Institute for Animal Health, Pirbright Laboratory, Woking, Surrey GU24 0NF, UK1
Nippon Institute for Biological Science, 9-2221-1 Shin-machi, Ome, Tokyo 198-00024, Japan2
Author for correspondence: Thomas Barrett. Fax +44 1483 232448. e-mail tom.barrett{at}bbsrc.ac.uk
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Abstract |
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Introduction |
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In recent years, vaccinia virus has been used widely as a vector to deliver immunogenic antigens from other pathogens and, as in the case of rabies virus, these recombinants have been shown to act as novel vaccines to protect against these diseases (Yamanouchi et al., 1998 ). Recombinant rinderpest vaccines based on poxvirus vectors have been shown to protect cattle against RPV infection (Yilma et al., 1988
; Belsham et al., 1989
; Giavedoni et al., 1991
; Romero et al., 1994
). We have developed a recombinant rinderpest vaccine (rRV) by using a highly attenuated strain of vaccinia virus (LC16mO) as a vector to express the virus haemagglutinin (H) protein, which also protects against RPV infection (Asano et al., 1991
; Yamanouchi et al., 1993
). The H protein is responsible for the attachment of the virus to the host cell receptor and neutralizing antibodies generated against this protein are thought to play an important role in protection (Giraudon & Wild, 1985
). A single subcutaneous inoculation of the rRV has been shown to give solid protective immunity in cattle for at least a year (Inui et al., 1995
). The safety of the vaccine has been demonstrated in cattle and laboratory animals and, in addition, its heat stability and genetic stability on passage in cattle have been confirmed in previous studies (Yamanouchi et al., 1993
; Yamanouchi & Barrett, 1994
). In this study, we report further on the duration of immunity to RPV afforded by this vaccine and present the results of 2 and 3 year trials to test its long-term efficacy in cattle. We have also examined the immune mechanisms involved in protection.
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Methods |
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Vaccination and challenge of animals.
Friesian cross Aberdeen Angus calves were inoculated subcutaneously with a single dose (108 p.f.u.) of rRV in a secure animal facility at the Compton Laboratory of the Institute for Animal Health (IAH), UK. After 6 weeks, they were released to normal pasture and kept for 2 (group I) or 3 years (group II). As controls to monitor for contact transmission of vaccinia virus, unvaccinated cattle were kept together in each group. At the time of challenge, the cattle were moved to the high-containment facility at the IAH Pirbright Laboratory and inoculated subcutaneously with 104 TCID50 of the virulent Saudi 1/81 strain of RPV (Taylor, 1986 ). This protocol was agreed with the local genetic manipulation safety committees at each laboratory and approved by the veterinary authorities at the Ministry of Agriculture, Fisheries and Food. All concerned were satisfied that the experiments posed no threat of contamination to the environment or to the health of those involved directly or indirectly in the experiments.
Clinical signs and leukocyte abnormalities.
After challenge, cattle were examined daily for clinical signs and rectal temperatures were recorded. Blood samples were taken and examined for leukopenia on days 4, 7, 9 and 12 after challenge in group I and on days 5, 8, 11, 14 and 21 in group II. As a measure of immunosuppression, proliferation of purified lymphocytes was assayed in response to the mitogen concanavalin A (ConA), in group II animals only, on days 5, 8, 14 and 21 after challenge according to a method described previously (Ohishi et al., 1999 ). Briefly, peripheral blood leukocytes (PBLs; 2x105 cells per well in 96-well microtitre plates) were cultured for 6 days in the presence of ConA (5 µg/ml). The cells were pulse-labelled with [3H]thymidine for the last 16 h and incorporation of label into cellular DNA was measured in a liquid scintillation counter.
Virus detection following challenge.
Virus isolation from PBLs was attempted by co-cultivation with B95a cells, which are highly sensitive hosts for the replication of RPV (Kobune et al., 1991 ). For this purpose, 106 PBLs purified from each blood sample were placed in one well of a 96-well microtitre plate along with 5x106 B95a cells, using five wells for each assay. Virus present in the eye secretions (collected by swabbing) was detected by RTPCR analysis of purified RNA according to a procedure described previously (Forsyth & Barrett, 1995
).
