Javier Ortego - Academia.edu (original) (raw)

Papers by Javier Ortego

Research paper thumbnail of IFNAR(-/-) Mice Constitute a Suitable Animal Model for Epizootic Hemorrhagic Disease Virus Study and Vaccine Evaluation

International journal of biological sciences, 2024

Epizootic hemorrhagic disease (EHD), caused by Epizootic hemorrhagic disease virus (EHDV), is an ... more Epizootic hemorrhagic disease (EHD), caused by Epizootic hemorrhagic disease virus (EHDV), is an emerging and severe livestock disease. Recent incursion and distribution of EHDV in Europe have outlined the emerging character of EHD. Despite its worldwide impact, numerous knowledge gaps exist. A range of inconveniences restricts utilization of natural hosts of EHDV. Here, we show that adult mice deficient in type I IFN receptor (IFNAR(-/-)) are highly susceptible to EHDV-6 and EHDV-8 infection when the virus is administered subcutaneously. Disease was characterized by ruffled hair, reluctance to move, dehydration and conjunctivitis, with viraemia detected from day 5 post-infection. A deeper characterization of EHDV-8 infection showed viral replication in the lung, liver, spleen, kidney, testis and ovaries. Importantly, increased expression levels of pro-inflammatory cytokines IL-1β, IL-6 and CXCL2 were observed in spleen after EHDV-8 infection. Furthermore, IFNAR(-/-) adult mice immunized with a EHDV-8 inactivated vaccine elicited neutralizing antibodies specific of EHDV-8 and full protection against challenge with a lethal dose of this virus. This study also explores the possibilities of this animal model for study of BTV and EHDV coinfection. In summary, the IFNAR(-/-) mouse model faithfully recapitulates EHD and can be applied for vaccine testing, which can facilitate progress in addressing the animal health challenge posed by this virus.

Research paper thumbnail of 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

The COVID-19 pandemic has underscored the importance of swift responses and the necessity of depe... more 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 report 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 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 fus...

Research paper thumbnail of Recombinant Rift Valley fever viruses encoding bluetongue virus (BTV) antigens: Immunity and efficacy studies upon a BTV-4 challenge

PLOS Neglected Tropical Diseases

Background Many ruminant diseases of viral aetiology can be effectively prevented using appropria... more Background Many ruminant diseases of viral aetiology can be effectively prevented using appropriate vaccination measures. For diseases such as Rift Valley fever (RVF) the long inter-epizootic periods make routine vaccination programs unfeasible. Coupling RVF prophylaxis with seasonal vaccination programmes by means of multivalent vaccine platforms would help to reduce the risk of new RVF outbreaks. Methodology/Principal findings In this work we generated recombinant attenuated Rift Valley fever viruses (RVFVs) encoding in place of the virulence factor NSs either the VP2 capsid protein or a truncated form of the non-structural NS1 protein of bluetongue virus serotype 4 (BTV-4). The recombinant viruses were able to carry and express the heterologous BTV genes upon consecutive passages in cell cultures. In murine models, a single immunization was sufficient to protect mice upon RVFV challenge and to elicit a specific immune response against BTV-4 antigens that was fully protective afte...

Research paper thumbnail of Nanoparticle- and Microparticle-Based Vaccines against Orbiviruses of Veterinary Importance

Vaccines

Bluetongue virus (BTV) and African horse sickness virus (AHSV) are widespread arboviruses that ca... more 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 micro...

Research paper thumbnail of The Combined Expression of the Nonstructural Protein NS1 and the N-Terminal Half of NS2 (NS2 1-180 ) by ChAdOx1 and MVA Confers Protection against Clinical Disease in Sheep upon Bluetongue Virus Challenge

Journal of Virology, 2022

Current BTV vaccines are effective but they do not allow to distinguish between vaccinated and in... more 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.

Research paper thumbnail of A protective bivalent vaccine against Rift Valley fever and bluetongue

npj Vaccines, 2020

Rift Valley fever (RVF) and bluetongue (BT) are two important ruminant diseases transmitted by ar... more 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, whi...

