A novel, recombinant myxoma virus-rabbit haemorrhagic disease virus (RHDV) vaccine has been developed for the prevention of myxomatosis and rabbit haemorrhagic disease (RHD). A number of laboratory studies are described illustrating the safety and efficacy of the vaccine following subcutaneous administration in laboratory rabbits from four weeks of age onwards. In these studies, both vaccinated and unvaccinated control rabbits were challenged using pathogenic strains of RHD and myxoma viruses, and 100 per cent of the vaccinated rabbits were protected against both myxomatosis and RHD.
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MYXOMATOSIS and rabbit haemorrhagic disease (RHD) are two viral diseases affecting the European rabbit. Both are almost invariably fatal in the naive animal; therefore, there is a real need for safe and efficacious vaccines.
Myxomatosis is caused by myxoma virus, a poxvirus of the genus Leporipoxvirus. Attempts were made to use myxomatosis as a form of rabbit control in the UK Denmark and Sweden in the 1930s (Hvass and Schmit-Jensen 1939, Schmit-Jensen 1939, Lockley 1940); however, the virus did not become established. It was not until the 1950s after the virus had been released in Australia and then in France that the virus became endemic in Western Europe. Biting insects are recognised as an important vector for the transmission of myxoma virus. The virus replicates in the skin at the inoculation site and spreads to the lymphoid tissue where it also replicates to high titres. The virus is then disseminated to the various organs in infected lymphocytes; the major sites of pathology are the lymphoid tissues and the skin - particularly around the eyes, ears and the ano-genital region, where the classical subcutaneous swellings (myxomas) are seen (Fenner and Marshall 1957). High virus titres are also observed in lachrymal and nasal secretions, and direct transmission of myxoma virus in the absence of insect vectors has also been documented (Houlihan and Lawson 1945, Chapuis and others 1994). A few years after the initial release of myxoma virus in Australia, it became clear that both the virus and the wild rabbit were each adapting to this host pathogen relationship (Fenner 1953, Marshall and Douglas 1961); the rabbits were acquiring a level of immunity and less virulent strains of the virus began to evolve. Although such detailed studies have not been performed in Europe, it is likely that a similar co-evolutionary situation exists here also. While there may be strains of myxoma virus of differing virulence circulating in the European wild rabbit population, these viruses may still be lethal for the domesticated rabbits which have little or no genetic immunity. In addition to the classical (nodular) form of myxomatosis, the strains of myxoma virus are circulating that do not produce the classical skin lesions or myxomas from which the virus derives its name (Marlier and others 1999). However, infection with the so-called amyxomatous forms of the virus still causes the typical swollen eyes, ears and genitalia seen with the classical strains. Severe acute respiratory disease, probably as a result of the secondary bacterial infection, is seen with both forms of the virus.
RHD is a highly contagious disease of the European rabbit with a high morbidity and mortality in susceptible animals. Although it, too, has been employed deliberately as a biological control agent in Australia and New Zealand, it has spread naturally throughout most of the rest of the world (Villafuerte and others 1994, Heneidi Zeckua and others 1997). The aetiological agent of the disease is a small positive strand RNA virus of the genus Lagovirus, a member of the family Caliciviridae (Koopmans and others 2005). It is a somewhat unusual disease in that animals younger than about 10 weeks of age are almost completely resistant. The mechanism of this resistance remains to be fully elucidated.
Vaccination against myxomatosis has been available for some time. All vaccines are live vaccines based either on attenuated myxoma virus strains or on a closely related poxvirus, Shope fibroma virus. Each has its advantages and disadvantages. Shope fibroma virus-based vaccines may be considered less immunogenic, while attenuated myxoma virus-based vaccines may be immunosuppressive, particularly in young rabbits (Fenner and Woodroofe 1954, McKercher and Saito 1964). Such immunosuppression in animals held in large rabbitries can lead to serious problems of bacterial respiratory infection. The duration of protection of live attenuated myxoma virus vaccines is usually around four to six months.