Detection of RPV-neutralizing antibodies.
Antibody titres were assayed by microneutralization tests with Vero cells as described previously (Sato et al., 1981 ). The assays were carried out in duplicate for group I sera and in quadruplicate for the group II sera.
RPV-specific lymphocyte proliferative responses.
Lymphocyte proliferation in response to RPV stimulation was assessed by [3H]thymidine incorporation into cellular DNA according to a method described previously (Ohishi et al., 1999 ). In brief, PBLs were cultured (2x105 per well) in 96-well microtitre plates for 6 days in the presence of UV-irradiated RPV (pre-UV titre of 103·9 TCID50 per well). All assays were carried out in triplicate. RPV-specific responses were expressed as the stimulation index (SI), which was calculated as the ratio of the mean c.p.m. of lymphocytes cultured in the presence of RPV to the mean c.p.m. in the absence of RPV. Values greater than 2·5 were considered significant.
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Results |
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Virus-neutralizing antibody in the vaccinated cattle
A single vaccination with rRV induced a low but significant level of anti-rinderpest neutralizing antibody in all animals. The titres reached maximal levels between 6 and 12 months after vaccination, after which they decreased slightly but then remained at the same levels until the experiment was completed at 3 years. A rapid rise in the level of anti-rinderpest neutralizing antibodies was observed in all the vaccinated cattle 12 weeks following challenge. No neutralizing antibody was produced in any of the control animals (Table 2).
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Discussion |
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The Saudi 1/81 strain of RPV used as a challenge virus is the most virulent strain among a number of well-characterized RPV isolates and can kill cattle within 3 days (Taylor, 1986 ). We have shown that a single subcutaneous vaccination with rRV provided either complete or partial protection against such a virulent virus for at least 3 years. Vaccinia virus replicates locally in the squamous epidermal cells without causing viraemia, and the optimal route for inoculation is intradermal rather than subcutaneous. However, safety considerations dictate that only the subcutaneous route of inoculation is acceptable when using vaccinia-vectored rinderpest vaccines in the field (Office International des Epizooties, 1989
). These results indicate that the rRV would be effective in protecting cattle from RPV infection under these conditions.
In contrast to the local replication of the rRV at the site of inoculation, RPV causes systemic infection with marked growth in the lymphoid tissues as its main targets. It is rather surprising that such limited local growth of the rRV provides long-lasting immunity against systemic infection with RPV, and it raises interesting questions concerning the immune mechanisms involved. Neutralizing antibodies were maintained at detectable levels for up to 3 years following a single vaccination in these cattle, and they increased rapidly following challenge. Specific lymphoproliferative responses to RPV were not detected before challenge in our assay system, but a marked increase in SI was observed after challenge in four of six animals tested. This indicates that specific immunological memory for both cell-mediated and humoral immunity persisted over this time-period and that they were activated rapidly by the antigenic stimulus provided by the limited growth of the challenge virus.
The relative importance of cell-mediated versus humoral immunity in the protective response remains to be defined clearly in the case of morbillivirus infections. In the present study, the cattle with solid immunity showed a higher neutralizing antibody titre compared with those with only partial protection. Overall, however, the levels of neutralizing antibody induced by rRV were low. This LC16mO-based vaccine system has been shown to be capable of inducing a protective cell-mediated immunity, including the ability to generate CTL responses in some cattle (Ohishi et al., 1991 , 1999
). A different recombinant vaccinia vaccine, expressing the H and F proteins of RPV, protected goats from peste des petits ruminants virus (PPRV) infection in the absence of PPRV-neutralizing antibodies in the vaccinated animals (Jones et al., 1993
). Furthermore, it was reported that the H antigen alone when expressed in a baculovirus recombinant system could induce antibodies to RPV; however, these did not protect cattle against RPV infection (Bassiri et al., 1993
). These observations indicate that there is probably a greater role for cell-mediated over humoral immunity in protection from RPV and that such an immunity is induced effectively by vaccinia-based vaccines.
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Acknowledgments |
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References |
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Received 19 November 1999;
accepted 3 March 2000.