Geração de imunógenos para herpesviroses bovinas utilizando a tecnologia dos nanotubos de carbono

Abstract

Bovine herpesviruses 1 and 5 (BoHV-1 and BoHV-5, respectively) are closely related alphaherpesviruses infecting cattle and co-infection is likely to occur. Both viruses are associated with neurological, respiratory and reproductive disease, causing great economic losses. Vaccination has been the recommended in control programs, although to date there is no vaccine capable of establishing a protective immune response against both viruses. Recombinant proteins have been widely used for production of helpful molecules employed in prevention and treatment of several diseases. Recently, our group created two recombinant multiepitope proteins containing, each one, four immunodominant linear epitopes of BoHV-1 or BoHV-5, respectively. Carbon Nanotubes (CNT) have been broadly studied due to their exceptional properties such as biocompatibility, high aspect ratio and cell internalization ability, and CNT functionalized with antigens have immunogenic potential, as shown in previous studies. In this work, we have used the CNT technology to build experimental immunogens against BoHV-1 and BoHV-5. The recombinant proteins were purified and covalently bound to multi-walled carbon nanotubes (MWNT). The functionalization efficacy was successfully verified by SDS-PAGE and Raman Spectroscopy. The recombinant proteins were recognized by IgG and IgM antibodies from sera of bovines naturally infected with both viruses, showing that the CNT doesnt interfere with the recognition profile of the recombinant proteins. These molecules, functionalized or not to the CNT, were used in a prime-boost immunization protocol in C57Bl-6 mice, comparing to recombinants alone or added with alum and inactivated commercial vaccine. Following that, mice TCD4+ and CD8+ lymphocytes activation were analyzed by flow citometry, quantifying the marker CD25. Mice immunized with the recombinant proteins associated to the CNT plus the adjuvant alum, showed a higher profile of activated CD4+ and CD8+ cells than the other groups. The recombinant proteins, in all tested formulations, induced a Th-2/Th-17 cytokine profile, particularly IL-6, IL-13 e TGF-1. Once our recombinant proteins were designed according to the bovine pattern recognition of viral epitopes, it was not possible to perform a comprehensive analysis of the humoral response, although the molecules were immunogenic in mouse model. Since the tools were successfully generated and the initial analyses were promising, further studies should be conducted, especially in other animal models, allowing a better assessment of the developed immunogens.