Vaccines for RHD currently consist of inactivated virus preparations derived from the infected rabbit livers and combined with an adjuvant. These products are able to stimulate the effective immune responses; however, the adjuvants used may sometimes cause unwanted site reactions particularly in dwarf and show animals. These vaccines may be used to vaccinate young rabbits several weeks before they become susceptible to the disease.
Here, the authors describe a number of safety and efficacy studies with a novel recombinant myxoma-rabbit haemorrhagic disease virus (RHDV) vaccine in specific pathogen-free (SPF) rabbits.
Materials and methods
The vaccine (Nobivac Myxo-RHD, MSD-Animal Health) was constructed from a laboratory-attenuated strain of myxoma virus and the capsid protein gene of a German isolate of RHDV (Meyers and others 1991). Recombinant virus was constructed using the standard laboratory methods and the RHDV capsid gene was inserted into the MGF/M11L locus of the myxoma virus genome. Vaccine material was prepared in rabbit kidney cells (RK-13). Vaccine dosages appropriate to the specific studies were used. Safety studies employed vaccine dosages of up to 107 focus-forming units (ffu). For efficacy studies, the minimal vaccine doses of 103 ffu were used.
As part of this series of studies, and to provide a relevant challenge, a myxoma virus was isolated in 1992 in Cambridgeshire UK, from a wild rabbit displaying the advanced signs of myxomatosis. This virus (precious strain) was characterised by its behaviour in vitro and in vivo together with the limited DNA sequence analysis (data not shown).
The virus was prepared in cultured RK-13 cells after no more than three passes in culture in order to limit any attenuation of pathogenicity. Virus titres were performed in RK-13 cells and expressed in plaque forming units (pfu). Both pfu and ffu maybe used to express the titres of myxoma virus. The values are more or less equivalent and derive from the visual appearance of the infected cell monolayer in the specific titration assay.
The RHDV Ascot strain was obtained from the Veterinary Laboratories Agency, Weybridge, UK.
Rabbits: husbandry and welfare
The rabbits were floor-housed in groups of about 10 to 20; hay and water were available ad libitum. Commercial food was available ad libitum until adult weight was attained where after it was provided once a day. The animals were vaccinated by the subcutaneous route. Lyophilised vaccine was reconstituted using an appropriate solvent and administered in a 1 ml dose into the neck/shoulder region. The animals were closely monitored for any adverse reaction to vaccination including signs of swelling at the site of injection, body temperature fluctuations and general health status.
Myxoma virus and RHDV challenges
Myxoma virus challenges were performed by the intradermal inoculation of virus into a site on the flank of the rabbit. A small area of the skin was shaved in order to clearly observe the administration; 50 µl of the culture medium containing 200 pfu of virulent virus was injected into the shaved area of the skin using a 25-gauge needle. The rabbits were monitored daily for clinical signs of myxomatosis such as swelling of eyes, genitals and ears; respiratory difficulties and skin lesions, and a clinical score was assigned according to the specific criteria (Table 1). Rabbits exhibiting severe signs of myxomatosis were euthanased.
RHDV challenges were carried out using diluted homogenates of the infected rabbit liver. The material was administered by intramuscular injection. Typically, the animals showed few, if any, clinical signs other than malaise before death. As the animals were floor-housed in open groups, the infected rabbits which were unable to avoid the capture and showed signs of acute malaise, were euthanased.
Study 1: efficacy against myxoma virus challenge
Eleven, 33-day-old rabbits with no pre-existing antibodies to either myxoma or RHDV were vaccinated with a single dose of vaccine (103 ffu) in 1.0 ml of the solvent. Five rabbits of identical age were kept as unvaccinated controls and were housed together with the vaccinated animals.
Blood samples were taken and assayed in order to confirm the negative serological status of the rabbits, and then 24 days postvaccination all the rabbits were challenged by the intradermal route with virulent myxoma virus
Study 2: efficacy against RHDV challenge
Sixteen, 31-day-old rabbits were divided into two groups of 11 vaccinates and five controls. The vaccinated group was inoculated with a single dose of vaccine (103 ffu) administered by the subcutaneous route, and the control group was not vaccinated. Blood samples were taken to monitor the antibody responses to the RHDV capsid protein. Vaccinated and control rabbits were housed together until the time of challenge. Since young rabbits are not sensitive to RHDV, the animals were challenged with virulent RHDV at 16 weeks of age when they were old enough to be sensitive.