Research paper thumbnail of Inhibition of Orbivirus Replication by Aurintricarboxylic Acid

International Journal of Molecular Sciences, 2020

Bluetongue virus (BTV) and African horse sickness virus (AHSV) are vector-borne viruses belonging... more 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 regulat...

Research paper thumbnail of Reverse genetics approaches: a novel strategy for African horse sickness virus vaccine design

Current Opinion in Virology, 2020

African horse sickness (AHS) is a devastating disease caused by African horse sickness virus (AHS... more 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.

Research paper thumbnail of Heterologous Combination of ChAdOx1 and MVA Vectors Expressing Protein NS1 as Vaccination Strategy to Induce Durable and Cross-Protective CD8+ T Cell Immunity to Bluetongue Virus

Vaccines, 2020

The sequence of non-structural protein NS1 of bluetongue virus (BTV), which contains immunodomina... more The sequence of non-structural protein NS1 of bluetongue virus (BTV), which contains immunodominant CD8+ T cell epitopes, is highly conserved among BTV serotypes, and has therefore become a major tool in the development of a universal BTV vaccine. In this work, we have engineered multiserotype BTV vaccine candidates based on recombinant chimpanzee adenovirus (ChAdOx1) and modified vaccinia virus Ankara (MVA) vectors expressing the NS1 protein of BTV-4 or its truncated form NS1-Nt. A single dose of ChAdOx1-NS1 or ChAdOx1-NS1-Nt induced a moderate CD8+ T cell response and protected IFNAR(-/-) mice against a lethal dose of BTV-4/MOR09, a reassortant strain between BTV-1 and BTV-4, although the animals showed low viremia after infection. Furthermore, IFNAR(-/-) mice immunized with a single dose of ChAdOx1-NS1 were protected after challenge with a lethal dose of BTV-8 in absence of viremia nor clinical signs. Additionally, the heterologous prime-boost ChAdOx1/MVA expressing NS1 or NS1-Nt...

Research paper thumbnail of MVA Vectored Vaccines Encoding Rift Valley Fever Virus Glycoproteins Protect Mice against Lethal Challenge in the Absence of Neutralizing Antibody Responses

Vaccines, 2020

In vitro neutralizing antibodies have been often correlated with protection against Rift Valley f... more In vitro neutralizing antibodies have been often correlated with protection against Rift Valley fever virus (RVFV) infection. We have reported previously that a single inoculation of sucrose-purified modified vaccinia Ankara (MVA) encoding RVFV glycoproteins (rMVAGnGc) was sufficient to induce a protective immune response in mice after a lethal RVFV challenge. Protection was related to the presence of glycoprotein specific CD8+ cells, with a low-level detection of in vitro neutralizing antibodies. In this work we extended those observations aimed to explore the role of humoral responses after MVA vaccination and to study the contribution of each glycoprotein antigen to the protective efficacy. Thus, we tested the efficacy and immune responses in BALB/c mice of recombinant MVA viruses expressing either glycoprotein Gn (rMVAGn) or Gc (rMVAGc). In the absence of serum neutralizing antibodies, our data strongly suggest that protection of vaccinated mice upon the RVFV challenge can be ac...

Research paper thumbnail of Increasing the Safety Profile of the Master Donor Live Attenuated Influenza Vaccine

Pathogens, 2020

Seasonal influenza epidemics remain one of the largest public health burdens nowadays. The best a... more Seasonal influenza epidemics remain one of the largest public health burdens nowadays. The best and most effective strategy to date in preventing influenza infection is a worldwide vaccination campaign. Currently, two vaccines are available to the public for the treatment of influenza infection, the chemically Inactivated Influenza Vaccine (IIV) and the Live Attenuated Influenza Vaccine (LAIV). However, the LAIV is not recommended for parts of the population, such as children under the age of two, immunocompromised individuals, the elderly, and pregnant adults. In order to improve the safety of the LAIV and make it available to more of the population, we sought to further attenuate the LAIV. In this study, we demonstrate that the influenza A virus (IAV) master donor virus (MDV) A/Ann Arbor/6/60 H2N2 LAIV can inhibit host gene expression using both the PA-X and NS1 proteins. Furthermore, we show that by removing PA-X, we can limit the replication of the MDV LAIV in a mouse model, whi...

