A number of vaccines are available to protect rabbits against myxomatosis and RHD. Immunization against myxomatosis relies on the use of cell culture-attenuated strains of MV (1
), whereas vaccination against RHD is achieved with inactivated RHDV (2
). These vaccines have proven effective in the control of both diseases among domestic rabbits, but they are not suited to be used for wild rabbit vaccination.
Immunization of wildlife is difficult to achieve because such animals are free ranging, thus precluding the use of vaccines that require individual administration by conventional veterinary practices. In these cases, the oral route is considered a feasible way of vaccine administration. For example, oral vaccination is being used to control enzootic sylvatic rabies in Europe and North America by means of a recombinant VV-rabies vaccine delivered by baiting (10
). Another possibility could be use of viral vectors capable of spreading within an animal population. This is a potentially useful approach for delivering antigens to wild animals, especially when the distribution, size, and turnover rate of a population preclude capture or baiting techniques as the only means for antigen delivery. The European rabbit (O. cuniculus
) is an example of such a population. With this in mind, we have explored the possibility of wild rabbit vaccination against both myxomatosis and RHD by using an MV-VP60 recombinant capable of spreading through rabbit populations by horizontal transmission. Hopefully, administration of the recombinant vaccine to a small number of rabbits will eventually lead to the immunization of a fraction of animals within the population which is sufficient to reduce the spread of both diseases.
Several facts led us to anticipate that the proposed transmissible vaccine could be useful for the control of myxomatosis and RHD among rabbit populations. First, recombinant poxvirus systems have been successfully developed as vectors for delivering a wide range of vaccine antigens to humans and animals. Viruses used include VV (38
), avipoxviruses (20
), raccoon poxvirus (21
), capripoxvirus (53
), swinepox virus (58
), and myxoma virus (4
). Second, the expression of RHDV VP60 capsid protein in several heterologous systems has been shown to induce protective immunity (4
). Remarkably, no indications of toxicity or side effects associated to the expression of VP60 have been reported. In addition, molecular epidemiology studies have revealed a low genomic variation (less than 10% nucleotide divergence) within isolates collected from different geographic areas and over a period of several years (29
). This result suggests that to date, a single RHDV serotype exists. Since this is also the case of MV, vaccination with a recombinant MV-VP60 is expected to provide effective protection against all currently circulating strains of MV and RHDV. Third, MV has some features that makes it a good choice for the development of transmissible recombinant vaccines for rabbits, with respect to efficacy and safety. Suitable insertion sites for heterologous genes have been described (23
), and the ability of MV-based recombinants to induce a strong immune response, including mucosal immunity, has been established (27
). Doses as low as 5 PFU of a recombinant MV induced high titers of specific antibodies in inoculated rabbits (27
), suggesting that the viral doses delivered by arthropod vectors in nature, which are in the range of 1 to 100 PFU (18
), can be sufficient to elicit adequate antibody responses against recombinant antigens. In addition, field trials carried out in Australia have addressed issues related to the introduction, spread, and monitoring of recombinant MVs released into wild rabbit populations in areas where MV is endemic (52
). Concerning safety issues, MV exhibits a very narrow host range, as it infects only rabbits (both Sylvilagus
spp.). The virus has been widely distributed throughout Europe, Australia, and the Americas for nearly 50 years with no evidence of infection of other species. Thus, the host-restricted nature of MV minimizes the risk of recombinant vaccine spreading to nontarget species in nature. Moreover, given the current widespread geographic distribution of MV, which is similar to the distribution of RHDV, the field use of a recombinant MV-RHDV vaccine would not involve the introduction of a virus that does not already exist in a particular area.
A critical step in the development of the transmissible vaccine was the choice of an appropriate parental MV strain for construction of the MV-VP60 recombinants, i.e., an MV strain with virtually no pathogenic potential and capable of horizontal transmission among rabbits. Results obtained so far by several authors indicate that we are unlikely to obtain such an MV strain by cell culture passage attenuation of virulent field strains, as these attenuated viruses usually lack the ability to spread through horizontal transmission (47
). Presumably, these attenuated MV variants have lost gene functions that are not essential for virus replication in cell cultures but are necessary for virus dissemination in vivo. This seems to be the case of MV strain SG33, a nontransmissible cell culture-attenuated strain used in France to immunize rabbits against myxomatosis (55
). SG33 has a deletion of approximately 13 kb (49
), which includes the Serp2
gene, which has been shown to be important in the pathobiology of MV (36
). As an alternative approach, we decided to select a suitable viral strain from among currently circulating field strains. The rationale was that if a suitable nonpathogenic strain could be isolated from the field, it should be capable of horizontal spread among rabbits, since the virus was circulating in nature. Indeed, an attenuated MV field strain, 6918, with remarkable biological characteristics was isolated (3a
). Strain 6918 causes a nonpathogenic infection comparable to that of attenuated MV strains derived from cell culture passages yet retains some extent of horizontal transmission potential. These features of strain 6918 make it an interesting MV variant with which to study the mechanisms involved in MV dissemination and pathobiology. Research aimed at its molecular characterization is in progress.
