)
ABSTRACT
Rossiter, P.B., 1982. Attempts to protect rabbits against challenge with virulent, cellassociated, malignant catarrhal fever virus. Vet. Microbiol., 7 : 419--425. Rabbits hyperimmunized with inactivated malignant catarrhal fever virus (MCFV) infected rabbit lymph node cells did not develop specific antibodies to the virus and succumbed to challenge with live MCFV-infected lymphoid cells. Rabbits hyperimmunized with either inactivated or live, cultured bovine kidney cells infected with MCFV developed antibodies to the virus, but also succumbed to challenge with live MCFV-infected rabbit lymphoid cells. Rabbits hyperimmunized with live cultured rabbit kidney cells infected with MCFV developed antibodies to the virus and resisted challenge with live MCFVinfected rabbit lymphoid tissues 47 weeks later. However, rechallenge of this group at 90 weeks post immunization resulted in the death of 2/4 rabbits suggesting a waning immunity.
INTRODUCTION A t t e n u a t e d a n d i n a c t i v a t e d cell-free a n d cell-associated m a l i g n a n t c a t a r r h a l fever virus ( M C F V ) p r e p a r a t i o n s have largely p r o v e d u n s u c c e s s f u l in p r o t e c t i n g c a t t l e f r o m cell-free, cell-associated or n a t u r a l challenge w i t h the virus ( R e i d a n d R o w e , 1 9 7 3 ; P l o w r i g h t , et al., 1975). R e c e n t d e s c r i p t i o n s o f partial (Russell, 1 9 8 0 ) a n d c o m p l e t e ( E d i n g t o n a n d P l o w r i g h t , 1 9 8 0 ) p r o t e c t i o n against i n t r a v e n o u s challenge w i t h v i r u l e n t cell-free M C F V in r a b b i t s i n o c u l a t e d w i t h i n a c t i v a t e d cell-free virus vaccines are a significant a d v a n c e . Nevertheless, t h e p r o t e c t e d r a b b i t s in o n e s t u d y ( E d i n g t o n and Plowright, 1 9 8 0 ) w e r e fully s u s c e p t i b l e t o challenge w i t h live M C F V - i n f e c t e d r a b b i t l y m p h o i d tissues i n d i c a t i n g t h a t t h e i r resistance d i f f e r e d t o t h a t o f r e c o v e r e d cases o f t h e disease in c a t t l e w h i c h are solidly i m m u n e to challenge w i t h infect i o u s tissues ( P l o w r i g h t et al., 1972). T h e r e f o r e , in o r d e r to m o r e fully u n d e r stand m e c h a n i s m s c o n t r i b u t i n g t o w a r d s i m m u n i t y t o M C F V , it s e e m e d logical to t r y t o r e p r o d u c e this solid i m m u n i t y . A t t e m p t s t o p r o d u c e such p r o t e c t i o n using w h o l e i n f e c t e d cells c a r r y i n g virus m e m b r a n e antigens, as successfully u s e d in the c o n t r o l o f M a r e k ' s disease ( K a a d e n et al., 1 9 7 4 ; Powell, 1 9 7 5 ) , are d e s c r i b e d here. 0378-1135182]0000--0000]$02.75 © 1982 Elsevier Scientific Publishing Company
420 MATERIALS AND METHODS Rabbits
New-Zealand white rabbits 3 to 6 m o n t h s old were used in all experiments. Virus strains
The cell~culture adapted WCII strain (Plowright et al., 1965) o f MCFV was used for 'in-vitro' infections, and the virulent C500 strain (Plowright et al., 1975) was used as challenge virus. Rabbits were challenged by intraperitoneal inoculation of 2 ml of 10% suspensions of infected rabbit spleen containing between 103.8 and 104.6 tissue culture infectious doses (TCID so) of MCFV. Serology
Sera were collected at weekly intervals t h r o u g h o u t the period of immunization and at 2 day intervals following challenge, and were assayed for antibodies to MCFV by indirect immunofluorescence (IIF) and VN, and in experiment 3 also by complement fixation and immunodiffusion, as previously described (Rossiter et al., 1980). Sk in tests
Each rabbit in experiment 3 received intradermal inoculations of 0.1 ml of MCFV complement fixation test antigen produced in rabbit kidney cells (RK13 cell line) (Rossiter et al., 1980), uninfected control antigen and phosphate buffered saline pH 7.2 (PBS), into separate shaved areas of their flank. The skin thickness was measured 72 h later. Experiment I
Single cell suspensions prepared from the popliteal lymph nodes of rabbits with clinical MCF were washed three times in PBS and fixed either in 0.125% paraformaldehyde or 0.125% glutaraldehyde as previously described (Powell, 1975). The pre-fixation titres between 104"s--10 s'7 TCIDs0/g of tissue and no infectivity was found in fixed cells. Four rabbits were inoculated 5 times at 3 to 5 week intervals with 4 X 109 paraformaldehyde fixed cells either s.c. or intramuscularly (i.m.) in an emulsion with adjuvant (Freund's complete adjuvant together with an equal volume of 2% Tween 80 was used as adjuvant in all experiments). Three rabbits were similarly inoculated 4 times at 4--8 week intervals with 5 × 108 glutaraldehyde fixed cells. F o u r control rabbits were similarly inoculated with glutaraldehyde fixed uninfected lymph node cells. All of the rabbits were challenged 4 weeks after their final inoculation.