Studies 3 and 4: virus dissemination and absence of shedding of vaccine virus
Four rabbits were vaccinated with an overdose of Nobivac Myxo-RHD (106.8 ffu). Six days following vaccination, the animals were euthanased and tissues samples were taken to establish the vaccine virus content.
Six rabbits were vaccinated with an overdose of Nobivac Myxo-RHD (106.8 ffu) and blood samples taken on a daily basis up to 14 days postvaccination. Lymphocytes were extracted from the whole blood samples (∼2.0 ml), and virus isolation assays were performed on the lymphocyte extracts.
The serum was separated from whole clotted blood and heat inactivated at 56°C for 30 minutes. Antibodies to myxoma virus were measured either by ELISA or by immunofluorescence (IF) using methanol-fixed myxoma virus infected cell monolayers. In the IF assay, the reactive rabbit antibodies to myxoma infected cells were detected using an FITC-labelled goat-antirabbit polyclonal antisera. In the ELISA, the rabbit antibodies were detected using an alkaline phosphatase conjugated goat-antirabbit antibody. Responses to the RHDV insert were assayed in a haemagglutination inhibition (HI) assay using human erythrocytes. Briefly, the diluted aliquots of sera were mixed with human O-type blood; these samples were then distributed in 96-well plates in the presence of a fixed amount of RHDV antigen. RHDV antigen causes agglutination of the red blood cells, a process which is inhibited by specific RHDV antibodies. The presence or absence of agglutination was determined by visual inspection.
Efficacy against myxoma virus challenge (study 1)
All of the control animals developed signs of severe myxomatosis and were euthanased 13 to 14 days postchallenge, whereas none of the vaccinated animals developed clinical disease (Table 2). Ten of the 11 vaccinates remained normal and healthy until the end of the study. The remaining vaccinated rabbit had to be euthanased due to acute enteritis (mucoid enteropathy), a condition unrelated to the challenge and seen sporadically in rabbits in other studies. Some vaccinated rabbits had rectal temperature readings which fell outside of the prescribed normal range on some days, and these were given a clinical score. However, none of these animals exhibited any of the recognised signs of myxomatosis.
Efficacy against RHDV challenge (study 2)
All vaccinates demonstrated a significant serum antibody response before challenge, whereas none of the unvaccinated rabbits showed any detectable rise in RHDV antibody (Table 3). All five non-vaccinates died from haemorrhagic disease within 72 hours of challenge whereas all vaccinated animals survived and showed no signs of disease (Table 4). Samples of liver were taken postmortem from the unvaccinated rabbits to confirm the cause of death and liver antigen levels were compared with those in liver samples taken at the end of the study from the vaccinates. All the non-vaccinates had high levels of RHDV in the liver while the vaccinates, in contrast, showed no or very low levels of RHDV (Table 5).
Virus dissemination and absence of shedding of vaccine virus (studies 3 and 4)
None of the non-vaccinated rabbits became seropositive to either myxoma virus or RHDV. In contrast to rabbits infected with wild-type myxoma virus and exhibiting signs of myxomatosis, the virus could not be detected in swabs taken from vaccinated animals (Table 6). Vaccine virus could occasionally be re-isolated (postmortem) from the draining lymph node and the skin immediately around the site of injection in some rabbits (Table 6). Tissues from a non-vaccinated rabbit were spiked with the vaccine virus and then processed alongside the test samples as positive controls in order to demonstrate the validity of the processing and assay methods. Virus recovery was shown to be greater than 75 per cent from all spiked tissues.
Virus could not be recovered from any lymphocyte preparations (data not shown).
Safety of Nobivac Myxo-RHD
In all studies, vaccinated rabbits remained healthy and no adverse signs were seen.
