IC-Tagging: plataforma universal para la producción de microesferas proteicas para aplicaciones biotecnológicas

La metodología del IC-Tagging se basa en el uso de una proteína, muNS-Mi, que por sí sola es capaz de formar estructuras esféricas en las células (microesferas, MS) que son fácilmente purificables. Además podemos etiquetar cualquier proteína con un tag denominado IC con el cual provocamos que se modifique la localización de forma que las proteínas etiquetadas quedan incluidas en las microesferas. En este estudio se utilizó este sistema para el desarrollo de un sensor de retrotranslocación de proteínas del retículo endoplasmático (RE). Para esto adaptamos el reensamblaje de fragmentos de EGFP al sistema IC-Tagging donde los resultados obtenidos mostraron que sería un sensor poco eficaz. También adaptamos el sistema al interior del retículo endoplasmático para intentar el desarrollo de vacunas contra virus envueltos, produciendo y purificando MS formadas en el interior del RE cargadas con glicoproteínas. Para intentar expresar proteínas difíciles, adaptamos el sistema para expresar y purificar proteínas en bacterias. Por último intentamos resolver la estructura de la proteína muNS-Mi y descubrir como las proteínas etiquetadas se distribuyen en las mismas

IC-Tagging methodology applied to the expression of viral glycoproteins and the difficult-to-express membrane-bound IGRP autoantigen

We have previously developed a methodology to produce protein microspheres (MS) that can be loaded with proteins of interest in living cells through their C or N-terminal tagging with the so-called IC-Tag. The IC-Tagging method has many applications ranging from the production of immobilized enzymes for industrial use to the production of subunit vaccines due to its intrinsic adjuvancy. Here we show the adaptation of the IC-Tagging to work inside the endoplasmic reticulum and bacteria, allowing us to produce properly modified viral glycoproteins. Additionally, we were able to express the Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), whose expression remained elusive to date possibly due to its toxicity when over-expressed. IGRP is an antigen of enormous pharmaceutical interest as it is specifically targeted during the autoimmune response taking place in both the Non-Obese Diabetic (NOD) mice and type 1 diabetes (T1D) patients leading to the destruction of insulin-producing beta cellsThis work was financed by the Spanish Ministerio de Economía y Competitividad, grant BFU2013-43513-R. Financial support from the Xunta de Galicia (Centro singular de investigación de Galicia accreditation 2016–2019) and the European Union (European Regional Development Fund - ERDF), is gratefully acknowledged. Irene Lostalé-Seijo was a recipient of a predoctoral FPU fellowship (Ministerio de Educación y Ciencia) and a Research Fellowship (Bolsa de Investigación; Deputación Provincial da Coruña)S

Nanoparticle- and Microparticle-Based Vaccines against Orbiviruses of Veterinary Importance

Bluetongue virus (BTV) and African horse sickness virus (AHSV) are widespread arboviruses that cause important economic losses in the livestock and equine industries, respectively. In addition to these, another arthropod-transmitted orbivirus known as epizootic hemorrhagic disease virus (EHDV) entails a major threat as there is a conducive landscape that nurtures its emergence in non-endemic countries. To date, only vaccinations with live attenuated or inactivated vaccines permit the control of these three viral diseases, although important drawbacks, e.g., low safety profile and effectiveness, and lack of DIVA (differentiation of infected from vaccinated animals) properties, constrain their usage as prophylactic measures. Moreover, a substantial number of serotypes of BTV, AHSV and EHDV have been described, with poor induction of cross-protective immune responses among serotypes. In the context of next-generation vaccine development, antigen delivery systems based on nano- or microparticles have gathered significant attention during the last few decades. A diversity of technologies, such as virus-like particles or self-assembled protein complexes, have been implemented for vaccine design against these viruses. In this work, we offer a comprehensive review of the nano- and microparticulated vaccine candidates against these three relevant orbiviruses. Additionally, we also review an innovative technology for antigen delivery based on the avian reovirus nonstructural protein muNS and we explore the prospective functionality of the nonstructural protein NS1 nanotubules as a BTV-based delivery platform

Cross-protective immune responses against African horse sickness virus after vaccination with protein NS1 delivered by avian reovirus muNS microspheres and modified vaccinia virus Ankara

8 Pág.African horse sickness virus (AHSV) is an insect-borne pathogen that causes acute disease in horses and other equids. In an effort to improve the safety of currently available vaccines and to acquire new knowledge about the determinants of AHSV immunogenicity, new generation vaccines are being developed. In this work we have generated and tested a novel immunization approach comprised of nonstructural protein 1 (NS1) of AHSV serotype 4 (AHSV-4) incorporated into avian reovirus muNS protein microspheres (MS-NS1) and/or expressed using recombinant modified vaccinia virus Ankara vector (MVA-NS1). The protection conferred against AHSV by a homologous MS-NS1 or heterologous MS-NS1 and MVA-NS1 prime/boost was evaluated in IFNAR (-/-) mice. Our results indicate that immunization based on MS-NS1 and MVA-NS1 afforded complete protection against the infection with homologous AHSV-4. Moreover, priming with MS-NS1 and boost vaccination with MVA-NS1 (MS-MVA-NS1) triggered NS1 specific cytotoxic CD8 + T cells and prevented AHSV disease in IFNAR (-/-) mice after challenge with heterologous serotype AHSV-9. Cross-protective immune responses are highly important since AHS can be caused by nine different serotypes, which means that a universal polyvalent vaccination would need to induce protective immunity against all serotypes.This work was supported by grants from the Ministerio de Ciencia, Innovación y Universidades, Spain (AGL-2014-57430-R, AGL2017-82570-R and BFU2013-43513-R). The Xunta de Galicia [Centro singular de investigación de Galicia accreditation 2016-2019 (ED431G/09) and ED431B 2018/04], and the European Regional Development Fund (ERDF) are gratefully acknowledged. We thank Rebeca Menaya for excellent technical assistance.Peer reviewe