RESUMEN
KEY MESSAGE: Expression of dengue-2 virus NS1 protein in Nicotiana tabacum plants for development of dengue immunodiagnostic kits. Dengue is one of the most important diseases caused by arboviruses in the world. A significant increase in its geographical distribution has been noticed over the last 20 years, with continuous transmission of several serotypes and emergence of the hemorrhagic fever in areas where the disease was previously not prevalent. Although the methodological processes for dengue diagnosis are in deep development and improvement, a limitation for the realization of dengue diagnostic tests is the difficulty of large-scale production of the antigen to be used in diagnostic tests. Due to this demand, the purpose of this study was to obtain the non-structural protein 1 (NS1) from dengue-2 serotype by heterologous expression in Nicotiana tabacum (Havana). After confirmation of the NS1 protein gene integration in the plant genome, the heterologous protein was characterized using SDS-PAGE and immunoblotting. In an immunoenzymatic test, the recombinant NS1 protein presents an antigen potential for development of dengue immunodiagnostic kits.
Asunto(s)
Dengue/diagnóstico , Nicotiana/genética , Proteínas Recombinantes/genética , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/inmunología , Aedes/virología , Agrobacterium tumefaciens/genética , Animales , Antígenos Virales/genética , Antígenos Virales/inmunología , Estudios de Casos y Controles , Virus del Dengue/inmunología , Virus del Dengue/patogenicidad , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Humanos , Immunoblotting , Plantas Modificadas Genéticamente , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Nicotiana/metabolismo , Proteínas no Estructurales Virales/metabolismoRESUMEN
To improve a DNA vaccine containing the truncated dengue virus serotype 2 (DENV-2) envelope (E) protein and evaluate the influence of precursor membrane (prM) glycoprotein polymorphism on E protein immunogenicity, two vaccine candidates have been constructed by upstream insertion of the DENV-2 and DENV-3 prM genes into the DENV-2 E gene, named pCID2EtD2prM and pCID2EtD3prM, respectively. Both constructs were able to induce antibody production, which were neutralizing against DENV-2 in a murine model. Splenocytes of immunized groups, when challenged with virus, demonstrated Th1 cytokine pattern and proliferation, in addition to the increase of specific T cells. Vaccine candidates pCID2EtD2prM and pCID2EtD3prM confer 70% and 90% protection against DENV-2, respectively. The pCID2EtD3prM plasmid conferred only 40% protection in the lethal challenge with DENV-2. The results demonstrate that DENV-3 prM has a greater influence on the immunogenicity of the E protein and, probably due to its role as a chaperone, these results may be related to the correct folding and, consequently, an increase in the presentation efficiency of produced transcripts.