Additionally, field safety studies conducted with farmed rabbits revealed no adverse clinical signs (data not shown). With intensively farmed rabbits, there is always an underlying respiratory disease; nevertheless, vaccination with Nobivac Myxo-RHD did not exacerbate any existing respiratory conditions, and therefore the vaccine is also considered safe for use in young, commercially farmed animals.
In further (unpublished Spibey and others) studies, doses of up to 107 ffu were administered to laboratory SPF and dwarf rabbits with no adverse reaction. It was notable that the vaccine virus did not disseminate to the typical sites of replication of the wild-type myxoma virus such as the skin around the eyes, ears and genitalia. Occasionally, at about a week after vaccination, small local reactions (1 to 2 cm in diameter) were observed at the site of injection; however, these resolved completely within a few days.
Myxomatosis and RHD are the two major diseases that afflict the European rabbit. Myxomatosis vaccines have been available for some time and are based on either Shope fibroma virus or attenuated strains of myxoma virus. RHDV vaccines comprise inactivated adjuvanted virus preparations. However, since the viral agent (RHDV) cannot be cultured in vivo, the vaccine has to be repeatedly prepared from the livers of infected rabbits, which is an undesirable method of vaccine production.
We demonstrate here that a novel bivalent myxomatosis/RHD vaccine is able to provide complete protection against both myxomatosis and RHD after a single dose immunisation. Further studies (unpublished) have shown that the immunity persists for at least 12 months following a single immunisation. It has been observed that the existing myxoma-based vaccines may have safety concerns, especially in very young rabbits where there is a significant bacterial burden. These safety issues are due to the immunosuppressive characteristic of the myxoma virus, which is not sufficiently attenuated in these vaccine strains. The authors have developed a vaccine strain which is unable to disseminate in the vaccinated rabbits beyond the local draining lymph node and the skin around the injection site. This inability to spread within the vaccinated animal probably accounts for the good safety profile as demonstrated by the absence of any adverse effects in very young rabbits, dwarf rabbits and rabbits from commercial meat farms with their typical bacterial load. Furthermore, studies were performed using vaccinated and non-vaccinated rabbits which were co-housed in animal rooms that allowed them to freely mix. These conditions favour the spread of the wild-type myxoma virus, but the vaccine virus was unable to spread from animal to animal. The favourable safety profile and the lack of capacity to disseminate within the animal do not appear to have resulted in any reduced immunity against the myxoma virus; on the contrary, in additional unpublished studies, vaccinated animals have remained protected against myxomatosis for a full 12 months, which is at least twice as long a period of protection as is claimed by the currently available myxoma virus-based vaccines. The removal of the two myxoma immunosuppressive factors (MGF and M11L) certainly contributes to the attenuation of the myxoma virus (Graham and others 1992, Opgenorth and others 1992). It has also been previously established that the M11L protein has an anti-apoptotic action and that in the absence of M11L the virus is unable to block apoptosis in infected lymphocytes (Everett and others 2000, Su and others 2006). As a consequence of this, the virus may well be unable to disseminate in the rabbit. Myxoma virus, in common with other members of the pox virus family, encodes for a large number of immunomodulatory functions, and mutations in a number of these could result in the observed phenotype.
Both humoral and cellular immune responses are important in mediating immunity to the myxoma virus infection. Immunity to RHDV is almost certainly antibody-mediated and rabbits vaccinated with Nobivac Myxo-RHD in the study described all showed a strong serological response to RHDV. The absence or very low level of RHDV in the liver of the vaccinated rabbits at the end of the study demonstrates that the immune response in these animals was very robust and either prevented any significant viral replication or allowed the rapid clearance of antigen.
Laboratory challenge studies are typically extreme demonstrations of immunity to viral pathogens; therefore, the immunity demonstrated in rabbits vaccinated with Nobivac Myxo-RHD to myxomatosis and RHD represents a significant level of protection and an improvement on many of the currently available vaccines.
Provenance not commissioned; externally peer reviewed
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