How are progressing the different WP of the project? The following series of texts will display you the advances of the work groups of VACDIVA during the last year.







The main goal of WP2 is generation of safe and efficacious vaccine candidates with DIVA characteristics for wild boar and domestic pigs against African swine fever (ASF). Three promising live attenuated vaccine (LAV) prototypes showing 92-100% protection against ASFV infection will be further attenuated and vested with differentiation of infected from vaccinated animals (DIVA) properties applying reverse genetic methods.

In the first year of the project, thorough sequence analysis of the candidates and wild type African swine fever virus (ASFV) strains was carried out in order to identify genetic regions contributing to further attenuation and DIVA characteristics. A novel sample processing method for ASFV full-genome sequencing was developed that can contribute to the analysis of the newly generated recombinant vaccine candidates.

A detailed protocol of ASFV mutant generation by CRISPR method was developed and distributed among VACDIVA partners. This protocol was tested on a wild-type ASFV, and mutants were successfully constructed and propagated. At present, this protocol is being applied to generate mutants. Highly immunogenic proteins of NH/P68 are being identified using wild boar and domestic pig sera from ASFV infected animals that can provide DIVA characteristics for the improved vaccine candidate to be constructed.

Finally, different cell lines were tested for propagation of the future vaccine candidates that can allow large scale vaccine production, and some cell lines seem to be appropriate to fulfil these requirements.





The aims of VACDIVA WP3 are to assess the efficacy and safety concerns of the LV17/WB/Rie1 vaccine prototype against African swine fever in wild boar through «in vivo» experiments, and to design the vaccination formula to be delivered in the field. Multiple experiment trials with wild boar from Spanish farms are being carried out at the biosafety level 3 (BSL3) animal facilities of VISAVET Health Surveillance Centre, University Complutense of Madrid. The protective efficacy of Lv17/WB/Rie1 vaccine prototype in wild boar has been already demonstrated after oral vaccination. However definition of the optimal dose and evaluation of the cross-protection level are being evaluated by oral route with the selected bait formula, at different doses, simulating the special circumstances of a potential field vaccination of this species. Overall, vaccination safety is the most important factor for the whole VACDIVA consortium, and it is evaluated in this WP in wild boar by clinical basis, immune response, virus shedding, post mortem analyses and virus dissemination tissues using standardized protocols. By other hand, oral baits compositions suitable to wild boar populations and administration systems are being evaluated in wild boar farms and game states of Spain. For this, several parameters  -detectability, palatability and uptake- are considered. Selective piglet feeders are distributed and monitored through digital motion-camera traps in the field to measure the factors influencing the potential success of different deployment strategies.»





The main goal of WP4 is to carry out in vivo experiments to get a safe and effective pilot vaccine/s based on the three VACDIVA prototypes for domestic pigs. The protective efficacy of Lv17/WB/Rie1 vaccine prototype in wild boar has been already demonstrated after oral vaccination. For this reason, this candidate has been selected during the first year of the project as the vaccine prototype to be evaluated also in domestic pigs within WP4. To select the required dose and route, a comparative “in vivo” safety study has been achieved using the Lv17/WB/Rie1 LAV prototype.By three different administration groups of domestic pigs (5 per group) were vaccinated with two different doses of the Latvian LAV. Vaccination efficiency has been measured in terms of the development of clinical signs, humoral immune response, and/or viremia and virus excretion. To assess the protective efficacy against a lethal dose of virulent strain, domestic pigs were challenged at 28 days post vaccination. The protection parameters have been evaluated in terms of survival, onset of the disease, gross lesions, humoral immune response, virus excretion and dissemination in tissues using standardized protocols. Different samples, including non- invasive samples have been collected for their use in the new diagnostic assays developed under WP6. Finally, in vivo experiments in domestic pigs are currently on –going to assess the protective efficacy of the MA104-NH/P68 -adapted strain compared with that of the original produced in PBMs to be used as vaccine prototype.





Assuming the future ASF vaccine is based on an attenuated modified-live virus strain, production of such a vaccine is going to be a challenge for two reasons. The first issue is that it is not sure at which containment level large scale production will have to take place. If the vaccine strain is absolutely safe for swine, there might be sufficient ground to manufacture the ASF vaccine in standard production facilities. However, authorities can still require high containment production in facilities that are for instance also used for the manufacture of FMD vaccines. The second problem is that a standard cell line has not been described that can support the efficient and stable growth of vaccine strains in large scale production, however as mentioned in WP2, different cell lines have been tested for propagation of the future vaccine candidates, and some cell lines seem to be appropriate to fulfil these requirements. Cells isolated from pig blood have been used so far to grow ASFV meaning that pigs have to be sacrificed to manufacture ASF vaccine. This is for several reasons not a sustainable and acceptable way of producing a vaccine. In Work Package 5 both issues are currently being addressed. We are looking into manufacturing options for the prototype vaccines while keeping in mind the current constraints. There is now a clear path forward and we will continue to focus on the aspects that we think will lead us to an efficient production process.





VACDIVA project will work on the production of several vaccine against ASFV. These candidate vaccines will stimulate an antibody response to a restricted ASFV antigen repertoire, lacking reactivity to those proteins not included in the vaccine, and therefore have theoretical DIVA (Differentiation of Infected from Vaccinated Animals) potential. Thus, WP6 is dedicated to the development of companion diagnostic DIVA tests in conjunction with the selected vaccine candidates. Both, molecular and immunological DIVA tests, will be developed under this WP to be used in domestic pigs and wild boars. For immunological tests, conventional ELISA, LateralFlow Assays (LFA) or microarrays will be explored as DIVA diagnosis assays. The target antigens have being expressed in different expression systems to be used in the newly developed tests.This WP will also include the evaluation of non- invasive methods and the use of alternative samples. Subsequently, a Proficiency Test will be carried out to fully validate the new assays.Finally, the developed tests will be adapted to commercial kits and bring into the market. For this purpose, Eurofins-INGENASA, an animal health world leader company, is leading this WP.





In WP7 we are investigating about control strategies for African swine fever (ASF) with and without vaccination, tailored to each of the epidemiological scenarios in which ASF is present or at high risk of consequence for the swine industry. In the EU, the identification of the different scenarios considers the risk of ASF exposure in domestic pigs, the risk that ASF is re-introduced or becomes endemic in a country, and the risk of consequence or impact in either the hunting sector or the swine industry. For each of these risks, several factors are taken into account, like historical and epidemiological evolution of ASF, species affected, geographical spread, biosecurity measures, acceptance of control measures and impact to trade or industry. We are considering 8 different scenarios in the EU, grouped by countries, namely: 1) Belgium; 2) Bulgaria & Romania; 3) Estonia & Latvia; 4) Germany; 5) Lithuania & Poland; 6) Sardinia; 7) Slovakia & Hungary; 8) Spain. Next, we will test and analyse movement, spread and transmission models of ASF which will help define the optimal strategy taking into account costs and benefits of vaccination to control ASF. All of these models will be supported, when possible, by field trials in wild boar and domestic pigs.

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