The combined expression of the nonstructural protein NS1 and the N-terminal half of NS2 (NS21-180) by ChAdOx1 and MVA confers protection against clinical disease in sheep upon bluetongue virus challenge

26 páginas, 2 tablas, 10 figuras.Bluetongue, caused by bluetongue virus (BTV), is a widespread arthropod-borne disease of ruminants that entails a recurrent threat to the primary sector of developed and developing countries. In this work, we report modified vaccinia virus Ankara (MVA) and ChAdOx1-vectored vaccines designed to simultaneously express the immunogenic NS1 protein and/or NS2-Nt, the N-terminal half of protein NS2 (NS21-180). A single dose of MVA or ChAdOx1 expressing NS1-NS2-Nt improved the protection conferred by NS1 alone in IFNAR(-/-) mice. Moreover, mice immunized with ChAdOx1/MVA-NS1, ChAdOx1/MVA-NS2-Nt, or ChAdOx1/MVA-NS1-NS2-Nt developed strong cytotoxic CD81 T-cell responses against NS1, NS2-Nt, or both proteins and were fully protected against a lethal infection with BTV serotypes 1, 4, and 8. Furthermore, although a single immunization with ChAdOx1-NS1-NS2-Nt partially protected sheep against BTV-4, the administration of a booster dose of MVA-NS1- NS2-Nt promoted a faster viral clearance, reduction of the period and level of viremia and also protected from the pathology produced by BTV infection. IMPORTANCE Current BTV vaccines are effective but they do not allow to distinguish between vaccinated and infected animals (DIVA strategy) and are serotype specific. In this work we have develop a DIVA multiserotype vaccination strategy based on adenoviral (ChAdOx1) and MVA vaccine vectors, the most widely used in current phase I and II clinical trials, and the conserved nonstructural BTV proteins NS1 and NS2. This immunization strategy solves the major drawbacks of the current marketed vaccines.This work was supported by grants AGL2017-82570-R and PID2020-112992RR-I00 from the Spanish Ministry of Science and by the EU Horizon 2020 Program (European Commission grant agreement no. 727393-PALE-Blu). S.U.T. was a recipient of a predoctoral fellowship from the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Centro de Investigación en Sanidad Animal (program FPI-SGIT2018). We declare no competing interests. J.O. conceived the study. S.U.T. and L.J.C. wrote the manuscript with contributions from J.O. and inputs from all other authors. S.U.T., L.J.C., S.G., and A.M.L. designed and developed all the vaccines used in this work. S.U.T., L.J.C., E.C.P., G.L., and S.M. conducted mice experiments and cytometric analysis. J.B. and P.S.C. conducted sheep immunizations, postmortem studies, and histopathological analysis. S.U.T. and L.J.C. analyzed antibodies, viremias, and hematological parameters. A.N. helped revising the articlePeer reviewe

Vaccinia Virus Strain MVA Expressing a Prefusion-Stabilized SARS-CoV-2 Spike Glycoprotein Induces Robust Protection and Prevents Brain Infection in Mouse and Hamster Models

24 Pág.The COVID-19 pandemic has underscored the importance of swift responses and the necessity of dependable technologies for vaccine development. Our team previously developed a fast cloning system for the modified vaccinia virus Ankara (MVA) vaccine platform. In this study, we reported on the construction and preclinical testing of a recombinant MVA vaccine obtained using this system. We obtained recombinant MVA expressing the unmodified full-length SARS-CoV-2 spike (S) protein containing the D614G amino-acid substitution (MVA-Sdg) and a version expressing a modified S protein containing amino-acid substitutions designed to stabilize the protein a in a pre-fusion conformation (MVA-Spf). S protein expressed by MVA-Sdg was found to be expressed and was correctly processed and transported to the cell surface, where it efficiently produced cell-cell fusion. Version Spf, however, was not proteolytically processed, and despite being transported to the plasma membrane, it failed to induce cell-cell fusion. We assessed both vaccine candidates in prime-boost regimens in the susceptible transgenic K18-human angiotensin-converting enzyme 2 (K18-hACE2) in mice and in golden Syrian hamsters. Robust immunity and protection from disease was induced with either vaccine in both animal models. Remarkably, the MVA-Spf vaccine candidate produced higher levels of antibodies, a stronger T cell response, and a higher degree of protection from challenge. In addition, the level of SARS-CoV-2 in the brain of MVA-Spf inoculated mice was decreased to undetectable levels. Those results add to our current experience and range of vaccine vectors and technologies for developing a safe and effective COVID-19 vaccine.This research was funded by Instituto de Salud Carlos III, Fondo COVID-19 de proyectos de investigación sobre SARS-CoV-2 y la enfermedad COVID-19 grant COV20/00901, and grant PID2021-128466OR-I00 funded by funded by MCIN/AEI/10.13039/501100011033 as part of Plan Estatal de Investigación Científica, Desarrollo e Innovación. This research work was also funded by the European Commission—NextGenerationEU, through CSIC’s Global Health Platform (PTI Salud Global). All experiments using bioluminescent imaging approach were supported by NIH grant to WM. Research on SARS-CoV-2 in L.M-S laboratory was partially supported by the San Antonio Partnership for Precision Therapeutics, the San Antonio Medical Foundation, and the Texas Biomedical Research Institute Forum Foundation.Peer reviewe