Research paper thumbnail of The immunogenicity of recombinant vaccines based on modified Vaccinia Ankara (MVA) viruses expressing African horse sickness virus VP2 antigens depends on the levels of expressed VP2 protein delivered to the host

Antiviral Research, 2018

Javier (2018) The immunogenicity of recombinant vaccines based on modified Vaccinia Ankara (MVA) ... more Javier (2018) The immunogenicity of recombinant vaccines based on modified Vaccinia Ankara (MVA) viruses expressing African horse sickness virus VP2 antigens depends on the levels of expressed VP2 protein delivered to the host. Antiviral Research, 154. pp. 132-139.

Research paper thumbnail of Efficacy of different DNA and MVA prime-boost vaccination regimens against a Rift Valley fever virus (RVFV) challenge in sheep 12 weeks following vaccination

Veterinary Research, 2018

The aim of this work was to evaluate the immunogenicity and efficacy of DNA and MVA vaccines enco... more The aim of this work was to evaluate the immunogenicity and efficacy of DNA and MVA vaccines encoding the RVFV glycoproteins Gn and Gc in an ovine model of RVFV infection. Adult sheep of both sexes were challenged 12 weeks after the last immunization and clinical, virological, biochemical and immunological consequences, were analyzed. Strategies based on immunization with homologous DNA or heterologous DNA/MVA prime-boost were able to induce a rapid in vitro neutralizing antibody response as well as IFNγ production after in vitro virus specific restimulation. In these animals we observed reduced viremia levels and less clinical signs when compared with mockimmunized controls. In contrast, sheep inoculated with a homologous MVA prime-boost showed increased viremia correlating with the absence of detectable neutralizing antibody responses, despite of inducing cellular responses after the last immunization. However, faster induction of neutralizing antibodies and IFNγ production after challenge were found in this group when compared to the mock vaccinated group, indicative of a primed immune response. In conclusion, these results suggest that vaccination strategies based on DNA priming were able to mount and maintain specific anti-RVFV glycoprotein immune responses upon homologous or heterologous booster doses, warranting further optimization in large animal models of infection.

Research paper thumbnail of Modeling Arboviral Infection in Mice Lacking the Interferon Alpha/Beta Receptor

Viruses, 2019

Arboviruses are arthropod-borne viruses that exhibit worldwide distribution and are a constant th... more Arboviruses are arthropod-borne viruses that exhibit worldwide distribution and are a constant threat, not only for public health but also for wildlife, domestic animals, and even plants. To study disease pathogenesis and to develop efficient and safe therapies, the use of an appropriate animal model is a critical concern. Adult mice with gene knockouts of the interferon α/β (IFN-α/β) receptor (IFNAR(−/−)) have been described as a model of arbovirus infections. Studies with the natural hosts of these viruses are limited by financial and ethical issues, and in some cases, the need to have facilities with a biosafety level 3 with sufficient space to accommodate large animals. Moreover, the number of animals in the experiments must provide results with statistical significance. Recent advances in animal models in the last decade among other gaps in knowledge have contributed to the better understanding of arbovirus infections. A tremendous advantage of the IFNAR(−/−) mouse model is the...

Research paper thumbnail of CD8 T Cell Responses to an Immunodominant Epitope within the Nonstructural Protein NS1 Provide Wide Immunoprotection against Bluetongue Virus in IFNAR −/− Mice

Journal of Virology, 2018

Conventional vaccines have controlled or limited BTV expansion in the past, but they cannot addre... more Conventional vaccines have controlled or limited BTV expansion in the past, but they cannot address the need for cross-protection among serotypes and do not allow distinguishing between infected and vaccinated animals (DIVA strategy). There is a need to develop universal vaccines that induce effective protection against multiple BTV serotypes. In this work we have shown the importance of the nonstructural protein NS1, conserved among all the BTV serotypes, in CD8 T cell-mediated protection against multiple BTV serotypes when vectorized as a recombinant MVA vaccine.