421
Experiment 2 Confluent cultures of bovine kidney (BK) cells were infected with MCFV strain WCII, maintained in serum-free medium and removed from the glass when c y t o p a t h i c effects affected 80--100% of the cells. More than 95% of these cells carried MCFV m e m br a ne antigens as det ect ed by IIF (Rossiter, 1980a). Half of the cells were fixed in 0.125% glutaraldehyde as described above and the remainder sham fixed in PBS. Three groups of four rabbits were inoculated i.m. with either 5 X 106 fixed infected cells, or 5 X 106 sham fixed infected cells or 5 × 106 uninfected cells, in adjuvant. Two rabbits in each group were challenged after 11 weeks. T he remainder received identical booster inoculations after 15 weeks and were challenged 5 weeks later.
Experiment 3 Rabbit kidney cells (RK13 cell-line) infected with MCFV strain WCII were p r o d u c e d as described in e x p e r i m e n t 2. More than 95% had MCFV m em brane antigens. F o u r rabbits received 3 i.m. inoculations of 107 live infected cells in adjuvant at 7--14 week intervals followed by 3 i.v. inoculations of 107 live infected cells at 5 to 10 weeks intervals. T w o cont rol rabbits were similarly immunized with uni nf e c t e d RK13 cells. All rabbits were challenged 4 weeks after their final inoculation and the survivors again 43 weeks later. RESULTS
Experiment 1 All o f the inoculated rabbits died from MCF following challenge, their mean disease time (+ standard deviation) being 19.3 (+ 2.0) days com pared with 18.7 (+ 1.9) days in the controls. None o f the immunized rabbits p r o d u c e d IIF or VN a n t i b o d y prior to challenge, and t h e y appeared n o t t o have been immunologically stimulated since their humoral response during the infection was similar to t h a t o f the controls, indicating a lack of an anamnestic response ( H u m p h r e y and White, 1970).
Experiment 2 All o f the test rabbits developed antibodies to MCFV after their first inoculation, the group mean IIF and VN titres in the group given live infected cells being 12 and 4.5 log~, respectively, c o m p a r e d to 9 and 3 log 2 in the group which received inactivated infected cells. In the group given the live preparation the b o o s ter inoculation did n o t significantly alter the IIF a n t i b o d y titre b u t increased the group mean VN titre to 6 log 2. In the group given the inactivated preparation both the mean IIF and VN titres were significantly boosted to 12 and 5.5 log2, respectively. All o f the immunized rabbits died
422
following MCFV challenge with a m e a n disease time o f 18.6 (-+ 2.2) days c o m p a r e d with 17.8 (-+ 1.7) days in the controls. T h e r e were n o significant differences b e t w e e n the disease times o f groups given either live or inactivated preparations or t h o s e which received o n e as o p p o s e d t o two inoculations.
Experiment 3 T h e i m m u n i z e d rabbits all d e v e l o p e d a n t i b o d i e s to MCFV {Fig. 1), and gave positive, MCFV-specific skin tests 4 weeks a f t e r p r i m a r y i m m u n i z a t i o n and resisted challenge at 47 weeks whereas the t w o c o n t r o l s died o f MCF a f t e r 18 and 23 days. At re-challenge, 43 weeks later, t w o o f the i m m u n i z e d rabbits died a f t e r 33 and 47 days c o m p a r e d with 9 and 10 days in t w o u n i m m u n i z e d controls.
.
j .~
I
/
/I
ll----g
I
t
.
