The data presented in this paper extend previous work showing that a vaccine based on attenuated AlHV-1 protected cattle challenged intranasally with pathogenic AlHV-1 C500 from developing clinical MCF . This previous study reported that protection could be obtained by immunisation with vaccine formulated with Freunds adjuvant, an unlicensed product. We have also demonstrated that the attenuated virus without adjuvant did not protect from intranasal challenge with pathogenic AlHV-1 (G.C. Russell, unpublished data). Here we show that the licensed oil-in-water adjuvant Emulsigen can facilitate protection from intranasal experimental AlHV-1 challenge given up to six months after primary immunisation.
Analysis of plasma and nasal secretion antibody responses to vaccination and challenge showed that (i) unvaccinated animals with MCF had no ELISA-detectable or neutralising antibody response to AlHV-1 following challenge; (ii) vaccinated animals that were protected from MCF had significantly higher titres of virus-neutralising antibodies in both plasma and nasal secretions at about 1 month after boost than unprotected animals and these titres did not significantly change after challenge (Figure 4); (iii) in contrast, vaccinated animals that succumbed to MCF showed significant increases in both total virus-specific antibody and virus-neutralising antibody during the development of clinical MCF (Figure 3 and Figure 4).
The lack of a detectable antibody response in unvaccinated cattle challenged with AlHV-1 could reflect the rapid onset of MCF that might prevent the induction of an immune response to combat the infection. However, in the experiments reported here, the appearance of clinical MCF in control animals ranged between 21 and 68 days after challenge (Table 1), suggesting that there was sufficient time for the development of an immune response in at least some of the infected cattle. These data confirm and extend the previous observation that control cattle challenged intranasally did not develop detectable titres of virus-neutralising antibody .
In field cases of MCF, virus-specific antibody responses are reported to be found in 70-80% of samples tested . The lack of antibody response in some MCF cases was confirmed in a comparative analysis of MCF-specific PCR and serological diagnostic testing where 14 of 39 OvHV-2 PCR-positive animals with clinical MCF were considered serologically borderline or negative . Additionally, sub-clinical infection with MCF viruses has been inferred from serological surveys in which animals from MCF-susceptible species (cattle, bison, deer) had detectable MCF-specific antibodies or viral DNA in the absence of disease [29–31]. More recently, experiments using OvHV-2 to infect cattle or bison showed sub-clinical infection of five cattle, with seroconversion, and of one bison without seroconversion; while evidence of previous infection was found in six bison of which two had both detectable anti-MCF antibodies and OvHV-2 DNA [32, 33]. Previous subclinical infection did not appear to reduce susceptibility to MCF following intranasal challenge.
These observations suggest that infection with MCF viruses and the ensuing immune response and disease may be more complex than previously thought, leading to the development of MCF in some animals without a detectable virus-specific antibody response. There is a lack of good transmission and epidemiological work on MCF that requires to be done to address such issues.
In contrast to the boost to AlHV-1-specific antibody responses found in vaccinated animals that developed MCF, vaccine-protected animals did not show a significant increase in antibody titres after challenge. This is likely to be because no virus in immunogenic quantity was able to penetrate the mucosal barrier of neutralising antibody to boost the response. This supports the contention that the establishment of a mucosal barrier of antibody is a mechanism of protection against MCF. This is reinforced by the observation that protected animals in groups 3 and 4 had high virus-neutralising antibody titres after immunisation compared to animals that succumbed to MCF. The higher titres are likely to be associated with longer duration of antibody responses and hence protection at later time points.
The development of high titre virus-specific antibody responses prior to the onset of MCF among vaccinated cattle in these experiments suggests that the development of clinical MCF includes the expression of virus gene products that stimulate an anamnestic immune response. Indeed, clinical MCF cases are often characterised by the development of a circulating antibody response which includes antibodies specific for virus structural antigens including the major glycoprotein complex of the viral envelope [34, 35]. This is in contrast to recent suggestions that MCF in rabbits and cattle is associated with a latent infection of lymphocytes and a lack of lytic gene expression that would include antigenic capsid and envelope proteins [11, 36, 37]. The presence of antibodies specific for virus structural proteins in MCF-affected animals suggests that infection results in a pattern of virus gene expression that includes a number of lytic cycle antigens but without the production of infectious virus. It may therefore be inappropriate to discuss MCF in terms of lytic or latent gene expression patterns since latency should be defined in the natural host rather than in MCF-susceptible species that cannot propagate these viruses.
Current work aims to improve the magnitude and duration of the protective immune response by the strategic inclusion of toll-like receptor (TLR) agonists in the MCF vaccine. In addition, the role, if any, of cytotoxic T cells in protection against MCF is currently not known and this is being investigated. The six month window of protection offered by the current immunisation regime should be adequate to protect cattle in Eastern and Southern Africa exposed to wildebeest during the calving period. This is being studied in field trials in Tanzania.