Research paper thumbnail of A single dose of African horse sickness virus (AHSV) VP2 based vaccines provides complete clinical protection in a mouse model

Vaccine, 2018

African horse sickness is a severe, often fatal, arboviral disease of equids. The control of Afri... more African horse sickness is a severe, often fatal, arboviral disease of equids. The control of African horse sickness virus (AHSV) in endemic countries is based currently on the use of live attenuated vaccines despite some biosafety concerns derived from its biological properties. Thus, experimental vaccination platforms have been developed over the years in order to avoid the biosafety concerns associated with the use of attenuated vaccines. Various studies showed that baculovirus-expressed AHSV-VP2 or modified Vaccinia Ankara virus expressing AHSV-VP2 (MVA-VP2) induced virus neutralising antibodies and protective immunity in small animals and horses. AHSV is an antigenically diverse pathogen and immunity against AHS is serotype-specific. Therefore, AHS vaccines for use in endemic countries need to induce an immune response capable of protecting against all existing serotypes. For this reason, current live attenuated vaccines are administered as polyvalent preparations comprising combinations of AHSV attenuated strains of different serotypes. Previous studies have shown that it is possible to induce cross-reactive virus neutralising antibodies against different serotypes of AHSV by using polyvalent vaccines comprising combinations of either different serotype-specific VP2 proteins, or MVA-VP2 viruses. However, these strategies could be difficult to implement if induction of protective immunity is highly dependent on using a two-dose vaccination regime for each serotype the vaccine intends to protect against. In our study, we have tested the protective capacity of MVA-VP2 and baculovirus-expressed VP2 vaccines when a single dose was used. Groups of interferon alpha receptor knockout mice were inoculated with either MVA-VP2 or baculovirus-expressed VP2 vaccines using one dose or the standard two-dose vaccination regime. After vaccination, all four vaccinated groups were challenged with AHSV and clinical responses, lethality and viraemia compared between the groups. Our results show that complete clinical protection was achieved after a single vaccination with either MVA-VP2 or baculovirus sub-unit VP2 vaccines.

Research paper thumbnail of Microspheres-prime/rMVA-boost vaccination enhances humoral and cellular immune response in IFNAR(-/-) mice conferring protection against serotypes 1 and 4 of bluetongue virus

Antiviral research, Jun 1, 2017

Bluetongue virus (BTV) is the causative agent of bluetongue disease (BT), which affects domestic ... more Bluetongue virus (BTV) is the causative agent of bluetongue disease (BT), which affects domestic and wild ruminants. At the present, 27 different serotypes have been documented. Vaccination has been demonstrated as one of the most effective methods to avoid viral dissemination. To overcome the drawbacks associated with the use of inactivated and attenuated vaccines we engineered a new recombinant BTV vaccine candidate based on proteins VP2, VP7, and NS1 of BTV-4 that were incorporated into avian reovirus muNS-Mi microspheres (MS-VP2/VP7/NS1) and recombinant modified vaccinia virus Ankara (rMVA). The combination of these two antigen delivery systems in a heterologous prime-boost vaccination strategy generated significant levels of neutralizing antibodies in IFNAR(-/-) mice. Furthermore, this immunization strategy increased the ratio of IgG2a/IgG1 in sera, indicating an induction of a Th1 response, and elicited a CD8 T cell response. Immunized mice were protected against lethal challe...

Research paper thumbnail of Pathological Characterization Of IFNAR(-/-) Mice Infected With Bluetongue Virus Serotype 4