O/
. /j .... Jz
o/
o
\
/"
\\
t
f TIME
f
t
t
C
I" / ~
C
IN WEEKS
Fig. 1. The mean development o f antibodies to malignant catarrhal fever virus (MCFV) in four rabbits successfully protected against cell-associated M C F V challenge. TIF (T0 =
indirect immunofluorescent antibodies to MCFV late antigen; VN = virus neutralising antibodies; CF = complement fixing antibodies, ID = immunodiffusion precipitating antibodies. Arrows indicate the time of immunizing inoculations and C indicates challenge. DISCUSSION As in m a n y virus infections, including t h o s e caused by herpesviruses {Rouse and Babiuk, 1978), successful i m m u n i t y to M C F V is p r o b a b l y c o m p r i s e d o f b o t h p r o t e c t i o n against i n f e c t i o n , in which h u m o r a l i m m u n e m e c h a n i s m s including those acting at mucosal surfaces are particularly i m p o r t a n t , and r e c o v e r y f r o m i n f e c t i o n , in which h u m o r a l and cell-mediated responses concert to eliminate i n f e c t e d cells. C o n s e q u e n t l y , it is p e r h a p s easier t o c o n s i d e r the results o f i m m u n i z a t i o n e x p e r i m e n t s against MCFV in this light; the results o f e x p e r i m e n t s e m p l o y i n g i n t r a v e n o u s cell-free challenge reflecting pro-
423 tection against infection and the results following cell-associated challenge with infected lymphoid cells, a situation caused by the difficulty of producing virulent cell-free MCFV, reflecting recovery from infection. Although the former approach, which would seem more feasible in view of the likelihood that natural infection is caused by cell-free virus (Mushi et al., 1980), has proved successful against intravenously administered cell-free MCFV in rabbits (Edington and Plowright, 1980; Russell, 1980), comparable experiments in cattle did n o t prove successful either in the laboratory or the field (Plowright et al, 1975). Therefore, an investigation of the mechanisms involved in recovery from infection, which have been hindered by the lack of naturally or experimentally induced, recovered animals, might contribute useful information towards an understanding of overall protective i m m u n i t y to MCFV. The main conclusion to be drawn from the results detailed here is that it is possible, using MCFV, to protectively immunize rabbits against MCFVinfected lymphoid cells, a finding which has not been reported previously. Obviously the successful immunization schedule used in experiment 3 is too cumbersome and expensive for regular laboratory use, let alone vaccination in the field, but future studies may refine if sufficiently to allow for its routine use. In order to do that efficiently it is necessary to try to assess which factor(s) in experiment 3 might have been responsible for its success. Since antibodies to MCFV developed in both experiment 2 and 3, it is unlikely that they had any protective role, though it is possible t h a t the hyperimmunization employed in the latter may have induced a response to a particular, serologically undefinable antigen associated with protection, and which is usually only present in very small quantities in infected cells. Alternatively, the presentation of MCFV antigens on allogeneic rabbit, as opposed to xenogeneic bovine cells may have assisted in inducing a strong protective response against infected rabbit cells. The subject of protective immunization induced by allogeneic materials, compared with xenogeneic materials, is little understood, since most attention has recently been focused on comparisons between allogeneic and syngeneic materials (Lawman et al., 1981). It would be interesting to compare immunizations made using autologous, allogeneic and xenogeneic cells, such as lymphocytes, which had been infected with MCFV 'in-vitro'. Another major difference between experiments 2 and 3 was the greater quantity of antigen and number of inoculations employed in the latter, suggesting that a dose response relationship may exist between the induction of protective i m m u n i t y to MCFV and the antigen dose used. This might have been expected to be the case if the successful immunization had been made with inactivated materials instead of live virus which, where successful, is often considered to confer long lasting i m m u n i t y together with rapidly developing high titres of antibody which tend to decrease slowly with time (Plotkin, 1975). The waning protection in experiment 3 suggests that the inocula in these experiments may have been acting as a dead antigen, which is n o t unlikely if the detergent Tween 80 present in the double emulsion adjuvant had
424 had c o n t a c t with the herpesvirus of MCF. In addition to this, there is no evidence th at any strains of wildebeest MCFV can multiply in rabbits or cattle w i t h o u t causing disease, and it is possible t hat the degree of at t enuat i on attributed to the cell-culture passaged, cell-free WCII strain of MCFV (Plowright, 1968; Plowright et al., 1975) m a y only reflect a failure to infect animals since no virus recovery has been reported from animals inoculated with this strain. In fact, strain WCII can be fully pathogenic as an earlier immunization a t t e m p t in this l abor a t or y ended when four rabbits which had each received an intravenous inoculation of 1 X 107 RK13 cells infected with MCFV strain WCII died from typical MCF within a normal disease period. Thus, the fact t h a t similar inoculations were used as boosters during the latter stages o f e x p e r i m e n t 3 is further evidence o f the protective i m m u n i t y which developed in t h a t experiment. Perhaps these boosters, whilst insufficiently virulent to cause disease in rabbits, which had already developed some forms of i m m u n i t y to MCFV antigens, m a y themselves have c o n t r i b u t e d to an imm u n i t y against infected cells as opposed to virus antigens alone (Witter et al, 1976; Sharma and Burmester, 1979). The role o f i m m u n i t y to MCFV virus antigens in p r o t e c t i o n against the disease is f u r th er complicated by the fact that the pathogenesis of the disease is p o o r ly u n d e r s t o o d . It is unlikely that the major disease process is caused by c y t o p a t h i c effects o f the virus in infected l y m p h o i d tissues (Patel and Edington, 1980; Rossiter, 1980b) a fact which is further supported by the results of ex p er ime nt 1 and a previous immunization trial (Plowright et al., 1975). Th er ef o r e, in the absence of strains of MCFV which infect and p r o t e c t cattle against virulent virus, it is h o p e d that further controlled experiments monitoring several parameters of i m m u n i t y will ext end this preliminary description o f the induction of protective i m m u n i t y to cell-associated MCFV using whole MCFV-infected cells. ACKNOWLEDGEMENTS The author is a staff m e m b e r of the Overseas D e v e l o p m e n t Administration, L o n d o n and part of the w or k was p e r f o r m e d at the Royal Veterinary College, L o n d o n University. This paper is published with the permission of the Director, Veterinary Research D e pa r t m e nt , Muguga.
