Rabies is still considered to be an important zoonoses in most of the developing countries and stray dogs represent the main vector and reservoir for this disease. The most efficient way to prevent the transmission of rabies to humans is by vaccinating dogs. In developing countries, the turnover rate of dogs is around 30% each year, mostly replaced by new born puppies . More than 42% of the reported rabid dogs in Thailand are younger than 6 months . During the vaccination campaigns, puppies younger than 3 months are not targeted. Owing to the yearly recurrence of the vaccination campaigns against rabies, unvaccinated puppies during the campaign keep that status for at least one year of their early lives. Consequently, we should persuade local populations to vaccinate their new born puppies against rabies. Furthermore, it is well known that ownerless and stray dogs are rarely administered a rabies vaccination. Thus, the vaccination of new born puppies before becoming ownerless and stray dogs allows residual rabies immunity; otherwise this will never be achieved. Unfortunately, it was well shown that during mass vaccination of dogs against rabies, a big proportion of the vaccinated puppies will die because they were highly exposed to infections with either canine parvovirus or distemper virus because of the intervention of the vaccinators. For this reason, puppy owners are reluctant to vaccinate them against rabies. To overcome this problem, we think it is necessary to include other valences in the inoculated vaccine against rabies, especially against CPV and CDV. We have already shown the efficiency of DNA-based vaccination against rabies in the mouse and dog models [12, 13]. Here we have further validated a monovalent DNA-based vaccine candidate (pCMV3ISS-CDVH) which encodes to the hemagglutinin glycoprotein of the canine distemper virus in the same animal models. Hence, multivalent DNA-based vaccination in dogs, including rabies and distemper valences, could represent a strong argument in raising vaccine coverage and efficacy.
We have designed different strategies of multivalent DNA-based vaccination and compared their efficiencies. The use of a cocktail of two plasmids, the first encoding to the rabies glycoprotein, and the second to the hemagglutinin glycoprotein of the CDV, allowed in vitro expression and induction in mice of specific virus-neutralizing antibodies against both valences. The induced humoral immune responses were identical to what was obtained when the inoculation was a single monovalent plasmid. This strategy associating two plasmids in the vaccine preparations has bioprocessing drawbacks, since there is a need for two separate processes, quality controls for each plasmid preparation, and mixing them.
The strategy of using a plasmid, which encodes to a fusion polyprotein without an internal cleavage element, induced in mice the same level of neutralizing rabies virus antibodies as the monovalent plasmid (pCMV3ISS-GPV), whether it was administered alone or in association with the plasmid pCMV3ISS-CDVH. Against the CDV valence, the induced humoral immune responses were rather depressed by the fusion of the two valences. In both cases (against rabies and CDV), and whether 50 μg or 100 μg of the plasmid pCMV3ISS-GPV-CDVH were inoculated to mice, the induced humoral immune responses did not change significantly. The introduction of the FMDV 2A gene between those of both antigens did not significantly improve the humoral responses against CDV. However, it seems that when the GPV is in vivo liberated from the fusion polyprotein, there is a boost of the immune responses against the rabies virus compared to the use of the monovalent plasmid. Hence, it can be postulated that when the CDVH native valence was expressed in the same cell as GPV, it was able to induce a cross boost effect. Similar results have been reported when a mixture of antigens was used [25, 26]. This can be explained by the induction of a more robust cytokine environment since it is reported that DNA-based vaccination is highly efficient in inducing T cell responses .
The last strategy based on IRES multicistronic plasmid seems to be the most efficient in inducing rabies virus and CDV-neutralizing antibodies. The beneficial cross effect of CDVH on rabies valence was at least the same with pCMV3ISS-GPV-IRES-CDVH as with pCMV3ISS-GPV-2A-CDVH. In addition, against CDV, mice inoculated with the bicistronic plasmid raised the same level of antibody response as those administered the monovalent plasmid. Compared to mice inoculated with the cocktail of the two monovalent plasmids (50 μg of each plasmid), those inoculated with the bicistronic plasmid gave better immune responses with a total quantity of 50 μg. Furthermore, it has been reported that a gene inserted upstream from the IRES is strongly cap-dependent expressed, while a gene placed downstream is IRES-dependent expressed at a lower level [16, 17]. Nevertheless, in our construction, even if there is a down expression of CDVH after the inoculation of mice with the bicistronic plasmid, the induced humoral immune responses were similar to that after the administration of the monovalent pCMV3ISS-CDVH plasmid. Therefore, the IRES strategy is not only beneficial at the immunogenecity level, but also at the bioprocessiong one. This multivalent approach is based on a single construct, allowing for a more straightforward processing and quality control. The cost of production is further restrained since only the half quantity of plasmid is needed for at least the same level of immune responses.
If we compare any of the DNA-based multivalent vaccine candidates with the use of cell culture vaccines, it is quite clear that they are more efficient, especially against the CDVH valence. Against the rabies valence, we have already reported that DNA-based vaccination is superior to the inoculation of commercial cell culture vaccines [21, 23]. With pCMV3ISS-GPV-IRES-CDVH, the induced immune responses are further improved compared to those induced with Tetradog® (p-value 0.0008, at day 28 post-vaccination) and Rabisin® (p-value 0.013, at day 28 post-vaccination).