International Journal of Biological Sciences, 2016

Bluetongue virus (BTV) replicates in lymphoid tissues where infected mononuclear leukocytes secre... more Bluetongue virus (BTV) replicates in lymphoid tissues where infected mononuclear leukocytes secrete proinflammatory and vasoactive mediators that can contribute to bluetongue (BT) pathogenesis. Using the well-characterized IFNAR(-/-) mice animal model, we have now studied the histopathology and dynamics of leukocyte populations in different target tissues (spleen, thymus, and lung) during BTV-4 infection by histological and immunohistochemical techniques. The spleen and thymus of BTV-4 infected mice showed severe lymphoid depletion on H&E stained sections. This finding was confirmed by IHC, showing moderate decreased immunopositivity against CD3 in the thymus, and scarce immunoreactivity against CD3 and CD79 in the rest of the white pulp in the spleen, together with an increase in MAC387 immunostaining. BTV-4 infection also induced the expression of active caspase-3 in the spleen, where apoptotic debris was observed by H&E. A dramatic increase in iNOS immunoreactivity associated to necrotic areas of the white pulp was observed, being less noticeable in the thymus and the lung. The induction of pro-inflammatory cytokines in tissues where BTV replicates was evaluated by measuring transcript levels by RT-qPCR. BTV-4 infection led to enhance transcription of IFN-γ, TNF, IL-6, IL-12-p40, and IL-1β mRNA in the thymus, spleen and lung, correlating with the level of virus replication in these tissues. Disease progression and pathogenesis in IFNAR(-/-) mice closely mimics hallmarks of bluetongue disease in ruminants. IFNAR(-/-) mice are a good choice to facilitate a faster advance in the field of orbiviruses.

Research paper thumbnail of Transmissible gastroenteritis virus (TGEV)-based vectors with engineered murine tropism express the rotavirus VP7 protein and immunize mice against rotavirus

A coronavirus vector based on the genome of the porcine transmissible gastroenteritis virus (TGEV... more A coronavirus vector based on the genome of the porcine transmissible gastroenteritis virus (TGEV) expressing the rotavirus VP7 protein was constructed to immunize and protect against rotavirus infections in a murine model. The tropism of this TGEV-derived vector was modified by replacing the spike S protein with the homologous protein from mouse hepatitis virus (MHV). The rotavirus gene encoding the VP7 protein was cloned into the coronavirus cDNA. BALB/c and STAT1-deficient mice were inoculated with the recombinant viral vector rTGEV S-MHV-VP7, which replicates in the intestine and spreads to other organs such as liver, spleen and lungs. TGEV-specific antibodies were detected in all the inoculated BALB/c mice, while rotavirusspecific antibodies were found only after immunization by the intraperitoneal route. Partial protection against rotavirus-induced diarrhea was achieved in suckling BALB/c mice born to dams immunized with the recombinant virus expressing VP7 when they were orally challenged with the homotypic rotavirus strain.

Research paper thumbnail of Antiserum from mice vaccinated with modified vaccinia Ankara virus expressing African horse sickness virus (AHSV) VP2 provides protection when it is administered 48h before, or 48h after challenge

Antiviral Research, 2015

Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on ... more Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre-including this research content-immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.

Research paper thumbnail of IFNAR(-/-) Mice Constitute a Suitable Animal Model for Epizootic Hemorrhagic Disease Virus Study and Vaccine Evaluation

International journal of biological sciences, 2024

Epizootic hemorrhagic disease (EHD), caused by Epizootic hemorrhagic disease virus (EHDV), is an ... more Epizootic hemorrhagic disease (EHD), caused by Epizootic hemorrhagic disease virus (EHDV), is an emerging and severe livestock disease. Recent incursion and distribution of EHDV in Europe have outlined the emerging character of EHD. Despite its worldwide impact, numerous knowledge gaps exist. A range of inconveniences restricts utilization of natural hosts of EHDV. Here, we show that adult mice deficient in type I IFN receptor (IFNAR(-/-)) are highly susceptible to EHDV-6 and EHDV-8 infection when the virus is administered subcutaneously. Disease was characterized by ruffled hair, reluctance to move, dehydration and conjunctivitis, with viraemia detected from day 5 post-infection. A deeper characterization of EHDV-8 infection showed viral replication in the lung, liver, spleen, kidney, testis and ovaries. Importantly, increased expression levels of pro-inflammatory cytokines IL-1β, IL-6 and CXCL2 were observed in spleen after EHDV-8 infection. Furthermore, IFNAR(-/-) adult mice immunized with a EHDV-8 inactivated vaccine elicited neutralizing antibodies specific of EHDV-8 and full protection against challenge with a lethal dose of this virus. This study also explores the possibilities of this animal model for study of BTV and EHDV coinfection. In summary, the IFNAR(-/-) mouse model faithfully recapitulates EHD and can be applied for vaccine testing, which can facilitate progress in addressing the animal health challenge posed by this virus.