REFERENCES Edington, N. and Plowright, W., 1980. The protection of rabbits against the herpesvirus of malignant catarrhal fever by inactivated vaccines. Res. Vet. Sci., 28: 384--386. Humphrey, J.H. and White, R.G., 1970. Immunology for Students of Medicine. Blackwell Scientific Publications, Oxford, 757 pp. Kaaden, O.R., Dietzschold, B. and Ueberschar, S., 1974. Vaccination against Marek's disease : Immunizing effect of purified turkey herpesvirus and cellular membranes from infected cells. Microbiol. Immunol., 159: 261--269.
425 Lawman, M.J.P., Naylor, P.T., Huang, L., Courtney, R.J. and Rouse, B.T., 1981. Cell mediated i m m u n i t y to herpes simplex virus: Induction of cytotoxic T Lymphocyte responses by viral antigens incorporated into liposomes. J. Immunol., 126: 304--308. Mushi, E.Z., Rossiter, P.B., Karstad, L. and Jessett, D.M., 1980. The demonstration of cell-free malignant catarrhal fever herpesvirus in wildebeest nasal secretions. J. Hyg. Camb., 85: 175--179. Patel, J. and Edington, N., 1980. The detection of the herpesvirus of bovine malignant catarrhal fever in rabbit lymphocytes 'in-vivo' and 'in-vitro'. J. Gen. Virol., 48: 437-444. Plotkin, S.A., 1975. Immunoprophylaxis and immunotherapy of virus infections. In: C. Kaprowski and H. Kaprowski (Editors), Viruses and Immunity. Academic Press, New-York, NY, pp. 117--127. Plowright, W., 1968. Malignant catarrhal fever. J. Am. Vet. Med. Ass., 152: 795--804. Plowright, W., Macadam, R.F. and Armstrong, J.S., 1965. Growth and characterization of the virus of bovine malignant catarrhal fever in East Africa. J. Gen. Microbiol., 39: 256--266. Plowright, W., Kalunda, M., Jessett, D.M. and Herniman, K.A.J., 1972. Congenital infection of cattle with the herpesvirus causing malignant catarrhal fever. Res. Vet. Sci., 13: 37--45. Plowright, W., Herniman, K.A.J., Jessett, D.M., Kalunda, M. and Rampton, C.S., 1975. Immunization of cattle against the herpesvirus of malignant catarrhal fever : failure of inactivated culture vaccines with adjuvant. Res. Vet. Sci., 19: 159--166. Powell, P.C., 1975. I m m u n i t y to Marek's disease induced by glutaraldehyde fixed cells of Marek's disease lymphoblastoid cell lines. Nature (London), 251: 79--80. Reid, H.E. and Rowe, L., 1973. The attenuation of a herpesvirus (Malignant catarrhal fever virus) isolated from hartebeest (Alcelaphus buselaphus cokei, Gunther). Res. Vet. Sci., 15: 144--146. Rossiter, P.B., 1980a. Serological and immunological investigations in malignant catarrhal fever. Ph.D. Thesis, University of London. Rossiter, P.B., 1980b. A lack of readily demonstrable virus antigens in the tissues of rabbits and cattle infected with malignant catarrhal fever virus. Br. Vet. J., 136: 478--483. Rossiter, P.B., Jessett, D.M., Mushi, E.Z. and Karstad, L., 1980. Antibodies to malignant catarrhal fever virus antigens in the sera of normal and naturally infected cattle in Kenya. Res. Vet. Sci., 29: 235--239. Rouse, B.T. and Babiuk, L.A., 1978. Mechanisms of recovery from herpesvirus infections -A review. Can. J. Comp. Med., 42: 414--427. Russell, P.H., 1980. Malignant catarrhal fever virus in rabbits -- reproduction of clinical disease by cell-free virus and partial protection against such disease by vaccination with inactivated virus. Vet. Microbiol., 5: 161--163. Sharma, J.M. and Burmester, B.R., 1979. I m m u n e mechanisms in Marek's disease. Comp. Immunol. Microbiol. Infect. Dis., 1 : 153--158. Witter, R.L., Sharma, J.M. and Offenbecker, L., 1976. Turkey herpesvirus infection in chickens. Induction of lymphoproliferative lesions and characterisation of vaccinal immunity against Marek's disease. Avian Dis., 20: 676---692.
