New strategies in the development of PRRS vaccines. Subunit vaccines and self-limiting vectors, based on defective coronavirusesJ. Plana-Durána, M. Mourinoa, E. Viaplanaa, M. Balascha, I. Casalb, M.J. Rodríguezb, L. Enjuanesc, A. Izetac, S. Alonsoc and I. Solác
a Fort Dodge Veterinaria S.A, Carretera Camprodón s/n- Finca "La Riba", 17813 Vall de Bianya, Gerona, Spain
b Inmunología y Genética Aplicada S.A. (INGENASA), Hermanos García Noblejas 41, 28037 Madrid, Spain
c Centro Nacional de Biotecnología (C.N.B.), Department of Molecular and Cell Biology, Campus Univ. Autónoma, Cantoblanco, 28049 Madrid, Spain
Abstract - Porcine reproductive and respiratory syndrome (PRRS) is an emerging viral disease that causes abortion and respiratory distress in swine. It was first reported in America in 1987, although its presence was previously described in the USA and Southeast Asia. The disease was first confirmed in Britain in 1991, coinciding with the establishment of its specific viral etiology. The causative agent, PRRS virus (PRRSV), has been classified as a member of the Arteviridae family. The disease has spread worldwide, becoming an economically significant swine health problem in Europe and North America. Different strategies to prevent economic losses caused by the virus have been designed such as nursery depopulation, control by means of vaccination and control without vaccination or depopulation. PRRSV in vitro propagation has been one of the most important events in PRRS vaccinology. At present, there are some available commercial PRRS vaccines based on modified live vaccines (MLV), autogenous inactivated vaccines (from MARC-145 cells) and inactivated vaccines (from porcine alveolar macrophages). The objective of our work is to explore new approaches to the development of a second generation of PRRS vaccines, such as subunit vaccines and defective viruses (self-limiting vectors). Subunit vaccines: The genomic region containing open reading frames (ORFs) 2 to 7 of the PRRSV Spanish isolate Olot/91 was cloned and sequenced. The genomic sequence showed a 95% homology with Lelystad and Tübingen isolates and 61-64% with the ORF7 region of American isolates. ORFs 2 to 7 were inserted into recombinant baculoviruses downstream of the polyhedrin promoter. To analyse the immunogenicity of the derived recombinant proteins and their ability to confer protection, Sf9 cells infected with the recombinant baculoviruses expressing gene products of ORFs 3, 5 and 7 (gp3, gp5 and N proteins respectively) were used to immunise pregnant sows, either individually or in combination. The results obtained indicated that gp3 and gp5 could be candidates for the development of a subunit vaccine against PRRS since they conferred partial protection (68.5 and 50% respectively), evaluated by the number of piglets born alive and healthy at the time of weaning. By contrast, piglets born from sows immunised with the N protein were not protected (16.6%). Despite the N protein being the most immunogenic protein of PRRSV, antibodies induced in sows are not protective. Other recombinant baculoviruses expressing ORF4 are also being studied. The final recombinant subunit vaccine will enable to distinguish vaccinated animals from infected animals. Defective viruses (self-limiting vectors): Mucosal surfaces are the site of entry of many pathogens. The objective of this part of the work was to describe a new strategy for vaccination using defective coronavirus particles comprising subgenomic defective RNAs (minigenomes) that would be able to produce heterologous recombinant proteins in gut-associated and lymphoid bronchus-associated lymphoid tissues. These antigens can be either viral antigens that develop a local active immune response or foreign recombinant monoclonal antibodies that neutralise infections in situ. Two types of expression vectors, based on transmissible gastroenteritis coronavirus (TGEV) minigenomes are being engineered. The first one is a helper virus-dependent expression system, and the second one is based on a single genome derived from TGEV, which can be modified by targeted recombination introducing heterologous genes at the 3' end of the genome. TGEV-derived vectors have other advantages. First, when inducing mucosal immunity, their tropism can be controlled by modifying the spike ( S) gene resulting in different tissue specific viruses. Secondly, non-pathogenic strains are available for the development of a safer helper virus. And thirdly, coronaviruses, as RNA cytoplasmic viruses, replicate without a DNA intermediary avoiding their integration into cellular chromosomes. This approach is being used to express ORF3, ORF4 and ORF5 from the PRRSV Olot/91 isolate in order to test the immunogenicity of the derived recombinant viruses in vivo.
Corresponding author: J. Plana-Durán Tel.: (34)-972 29 00 01; fax: (34)-972 29 01 02;
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