Research paper thumbnail of 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

The COVID-19 pandemic has underscored the importance of swift responses and the necessity of depe... more 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 report 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 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 fus...

Research paper thumbnail of Recombinant Rift Valley fever viruses encoding bluetongue virus (BTV) antigens: Immunity and efficacy studies upon a BTV-4 challenge

PLOS Neglected Tropical Diseases

Background Many ruminant diseases of viral aetiology can be effectively prevented using appropria... more Background Many ruminant diseases of viral aetiology can be effectively prevented using appropriate vaccination measures. For diseases such as Rift Valley fever (RVF) the long inter-epizootic periods make routine vaccination programs unfeasible. Coupling RVF prophylaxis with seasonal vaccination programmes by means of multivalent vaccine platforms would help to reduce the risk of new RVF outbreaks. Methodology/Principal findings In this work we generated recombinant attenuated Rift Valley fever viruses (RVFVs) encoding in place of the virulence factor NSs either the VP2 capsid protein or a truncated form of the non-structural NS1 protein of bluetongue virus serotype 4 (BTV-4). The recombinant viruses were able to carry and express the heterologous BTV genes upon consecutive passages in cell cultures. In murine models, a single immunization was sufficient to protect mice upon RVFV challenge and to elicit a specific immune response against BTV-4 antigens that was fully protective afte...

Research paper thumbnail of Nanoparticle- and Microparticle-Based Vaccines against Orbiviruses of Veterinary Importance

Vaccines

Bluetongue virus (BTV) and African horse sickness virus (AHSV) are widespread arboviruses that ca... more 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 micro...

Research paper thumbnail of The Combined Expression of the Nonstructural Protein NS1 and the N-Terminal Half of NS2 (NS2 1-180 ) by ChAdOx1 and MVA Confers Protection against Clinical Disease in Sheep upon Bluetongue Virus Challenge

Journal of Virology, 2022

Current BTV vaccines are effective but they do not allow to distinguish between vaccinated and in... more 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.

Research paper thumbnail of A protective bivalent vaccine against Rift Valley fever and bluetongue

npj Vaccines, 2020

Rift Valley fever (RVF) and bluetongue (BT) are two important ruminant diseases transmitted by ar... more 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, whi...

Research paper thumbnail of Inhibition of Orbivirus Replication by Aurintricarboxylic Acid

International Journal of Molecular Sciences, 2020

Bluetongue virus (BTV) and African horse sickness virus (AHSV) are vector-borne viruses belonging... more 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 regulat...

Research paper thumbnail of Reverse genetics approaches: a novel strategy for African horse sickness virus vaccine design

Current Opinion in Virology, 2020

African horse sickness (AHS) is a devastating disease caused by African horse sickness virus (AHS... more 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.

Research paper thumbnail of Heterologous Combination of ChAdOx1 and MVA Vectors Expressing Protein NS1 as Vaccination Strategy to Induce Durable and Cross-Protective CD8+ T Cell Immunity to Bluetongue Virus

Vaccines, 2020

The sequence of non-structural protein NS1 of bluetongue virus (BTV), which contains immunodomina... more The sequence of non-structural protein NS1 of bluetongue virus (BTV), which contains immunodominant CD8+ T cell epitopes, is highly conserved among BTV serotypes, and has therefore become a major tool in the development of a universal BTV vaccine. In this work, we have engineered multiserotype BTV vaccine candidates based on recombinant chimpanzee adenovirus (ChAdOx1) and modified vaccinia virus Ankara (MVA) vectors expressing the NS1 protein of BTV-4 or its truncated form NS1-Nt. A single dose of ChAdOx1-NS1 or ChAdOx1-NS1-Nt induced a moderate CD8+ T cell response and protected IFNAR(-/-) mice against a lethal dose of BTV-4/MOR09, a reassortant strain between BTV-1 and BTV-4, although the animals showed low viremia after infection. Furthermore, IFNAR(-/-) mice immunized with a single dose of ChAdOx1-NS1 were protected after challenge with a lethal dose of BTV-8 in absence of viremia nor clinical signs. Additionally, the heterologous prime-boost ChAdOx1/MVA expressing NS1 or NS1-Nt...