419
ATTEMPTS TO PROTECT RABBITS AGAINST CHALLENGE WITH VIRULENT, CELL-ASSOCIATED, MALIGNANT CATARRHAL FEVER VIRUS
P.B. ROSSITER
Division of Virology, Veterinary Research Department, Muguga, P.O. Box 32, Kikuyu (Kenya) (Accepted 10 July 1982)
ABSTRACT
Rossiter, P.B., 1982. Attempts to protect rabbits against challenge with virulent, cellassociated, malignant catarrhal fever virus. Vet. Microbiol., 7 : 419--425. Rabbits hyperimmunized with inactivated malignant catarrhal fever virus (MCFV) infected rabbit lymph node cells did not develop specific antibodies to the virus and succumbed to challenge with live MCFV-infected lymphoid cells. Rabbits hyperimmunized with either inactivated or live, cultured bovine kidney cells infected with MCFV developed antibodies to the virus, but also succumbed to challenge with live MCFV-infected rabbit lymphoid cells. Rabbits hyperimmunized with live cultured rabbit kidney cells infected with MCFV developed antibodies to the virus and resisted challenge with live MCFVinfected rabbit lymphoid tissues 47 weeks later. However, rechallenge of this group at 90 weeks post immunization resulted in the death of 2/4 rabbits suggesting a waning immunity.
INTRODUCTION A t t e n u a t e d a n d i n a c t i v a t e d cell-free a n d cell-associated m a l i g n a n t c a t a r r h a l fever virus ( M C F V ) p r e p a r a t i o n s have largely p r o v e d u n s u c c e s s f u l in p r o t e c t i n g c a t t l e f r o m cell-free, cell-associated or n a t u r a l challenge w i t h the virus ( R e i d a n d R o w e , 1 9 7 3 ; P l o w r i g h t , et al., 1975). R e c e n t d e s c r i p t i o n s o f partial (Russell, 1 9 8 0 ) a n d c o m p l e t e ( E d i n g t o n a n d P l o w r i g h t , 1 9 8 0 ) p r o t e c t i o n against i n t r a v e n o u s challenge w i t h v i r u l e n t cell-free M C F V in r a b b i t s i n o c u l a t e d w i t h i n a c t i v a t e d cell-free virus vaccines are a significant a d v a n c e . Nevertheless, t h e p r o t e c t e d r a b b i t s in o n e s t u d y ( E d i n g t o n and Plowright, 1 9 8 0 ) w e r e fully s u s c e p t i b l e t o challenge w i t h live M C F V - i n f e c t e d r a b b i t l y m p h o i d tissues i n d i c a t i n g t h a t t h e i r resistance d i f f e r e d t o t h a t o f r e c o v e r e d cases o f t h e disease in c a t t l e w h i c h are solidly i m m u n e to challenge w i t h infect i o u s tissues ( P l o w r i g h t et al., 1972). T h e r e f o r e , in o r d e r to m o r e fully u n d e r stand m e c h a n i s m s c o n t r i b u t i n g t o w a r d s i m m u n i t y t o M C F V , it s e e m e d logical to t r y t o r e p r o d u c e this solid i m m u n i t y . A t t e m p t s t o p r o d u c e such p r o t e c t i o n using w h o l e i n f e c t e d cells c a r r y i n g virus m e m b r a n e antigens, as successfully u s e d in the c o n t r o l o f M a r e k ' s disease ( K a a d e n et al., 1 9 7 4 ; Powell, 1 9 7 5 ) , are d e s c r i b e d here. 0378-1135182]0000--0000]$02.75 © 1982 Elsevier Scientific Publishing Company
420 MATERIALS AND METHODS Rabbits
New-Zealand white rabbits 3 to 6 m o n t h s old were used in all experiments. Virus strains
The cell~culture adapted WCII strain (Plowright et al., 1965) o f MCFV was used for 'in-vitro' infections, and the virulent C500 strain (Plowright et al., 1975) was used as challenge virus. Rabbits were challenged by intraperitoneal inoculation of 2 ml of 10% suspensions of infected rabbit spleen containing between 103.8 and 104.6 tissue culture infectious doses (TCID so) of MCFV. Serology
Sera were collected at weekly intervals t h r o u g h o u t the period of immunization and at 2 day intervals following challenge, and were assayed for antibodies to MCFV by indirect immunofluorescence (IIF) and VN, and in experiment 3 also by complement fixation and immunodiffusion, as previously described (Rossiter et al., 1980). Sk in tests
Each rabbit in experiment 3 received intradermal inoculations of 0.1 ml of MCFV complement fixation test antigen produced in rabbit kidney cells (RK13 cell line) (Rossiter et al., 1980), uninfected control antigen and phosphate buffered saline pH 7.2 (PBS), into separate shaved areas of their flank. The skin thickness was measured 72 h later. Experiment I
Single cell suspensions prepared from the popliteal lymph nodes of rabbits with clinical MCF were washed three times in PBS and fixed either in 0.125% paraformaldehyde or 0.125% glutaraldehyde as previously described (Powell, 1975). The pre-fixation titres between 104"s--10 s'7 TCIDs0/g of tissue and no infectivity was found in fixed cells. Four rabbits were inoculated 5 times at 3 to 5 week intervals with 4 X 109 paraformaldehyde fixed cells either s.c. or intramuscularly (i.m.) in an emulsion with adjuvant (Freund's complete adjuvant together with an equal volume of 2% Tween 80 was used as adjuvant in all experiments). Three rabbits were similarly inoculated 4 times at 4--8 week intervals with 5 × 108 glutaraldehyde fixed cells. F o u r control rabbits were similarly inoculated with glutaraldehyde fixed uninfected lymph node cells. All of the rabbits were challenged 4 weeks after their final inoculation.
