Explanation of the objectives. The development of more efficacious vaccines was one of the objectives for NPB 2007 funding. The work reported here is focused on the engineering of a modified live vaccine using transmissible gastroenteritis virus (TGEV) as a vector, expressing PRRSV antigens of interest in protection. Furthermore, the work planned with TGEV-based vectors will allow the identification of protective epitopes, which was also an objective of NPB 2007 funding. As a vector, TGEV is a potent inducer of interferon, elicits strong mucosal and systemic immune responses and could be engineered to develop safe vectors. Therefore, TGEV represents a new strategy to achieve protection against PRRSV. The objectives planned for this year consist in the generation of a set of TGEV vectors expressing PRRSV proteins and modified PRRSV proteins that will allow high specific immune responses anti-PRRSV. Objectives include the quality control of the obtained TGEV vectors and the analysis of the immune response induced by some of the generated TGEV vectors. How research was conducted. Our group previously generated a TGEV vector co-expressing two PRRSV proteins: GP5, the main inducer of neutralizing antibodies, and M, involved in the cellular immune response against PRRSV. Piglets were inoculated with this vector and challenged with a virulent PRRSV strain. The immune response and protection of vaccinated animals was compared to that of non-vaccinated animals. Using this TGEV vector, a killed vaccine was also formulated that was also tested in the weaned pigs system. The results indicated that, although a certain degree of protection was achieved, it was not enough for a good candidate vaccine (see bellow). Therefore, a new set of TGEV vectors co-expressing PRRSV M protein and different modified GP5 proteins were generated. Only some of the planned vectors were successfully obtained, fulfilling all the lab quality controls previous to their animal testing. Discussion of research findings. Concerning the animal experiments using the TGEV vector expressing PRRSV GP5 and M proteins, it was found that all animals present a high antibody response against TGEV, therefore, the vector infected target tissues as expected. Also, vaccinated animals showed a clear antibody response against the PRRSV antigens (i.e., GP5 and M proteins). After a challenge with a virulent PRRSV isolate, a fast recall response was observed, as vaccinated animals induced higher antibody titers against PRRSV antigens and earlier than control animals. Nevertheless, the immune response was not strong enough to provide full protection against PRRSV. That was likely due to the low levels of neutralizing antibodies produced before challenge. Using TGEV vector, a killed vaccine was also formulated and tested in piglets. The results were similar to those obtained with the live TGEV-based vaccine. Vaccinated animals induced higher and faster antibody levels against PRRSV antigens than control animals. Using this experimental approach, a clear degree of protection was observed, as the lungs from vaccinated animals showed a lower degree of lung damage and PRRSV titers in the pulmonary lavages were significantly lower than those obtained in the non-vaccinated animals. Therefore, results using rTGEV as a platform were promising, as antibodies against PRRSV antigens was elicited and a certain degree of protection was observed. Nevertheless, our objective is to improve the protection elicited by our vaccine against PRRSV over currently available commercial modified live vaccines. To improve the immunogenicity of GP5, several modifications were introduced in the GP5 expressed by TGEV vectors. One of them was the elimination of glycosylation sites of the protein. It was proposed that the glycosylation of GP5 probably prevents the exposure of the protein domain responsible of the induction of neutralizing antibodies (neutralizing epitope). Among several generated TGEV vectors expressing GP5 mutants, one stably expressed the GP5 mutant lacking the glycosylation site closer to the neutralizing epitope. A second approach was the elimination of an immunodominant domain in the GP5 that induces non-neutralizing antibodies (decoy epitope) that is close to the neutralizing epitope. A TGEV vector stably expressing a GP5 lacking both the glycosylation site and the decoy epitope was also obtained. These TGEV vectors expressing modified GP5 proteins will be tested in animals during the second year of the project, funded by NPB 2008 (NPB #08-197). Explanation of what these findings mean to the industry. Pork producers are hindered, among others, by infectious disease problems that increase production costs. PRRSV is the agent causing the most important infectious disease affecting swine, resulting in more than $600 million economic loss to US pork producers annually. Therefore, an improvement of vaccination strategies is required to significantly reduce the production costs and improve the performance of the herds. Current vaccines against PRRSV have limited efficacy. Best results have been obtained using modified live vaccines, although they have several problems such as incomplete protection, virus shedding and possible reversion to virulence. This fact has led to an increase in the use of potentially hazardous methods to control the disease, such as using live field virus to vaccinate pigs. Vector- based vaccines could represent and advantage to stimulate both humoral and cell immune responses against PRRSV. Nevertheless, the results reported to date using viral vectors are not fully satisfactory and new vectors must be explored. The main novelty of the reported work derives from the use of the TGEV-based vector to express different PRRSV antigenic combinations. The proposed vaccine may represent a candidate that could provide protection against two viruses: PRRSV and TGEV. As this is a live vaccine, its efficacy should be high, whereas its cost should be competitive. Contact information. Luis Enjuanes. Centro Nacional de Biotecnologia, CSIC. Department of Molecular and Cell Biology. Darwin 3. Campus Univ. Autonoma, Cantoblanco. 28049 Madrid, Spain. Fax: 34-91- 585 4915 or 34-91- 585 4506 Ph: 34-91- 585 45 55 Email: L.Enjuanes@cnb.csic.es