Research paper thumbnail of MVA Vectored Vaccines Encoding Rift Valley Fever Virus Glycoproteins Protect Mice against Lethal Challenge in the Absence of Neutralizing Antibody Responses

Vaccines, 2020

In vitro neutralizing antibodies have been often correlated with protection against Rift Valley f... more In vitro neutralizing antibodies have been often correlated with protection against Rift Valley fever virus (RVFV) infection. We have reported previously that a single inoculation of sucrose-purified modified vaccinia Ankara (MVA) encoding RVFV glycoproteins (rMVAGnGc) was sufficient to induce a protective immune response in mice after a lethal RVFV challenge. Protection was related to the presence of glycoprotein specific CD8+ cells, with a low-level detection of in vitro neutralizing antibodies. In this work we extended those observations aimed to explore the role of humoral responses after MVA vaccination and to study the contribution of each glycoprotein antigen to the protective efficacy. Thus, we tested the efficacy and immune responses in BALB/c mice of recombinant MVA viruses expressing either glycoprotein Gn (rMVAGn) or Gc (rMVAGc). In the absence of serum neutralizing antibodies, our data strongly suggest that protection of vaccinated mice upon the RVFV challenge can be ac...

Research paper thumbnail of Increasing the Safety Profile of the Master Donor Live Attenuated Influenza Vaccine

Pathogens, 2020

Seasonal influenza epidemics remain one of the largest public health burdens nowadays. The best a... more Seasonal influenza epidemics remain one of the largest public health burdens nowadays. The best and most effective strategy to date in preventing influenza infection is a worldwide vaccination campaign. Currently, two vaccines are available to the public for the treatment of influenza infection, the chemically Inactivated Influenza Vaccine (IIV) and the Live Attenuated Influenza Vaccine (LAIV). However, the LAIV is not recommended for parts of the population, such as children under the age of two, immunocompromised individuals, the elderly, and pregnant adults. In order to improve the safety of the LAIV and make it available to more of the population, we sought to further attenuate the LAIV. In this study, we demonstrate that the influenza A virus (IAV) master donor virus (MDV) A/Ann Arbor/6/60 H2N2 LAIV can inhibit host gene expression using both the PA-X and NS1 proteins. Furthermore, we show that by removing PA-X, we can limit the replication of the MDV LAIV in a mouse model, whi...

Research paper thumbnail of The immunogenicity of recombinant vaccines based on modified Vaccinia Ankara (MVA) viruses expressing African horse sickness virus VP2 antigens depends on the levels of expressed VP2 protein delivered to the host

Antiviral Research, 2018

Javier (2018) The immunogenicity of recombinant vaccines based on modified Vaccinia Ankara (MVA) ... more Javier (2018) The immunogenicity of recombinant vaccines based on modified Vaccinia Ankara (MVA) viruses expressing African horse sickness virus VP2 antigens depends on the levels of expressed VP2 protein delivered to the host. Antiviral Research, 154. pp. 132-139.

Research paper thumbnail of Efficacy of different DNA and MVA prime-boost vaccination regimens against a Rift Valley fever virus (RVFV) challenge in sheep 12 weeks following vaccination

Veterinary Research, 2018

The aim of this work was to evaluate the immunogenicity and efficacy of DNA and MVA vaccines enco... more The aim of this work was to evaluate the immunogenicity and efficacy of DNA and MVA vaccines encoding the RVFV glycoproteins Gn and Gc in an ovine model of RVFV infection. Adult sheep of both sexes were challenged 12 weeks after the last immunization and clinical, virological, biochemical and immunological consequences, were analyzed. Strategies based on immunization with homologous DNA or heterologous DNA/MVA prime-boost were able to induce a rapid in vitro neutralizing antibody response as well as IFNγ production after in vitro virus specific restimulation. In these animals we observed reduced viremia levels and less clinical signs when compared with mockimmunized controls. In contrast, sheep inoculated with a homologous MVA prime-boost showed increased viremia correlating with the absence of detectable neutralizing antibody responses, despite of inducing cellular responses after the last immunization. However, faster induction of neutralizing antibodies and IFNγ production after challenge were found in this group when compared to the mock vaccinated group, indicative of a primed immune response. In conclusion, these results suggest that vaccination strategies based on DNA priming were able to mount and maintain specific anti-RVFV glycoprotein immune responses upon homologous or heterologous booster doses, warranting further optimization in large animal models of infection.