Since preservation of the biological properties of the original MV strain was of major importance, we considered the effect of insertion of foreign DNA into the MV genome. The conventional procedure for the isolation of recombinant poxviruses based on TK gene disruption results in a severe attenuation of the virus (11
). Provided that strain 6918 was already a nonvirulent MV strain, further attenuation was not desirable since this could result in a loss of vaccine efficacy and affect the virus transmissibility. Thus, we decided not to disrupt any viral gene. The different VP60 constructs were inserted in the intergenic site between the ORFs MJ2 (TK gene) and MJ2a, as recombinant MVs with a foreign gene inserted at this site had been previously shown to retain overall wild-type biological properties (24
The TDS two-step selection process (17
) was used for the isolation of the recombinant viruses. This procedure enabled the construction of three MV-VP60 recombinants without any marker genes inserted in the final recombinant viral genomes. This is a very desirable feature for recombinant viruses intended for field release, since the environmental risks associated to selectable marker genes (i.e., antibiotic resistance genes or genes encoding metabolic enzymes such as β-galactosidase) is a problem of major concern.
Recently, two MV-VP60 recombinants, obtained by coinsertion of the RHDV VP60 gene and the E. coli lacZ
(β-galactosidase) marker gene into the genome of MV, have been reported to simultaneously protect domestic rabbits against myxomatosis and RHD (4
). However, these recombinant viruses are not well suited for vaccination of wild rabbit populations since they are based on the nontransmissible MV strain SG33, which was further attenuated by deletion of the TK gene (22
) or the MV virulence factors myxoma growth factor and M11L (45
). In addition, there was no indication of effective protective immunization by the oral route (4
The MV-VP60 recombinants described in this work expressed the different VP60 gene constructs to high levels in infected cells (Fig. ). Upon s.c. injection of wild rabbits, the recombinant viruses induced specific antibody responses against MV (Fig. A) and RHDV (Fig. B). In addition, the peptide tag present in 6918VP60-T1 and 6918VP60-T2 elicited a specific antibody response (Fig. C). The time course of the antibody response against the peptide tag differed from that against MV and RHDV. This difference may be due to the difference in nature of the antigens. In the case of the peptide tag (11 amino acids), only one (or few) epitopes are exposed to the immune system of rabbits upon inoculation with the recombinant virus, whereas the kinetics of the antibody responses against MV and RHDV result from the concurrent presentation of multiple epitopes (several viral proteins and the whole VP60, respectively).
Direct immunization of rabbits with recombinant 6918VP60-T2 either by a single s.c. injection or by oral administration induced complete protection against a lethal challenge with RHDV. Furthermore, protection could be transmitted to unvaccinated rabbits by contact transmission (Table ) or by flea-mediated transmission (Table ). On the whole, 50% of the rabbits in a first passage and around 10% in a second passage were protected from RHDV challenge. Indeed, all rabbits with detectable anti-VP60 antibody titers resisted a lethal RHDV challenge, in agreement with previous results (8
), which indicated that protective immunity against RHD is efficient as soon as antibodies against VP60 can be detected in animal sera. Similar results were obtained when rabbits were challenged with virulent MV, with nearly complete protection of directly immunized rabbits, 50% protection of the first-passage rabbits, and around 10% protection in second-passage rabbits.
The fact that the antibody titers against MV and RHDV begin to drop by 56 dpi (Fig. ) raises questions about the efficiency of 6918VP60-T2 in long-term protection. Ongoing experiments indicate that antibody titers against MV and RHDV continue to drop over time but are still detectable at least 8 months after inoculation (data not shown), suggesting that inoculated rabbits may be protected over this period of time. On the other hand, it was shown that infection of immunized rabbits with virulent MV or RHDV induced a high increase in antibody titers. This result indicates that the immunity evoked by 6918VP60-T2 is readily reinforced by exposure to virulent virus. In areas where myxomatosis or RHD is endemic, vaccinated rabbits will be readily reexposed to the viruses. Therefore, a high level of immunity is likely to be maintained in vaccinated rabbits over a prolonged period of time. It should be borne in mind that the virulent challenges were carried out with a high dose (1,000 PFU) of the Laussane strain (virulence grade I) or RHDV (100 LD50). Thus, the challenge conditions used in the experiments reported in this work were by far more severe than those occurring in the field.
Interestingly, oral administration of a high dose of 6918VP60-T2 was able to induce transmissible protection against a virulent challenge with MV and RHDV (Table ). This result opens the possibility of combining both characteristics of the 6918VP60-T2 vaccine, i.e., oral administration and horizontal transmissibility among rabbits, in field immunization of wild rabbits to control myxomatosis and RHD.
On the basis of the results presented in this paper, along with experimental data addressing further safety and efficacy issues (to be published elsewhere), the recombinant 6918VP60-T2 has been subjected to the mandatory risk assessment process relative to the release of genetically modified organisms. Authorization of a limited field trial is currently being considered by the Spanish National Committee of Biosafety to assess the efficacy and safety of the vaccine under controlled field conditions, with respect to its use in a large-scale program for the control of myxomatosis and RHD among wild rabbit populations.