Epizootic Hemorrhagic Disease Virus: Current Knowledge and Emerging Perspectives

27 Pág.Epizootic Hemorrhagic Disease (EHD) of ruminants is a viral pathology that has significant welfare, social, and economic implications. The causative agent, epizootic hemorrhagic disease virus (EHDV), belongs to the Orbivirus genus and leads to significant regional disease outbreaks among livestock and wildlife in North America, Asia, Africa, and Oceania, causing significant morbidity and mortality. During the past decade, this viral disease has become a real threat for countries of the Mediterranean basin, with the recent occurrence of several important outbreaks in livestock. Moreover, the European Union registered the first cases of EHDV ever detected within its territory. Competent vectors involved in viral transmission, Culicoides midges, are expanding its distribution, conceivably due to global climate change. Therefore, livestock and wild ruminants around the globe are at risk for this serious disease. This review provides an overview of current knowledge about EHDV, including changes of distribution and virulence, an examination of different animal models of disease, and a discussion about potential treatments to control the disease.J.O., G.L. and E.C.-P. are funded by grant PID2020-112992RR-I00 from the Spanish Ministry of Science. S.U.-T. and L.J.-C. were awarded grants by FPI SGIT 2018 and PRE2021 097320 funded by MCIN/AEI/10.13039/501100011033 and by “ESF Investing in your future”.Peer reviewe

Reverse genetics approaches: a novel strategy for African horse sickness virus vaccine design

9 Pág.African horse sickness (AHS) is a devastating disease caused by African horse sickness virus (AHSV) and transmitted by arthropods between its equine hosts. AHSV is endemic in sub-Saharan Africa, where polyvalent live attenuated vaccine is in use even though it is associated with safety risks. This review article summarizes and compares new strategies to generate safe and effective AHSV vaccines based on protein, virus like particles, viral vectors and reverse genetics technology. Manipulating the AHSV genome to generate synthetic viruses by means of reverse genetic systems has led to the generation of potential safe vaccine candidates that are under investigation.This work was supported by the Spanish Ministry of Science ( AGL2017-82570-R ) and EU Horizon 2020 Program (NO. 727393-PALE-Blu ). Sergio Utrilla-Trigo was a recipient of a predoctoral fellowship from the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria , Centro de Investigación en Sanidad Animal (program FPI-SGIT-2018).Peer reviewe

Mosquito Salivary Proteins and Arbovirus Infection: From Viral Enhancers to Potential Targets for Vaccines