421
Experiment 2 Confluent cultures of bovine kidney (BK) cells were infected with MCFV strain WCII, maintained in serum-free medium and removed from the glass when c y t o p a t h i c effects affected 80--100% of the cells. More than 95% of these cells carried MCFV m e m br a ne antigens as det ect ed by IIF (Rossiter, 1980a). Half of the cells were fixed in 0.125% glutaraldehyde as described above and the remainder sham fixed in PBS. Three groups of four rabbits were inoculated i.m. with either 5 X 106 fixed infected cells, or 5 X 106 sham fixed infected cells or 5 × 106 uninfected cells, in adjuvant. Two rabbits in each group were challenged after 11 weeks. T he remainder received identical booster inoculations after 15 weeks and were challenged 5 weeks later.
Experiment 3 Rabbit kidney cells (RK13 cell-line) infected with MCFV strain WCII were p r o d u c e d as described in e x p e r i m e n t 2. More than 95% had MCFV m em brane antigens. F o u r rabbits received 3 i.m. inoculations of 107 live infected cells in adjuvant at 7--14 week intervals followed by 3 i.v. inoculations of 107 live infected cells at 5 to 10 weeks intervals. T w o cont rol rabbits were similarly immunized with uni nf e c t e d RK13 cells. All rabbits were challenged 4 weeks after their final inoculation and the survivors again 43 weeks later. RESULTS
Experiment 1 All o f the inoculated rabbits died from MCF following challenge, their mean disease time (+ standard deviation) being 19.3 (+ 2.0) days com pared with 18.7 (+ 1.9) days in the controls. None o f the immunized rabbits p r o d u c e d IIF or VN a n t i b o d y prior to challenge, and t h e y appeared n o t t o have been immunologically stimulated since their humoral response during the infection was similar to t h a t o f the controls, indicating a lack of an anamnestic response ( H u m p h r e y and White, 1970).
Experiment 2 All o f the test rabbits developed antibodies to MCFV after their first inoculation, the group mean IIF and VN titres in the group given live infected cells being 12 and 4.5 log~, respectively, c o m p a r e d to 9 and 3 log 2 in the group which received inactivated infected cells. In the group given the live preparation the b o o s ter inoculation did n o t significantly alter the IIF a n t i b o d y titre b u t increased the group mean VN titre to 6 log 2. In the group given the inactivated preparation both the mean IIF and VN titres were significantly boosted to 12 and 5.5 log2, respectively. All o f the immunized rabbits died
422
following MCFV challenge with a m e a n disease time o f 18.6 (-+ 2.2) days c o m p a r e d with 17.8 (-+ 1.7) days in the controls. T h e r e were n o significant differences b e t w e e n the disease times o f groups given either live or inactivated preparations or t h o s e which received o n e as o p p o s e d t o two inoculations.
Experiment 3 T h e i m m u n i z e d rabbits all d e v e l o p e d a n t i b o d i e s to MCFV {Fig. 1), and gave positive, MCFV-specific skin tests 4 weeks a f t e r p r i m a r y i m m u n i z a t i o n and resisted challenge at 47 weeks whereas the t w o c o n t r o l s died o f MCF a f t e r 18 and 23 days. At re-challenge, 43 weeks later, t w o o f the i m m u n i z e d rabbits died a f t e r 33 and 47 days c o m p a r e d with 9 and 10 days in t w o u n i m m u n i z e d controls.
.
j .~
I
/
/I
ll----g
I
t
.
