• Determination of the best conditions for the production and purification of VLPs in terms of yield and production costs;

The production and purification process of RHDV2 VP60-VLPs was designed to generate vaccine preparations in sufficient quantity and acceptable quality for testing on animal models. It is expected that a reduction in production costs will be achieved by simplifying the production process, the VLP purification process, namely due to the lower purity requirement when compared to human vaccines, and the high immunogenicity of these particles.

• Obtaining VLPs

Obtaining a sufficient amount of VLPs is a critical result for the development of the remaining stages of the project. Hence, strategies were designed to guarantee the success of this stage.

Once obtained, the RHDV2 VP60-VLPs vaccine candidate will be subjected to a structural and functional evaluation, and their stability after being subjected to different conditions of temperature, humidity and lyophilisation will also be evaluated.

• Implementation of indirect ELISA to detect antibodies to RHDV2

The recombinant VLP proteins will be used to develop an indirect ELISA (I-ELISA). In the absence of a commercial test, the development of this test is essential since it will allow, to assess the presence of antibodies to RHDV2 before the oral administration of VLPs to experimental animals and to follow the rabbits' immune response to oral intake of the VLPs.

• Determination of the dose of VLPs (in µg) required for the oral immunization of domestic rabbits and wild rabbits.

It is expected that, at the end of the animal experimentation phase under laboratory conditions, the immunogenic dose of RHDV2 VP60-VLPs necessary for the immunization of wild rabbits has been determined.

As it is not possible to perform it in vitro, the purified VP60-VLPs ability to induce a protective humoral response against the type 2 haemorrhagic disease virus (RHDV2) will be assessed in vivo. The animal experimentation stage will be conducted in accordance with national regulations for animals for experimental purposes (DL 113/2013, of 7 August). At the start of this stage, a pilot test will be carried out, under laboratory conditions, on domestic SPF rabbits to determine the minimum amount of viral protein to induce the production of high antibody titers. Once the immunogenic dose of VLPs has been determined, the experiment will be extended to the wild rabbit, also under laboratory conditions.

• Determination of the immunogenic and protective capacity of VLPs

In the course of this project, it is expected to determine, and to know in detail, the immunogenic and protective capacity of the RHDV2 VP60-VLPs.

Immunological protection will be assessed by performing a challenge in 50% of the animals, which will be inoculated with a virulent strain of RHDV2. Challenge will be carried out under laboratory conditions.

The duration of immunity induced by oral vaccination will be monitored for 6 months in the other 50% of the animals, allowing to assess the influence of a possible heterogeneity of the bait ingestion (feed containing the vaccine) on the efficiency of the immunization and on the duration of the immunity.

• Obtaining a vaccine prototype

The expected end result will be to obtain a prototype of the patentable vaccine.