16 Pág.Arthropod-borne viruses present important public health challenges worldwide. Viruses such as DENV, ZIKV, and WNV are of current concern due to an increasing incidence and an expanding geographic range, generating explosive outbreaks even in non-endemic areas. The clinical signs associated with infection from these arboviruses are often inapparent, mild, or nonspecific, but occasionally develop into serious complications marked by rapid onset, tremors, paralysis, hemorrhagic fever, neurological alterations, or death. They are predominately transmitted to humans through mosquito bite, during which saliva is inoculated into the skin to facilitate blood feeding. A new approach to prevent arboviral diseases has been proposed by the observation that arthropod saliva facilitates transmission of pathogens. Viruses released within mosquito saliva may more easily initiate host invasion by taking advantage of the host's innate and adaptive immune responses to saliva. This provides a rationale for creating vaccines against mosquito salivary proteins, especially because of the lack of licensed vaccines against most of these viruses. This review aims to provide an overview of the effects on the host immune response by the mosquito salivary proteins and how these phenomena alter the infection outcome for different arboviruses, recent attempts to generate mosquito salivary-based vaccines against flavivirus including DENV, ZIKV, and WNV, and the potential benefits and pitfalls that this strategy involves.This work was supported by grants from the NIH (AI126033, AI138949, AI152904) and the Steven and Alexandra Cohen Foundation. This research was also supported in part by the Howard Hughes Medical Institute Emerging Pathogens Initiative.Peer reviewe

Inhibition of Orbivirus Replication by Aurintricarboxylic Acid

14 Pág.Bluetongue virus (BTV) and African horse sickness virus (AHSV) are vector-borne viruses belonging to the Orbivirus genus, which are transmitted between hosts primarily by biting midges of the genus Culicoides. With recent BTV and AHSV outbreaks causing epidemics and important economy losses, there is a pressing need for efficacious drugs to treat and control the spread of these infections. The polyanionic aromatic compound aurintricarboxylic acid (ATA) has been shown to have a broad-spectrum antiviral activity. Here, we evaluated ATA as a potential antiviral compound against Orbivirus infections in both mammalian and insect cells. Notably, ATA was able to prevent the replication of BTV and AHSV in both cell types in a time- and concentration-dependent manner. In addition, we evaluated the effect of ATA in vivo using a mouse model of infection. ATA did not protect mice against a lethal challenge with BTV or AHSV, most probably due to the in vivo effect of ATA on immune system regulation. Overall, these results demonstrate that ATA has inhibitory activity against Orbivirus replication in vitro, but further in vivo analysis will be required before considering it as a potential therapy for future clinical evaluation.This work was supported by the EU Horizon 2020 Program (European Commission grant agreement no. 727393-PALE-Blu [to J.O.]) and the Spanish Ministry of Economy and Competitiveness (MINECO) grant AGL2014-57430-R (to J.O. and A.N.). A.N. is supported by a MINECO “Ramon y Cajal” Incorporación grant (RYC-2017). S.U-T. is supported by an INIA SGIT-FPI predoctoral fellowship.Peer reviewe

A protective bivalent vaccine against Rift Valley fever and bluetongue

12 páginas, 9 figuras.Rift Valley fever (RVF) and bluetongue (BT) are two important ruminant diseases transmitted by arthropods. Both viruses have shown important geographic spread leading to endemicity of BT virus (BTV) in Africa and Europe. In this work, we report a dual vaccine that simultaneously induces protective immune responses against BTV and RVFV based on modified vaccinia Ankara virus (MVA) expressing BTV proteins VP2, NS1, or a truncated form of NS1 (NS1-Nt), and RVFV Gn and Gc glycoproteins. IFNAR mice immunized with two doses of MVA-GnGc-VP2 developed a significant neutralizing antibody response against BTV-4 and RVFV. Furthermore, the homologous prime-boost immunization with MVA-GnGc-NS1 or MVA-GnGc-NS1-Nt triggered neutralizing antibodies against RVFV and NS1-specific cytotoxic CD8+ T cells in mice. Moreover, all mice immunized with MVA-GnGc-NS1 or MVA-GnGc-NS1-Nt remained healthy after lethal challenge with RVFV or BTV-4. The homologous prime-boost vaccination with MVA-GnGc-NS1, which was the best immunization strategy observed in mice, was assayed in sheep. Clinical signs and viremia were absent or highly reduced in vaccinated sheep after challenge with BTV-4 or RVFV. These results indicate that MVA-GnGc-NS1 vaccination elicits immune protection against RVFV and BTV in sheep.This work was supported by grants AGL-2014-57430R, AGL2017-83226R, and AGL2017-82570-R from the Spanish Ministry of Science and EU Horizon 2020 Program (European Comission Grant Agreement No. 727393-PALE-Blu). SUT was a recipient of a predoctoral fellowship from the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Centro de Investigación en Sanidad Animal (program FPI-SGIT-2018

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