Vírus influenza recombinantes como vetores vacinais: desenvolvimento e aplicação no modelo de doença de chagas

Abstract

Vaccines are considered one of the most successful and effective medical interventions to prevent infectious diseases. It is estimated that up to 3 million human lives are spared through vaccination per year, in addition to preventing illness of more than 100 million people and saving costs for productivity and health systems. The use of recombinant viruses as vectors is one of the most viable strategies for the development of new vaccines. In 2010, our group has demonstrated the feasibility of the generation and use of influenza recombinant viruses in combination with recombinant adenoviruses to protect against Toxoplasma gondii infection in mice. Chagas disease is an important neglected disease. It is caused by infection with the protozoan Trypanosoma cruzi and is prevalent in Latin America. The treatment has higher efficacy in the acute phase of the disease, yet causes undesired side effects. There are currently no vaccines Chagas disease in humans, which makes the study and development of an optimized vaccination protocol an important research subject. Using reverse genetics we generated recombinant influenza viruses encoding the C-terminus and Medial portions of the T. cruzi protein ASP-2 and further characterized the phenotype and genotype of those viruses. Immunization using Influenza-ASP-2 as prime and Ad-ASP-2 as boost induced a potent immunodominant CD8+ polyfunctional T cell response, which was mostly immunodominant. This immune response was able to protect C57BL/6 and C3H/He mice challenged with Y strain of T. cruzi. However, the generated influenza-ASP-2 viruses retained virulence when given at a higher inoculation dose. Thus, we modified the vector by deleting most of the coding region of the neuraminidase segment, vNA-, making them unable to escape from the cells they were infecting. We confirmed attenuation of the modified viruses by measuring weigh and monitoring mortality after intranasal inoculation of C57BL/6 mice. Vírus influenza recombinantes como vetores vacinais: desenvolvimento e aplicação no modelo de doença de Chagas| 12 Next, we demonstrated that those viruses caused mild inflammation and cellular influx into the lungs of inoculated mice. It is noteworthy that proinflammatory cytokines were strongly reduced in the sera and lungs of mice upon inoculation with vNA-. Those viruses induced strong humoral and cellular immune responses specific against influenza, which was able to protect C57BL/6 mice against lethal challenge with wild-type PR8 virus in a dose-dependent fashion. Finally, we have shown that vNA- are attenuated even in severely immunocompromised mice both to innate (MyD88-/-) or adaptive immune response (RAG-/-). In conclusion, we were able to generate and characterize recombinant influenza viruses that when in combination with recombinant adenoviruses were able to induce a potent cellular immune response and protect against lethal infection in mice challenged with T. cruzi. Furthermore, we generated an attenuated influenza virus to be used as a safer vector. Upon intranasal inoculation, we characterized those attenuated viruses with respect to their inflammatory profile, specific immunological response and protection against wild-type lethal infection. Finally, those viruses were attenuated even in immunodeficient mice.