O/
. /j .... Jz
o/
o
\
/"
\\
t
f TIME
f
t
t
C
I" / ~
C
IN WEEKS
Fig. 1. The mean development o f antibodies to malignant catarrhal fever virus (MCFV) in four rabbits successfully protected against cell-associated M C F V challenge. TIF (T0 =
indirect immunofluorescent antibodies to MCFV late antigen; VN = virus neutralising antibodies; CF = complement fixing antibodies, ID = immunodiffusion precipitating antibodies. Arrows indicate the time of immunizing inoculations and C indicates challenge. DISCUSSION As in m a n y virus infections, including t h o s e caused by herpesviruses {Rouse and Babiuk, 1978), successful i m m u n i t y to M C F V is p r o b a b l y c o m p r i s e d o f b o t h p r o t e c t i o n against i n f e c t i o n , in which h u m o r a l i m m u n e m e c h a n i s m s including those acting at mucosal surfaces are particularly i m p o r t a n t , and r e c o v e r y f r o m i n f e c t i o n , in which h u m o r a l and cell-mediated responses concert to eliminate i n f e c t e d cells. C o n s e q u e n t l y , it is p e r h a p s easier t o c o n s i d e r the results o f i m m u n i z a t i o n e x p e r i m e n t s against MCFV in this light; the results o f e x p e r i m e n t s e m p l o y i n g i n t r a v e n o u s cell-free challenge reflecting pro-
423 tection against infection and the results following cell-associated challenge with infected lymphoid cells, a situation caused by the difficulty of producing virulent cell-free MCFV, reflecting recovery from infection. Although the former approach, which would seem more feasible in view of the likelihood that natural infection is caused by cell-free virus (Mushi et al., 1980), has proved successful against intravenously administered cell-free MCFV in rabbits (Edington and Plowright, 1980; Russell, 1980), comparable experiments in cattle did n o t prove successful either in the laboratory or the field (Plowright et al, 1975). Therefore, an investigation of the mechanisms involved in recovery from infection, which have been hindered by the lack of naturally or experimentally induced, recovered animals, might contribute useful information towards an understanding of overall protective i m m u n i t y to MCFV. The main conclusion to be drawn from the results detailed here is that it is possible, using MCFV, to protectively immunize rabbits against MCFVinfected lymphoid cells, a finding which has not been reported previously. Obviously the successful immunization schedule used in experiment 3 is too cumbersome and expensive for regular laboratory use, let alone vaccination in the field, but future studies may refine if sufficiently to allow for its routine use. In order to do that efficiently it is necessary to try to assess which factor(s) in experiment 3 might have been responsible for its success. Since antibodies to MCFV developed in both experiment 2 and 3, it is unlikely that they had any protective role, though it is possible t h a t the hyperimmunization employed in the latter may have induced a response to a particular, serologically undefinable antigen associated with protection, and which is usually only present in very small quantities in infected cells. Alternatively, the presentation of MCFV antigens on allogeneic rabbit, as opposed to xenogeneic bovine cells may have assisted in inducing a strong protective response against infected rabbit cells. The subject of protective immunization induced by allogeneic materials, compared with xenogeneic materials, is little understood, since most attention has recently been focused on comparisons between allogeneic and syngeneic materials (Lawman et al., 1981). It would be interesting to compare immunizations made using autologous, allogeneic and xenogeneic cells, such as lymphocytes, which had been infected with MCFV 'in-vitro'. Another major difference between experiments 2 and 3 was the greater quantity of antigen and number of inoculations employed in the latter, suggesting that a dose response relationship may exist between the induction of protective i m m u n i t y to MCFV and the antigen dose used. This might have been expected to be the case if the successful immunization had been made with inactivated materials instead of live virus which, where successful, is often considered to confer long lasting i m m u n i t y together with rapidly developing high titres of antibody which tend to decrease slowly with time (Plotkin, 1975). The waning protection in experiment 3 suggests that the inocula in these experiments may have been acting as a dead antigen, which is n o t unlikely if the detergent Tween 80 present in the double emulsion adjuvant had
424 had c o n t a c t with the herpesvirus of MCF. In addition to this, there is no evidence th at any strains of wildebeest MCFV can multiply in rabbits or cattle w i t h o u t causing disease, and it is possible t hat the degree of at t enuat i on attributed to the cell-culture passaged, cell-free WCII strain of MCFV (Plowright, 1968; Plowright et al., 1975) m a y only reflect a failure to infect animals since no virus recovery has been reported from animals inoculated with this strain. In fact, strain WCII can be fully pathogenic as an earlier immunization a t t e m p t in this l abor a t or y ended when four rabbits which had each received an intravenous inoculation of 1 X 107 RK13 cells infected with MCFV strain WCII died from typical MCF within a normal disease period. Thus, the fact t h a t similar inoculations were used as boosters during the latter stages o f e x p e r i m e n t 3 is further evidence o f the protective i m m u n i t y which developed in t h a t experiment. Perhaps these boosters, whilst insufficiently virulent to cause disease in rabbits, which had already developed some forms of i m m u n i t y to MCFV antigens, m a y themselves have c o n t r i b u t e d to an imm u n i t y against infected cells as opposed to virus antigens alone (Witter et al, 1976; Sharma and Burmester, 1979). The role o f i m m u n i t y to MCFV virus antigens in p r o t e c t i o n against the disease is f u r th er complicated by the fact that the pathogenesis of the disease is p o o r ly u n d e r s t o o d . It is unlikely that the major disease process is caused by c y t o p a t h i c effects o f the virus in infected l y m p h o i d tissues (Patel and Edington, 1980; Rossiter, 1980b) a fact which is further supported by the results of ex p er ime nt 1 and a previous immunization trial (Plowright et al., 1975). Th er ef o r e, in the absence of strains of MCFV which infect and p r o t e c t cattle against virulent virus, it is h o p e d that further controlled experiments monitoring several parameters of i m m u n i t y will ext end this preliminary description o f the induction of protective i m m u n i t y to cell-associated MCFV using whole MCFV-infected cells. ACKNOWLEDGEMENTS The author is a staff m e m b e r of the Overseas D e v e l o p m e n t Administration, L o n d o n and part of the w or k was p e r f o r m e d at the Royal Veterinary College, L o n d o n University. This paper is published with the permission of the Director, Veterinary Research D e pa r t m e nt , Muguga.
REFERENCES Edington, N. and Plowright, W., 1980. The protection of rabbits against the herpesvirus of malignant catarrhal fever by inactivated vaccines. Res. Vet. Sci., 28: 384--386. Humphrey, J.H. and White, R.G., 1970. Immunology for Students of Medicine. Blackwell Scientific Publications, Oxford, 757 pp. Kaaden, O.R., Dietzschold, B. and Ueberschar, S., 1974. Vaccination against Marek's disease : Immunizing effect of purified turkey herpesvirus and cellular membranes from infected cells. Microbiol. Immunol., 159: 261--269.
425 Lawman, M.J.P., Naylor, P.T., Huang, L., Courtney, R.J. and Rouse, B.T., 1981. Cell mediated i m m u n i t y to herpes simplex virus: Induction of cytotoxic T Lymphocyte responses by viral antigens incorporated into liposomes. J. Immunol., 126: 304--308. Mushi, E.Z., Rossiter, P.B., Karstad, L. and Jessett, D.M., 1980. The demonstration of cell-free malignant catarrhal fever herpesvirus in wildebeest nasal secretions. J. Hyg. Camb., 85: 175--179. Patel, J. and Edington, N., 1980. The detection of the herpesvirus of bovine malignant catarrhal fever in rabbit lymphocytes 'in-vivo' and 'in-vitro'. J. Gen. Virol., 48: 437-444. Plotkin, S.A., 1975. Immunoprophylaxis and immunotherapy of virus infections. In: C. Kaprowski and H. Kaprowski (Editors), Viruses and Immunity. Academic Press, New-York, NY, pp. 117--127. Plowright, W., 1968. Malignant catarrhal fever. J. Am. Vet. Med. Ass., 152: 795--804. Plowright, W., Macadam, R.F. and Armstrong, J.S., 1965. Growth and characterization of the virus of bovine malignant catarrhal fever in East Africa. J. Gen. Microbiol., 39: 256--266. Plowright, W., Kalunda, M., Jessett, D.M. and Herniman, K.A.J., 1972. Congenital infection of cattle with the herpesvirus causing malignant catarrhal fever. Res. Vet. Sci., 13: 37--45. Plowright, W., Herniman, K.A.J., Jessett, D.M., Kalunda, M. and Rampton, C.S., 1975. Immunization of cattle against the herpesvirus of malignant catarrhal fever : failure of inactivated culture vaccines with adjuvant. Res. Vet. Sci., 19: 159--166. Powell, P.C., 1975. I m m u n i t y to Marek's disease induced by glutaraldehyde fixed cells of Marek's disease lymphoblastoid cell lines. Nature (London), 251: 79--80. Reid, H.E. and Rowe, L., 1973. The attenuation of a herpesvirus (Malignant catarrhal fever virus) isolated from hartebeest (Alcelaphus buselaphus cokei, Gunther). Res. Vet. Sci., 15: 144--146. Rossiter, P.B., 1980a. Serological and immunological investigations in malignant catarrhal fever. Ph.D. Thesis, University of London. Rossiter, P.B., 1980b. A lack of readily demonstrable virus antigens in the tissues of rabbits and cattle infected with malignant catarrhal fever virus. Br. Vet. J., 136: 478--483. Rossiter, P.B., Jessett, D.M., Mushi, E.Z. and Karstad, L., 1980. Antibodies to malignant catarrhal fever virus antigens in the sera of normal and naturally infected cattle in Kenya. Res. Vet. Sci., 29: 235--239. Rouse, B.T. and Babiuk, L.A., 1978. Mechanisms of recovery from herpesvirus infections -A review. Can. J. Comp. Med., 42: 414--427. Russell, P.H., 1980. Malignant catarrhal fever virus in rabbits -- reproduction of clinical disease by cell-free virus and partial protection against such disease by vaccination with inactivated virus. Vet. Microbiol., 5: 161--163. Sharma, J.M. and Burmester, B.R., 1979. I m m u n e mechanisms in Marek's disease. Comp. Immunol. Microbiol. Infect. Dis., 1 : 153--158. Witter, R.L., Sharma, J.M. and Offenbecker, L., 1976. Turkey herpesvirus infection in chickens. Induction of lymphoproliferative lesions and characterisation of vaccinal immunity against Marek's disease. Avian Dis., 20: 676---692.