Research paper thumbnail of Modeling Arboviral Infection in Mice Lacking the Interferon Alpha/Beta Receptor

Viruses, 2019

Arboviruses are arthropod-borne viruses that exhibit worldwide distribution and are a constant th... more Arboviruses are arthropod-borne viruses that exhibit worldwide distribution and are a constant threat, not only for public health but also for wildlife, domestic animals, and even plants. To study disease pathogenesis and to develop efficient and safe therapies, the use of an appropriate animal model is a critical concern. Adult mice with gene knockouts of the interferon α/β (IFN-α/β) receptor (IFNAR(−/−)) have been described as a model of arbovirus infections. Studies with the natural hosts of these viruses are limited by financial and ethical issues, and in some cases, the need to have facilities with a biosafety level 3 with sufficient space to accommodate large animals. Moreover, the number of animals in the experiments must provide results with statistical significance. Recent advances in animal models in the last decade among other gaps in knowledge have contributed to the better understanding of arbovirus infections. A tremendous advantage of the IFNAR(−/−) mouse model is the...

Research paper thumbnail of CD8 T Cell Responses to an Immunodominant Epitope within the Nonstructural Protein NS1 Provide Wide Immunoprotection against Bluetongue Virus in IFNAR −/− Mice

Journal of Virology, 2018

Conventional vaccines have controlled or limited BTV expansion in the past, but they cannot addre... more Conventional vaccines have controlled or limited BTV expansion in the past, but they cannot address the need for cross-protection among serotypes and do not allow distinguishing between infected and vaccinated animals (DIVA strategy). There is a need to develop universal vaccines that induce effective protection against multiple BTV serotypes. In this work we have shown the importance of the nonstructural protein NS1, conserved among all the BTV serotypes, in CD8 T cell-mediated protection against multiple BTV serotypes when vectorized as a recombinant MVA vaccine.

Research paper thumbnail of A single dose of African horse sickness virus (AHSV) VP2 based vaccines provides complete clinical protection in a mouse model

Vaccine, 2018

African horse sickness is a severe, often fatal, arboviral disease of equids. The control of Afri... more African horse sickness is a severe, often fatal, arboviral disease of equids. The control of African horse sickness virus (AHSV) in endemic countries is based currently on the use of live attenuated vaccines despite some biosafety concerns derived from its biological properties. Thus, experimental vaccination platforms have been developed over the years in order to avoid the biosafety concerns associated with the use of attenuated vaccines. Various studies showed that baculovirus-expressed AHSV-VP2 or modified Vaccinia Ankara virus expressing AHSV-VP2 (MVA-VP2) induced virus neutralising antibodies and protective immunity in small animals and horses. AHSV is an antigenically diverse pathogen and immunity against AHS is serotype-specific. Therefore, AHS vaccines for use in endemic countries need to induce an immune response capable of protecting against all existing serotypes. For this reason, current live attenuated vaccines are administered as polyvalent preparations comprising combinations of AHSV attenuated strains of different serotypes. Previous studies have shown that it is possible to induce cross-reactive virus neutralising antibodies against different serotypes of AHSV by using polyvalent vaccines comprising combinations of either different serotype-specific VP2 proteins, or MVA-VP2 viruses. However, these strategies could be difficult to implement if induction of protective immunity is highly dependent on using a two-dose vaccination regime for each serotype the vaccine intends to protect against. In our study, we have tested the protective capacity of MVA-VP2 and baculovirus-expressed VP2 vaccines when a single dose was used. Groups of interferon alpha receptor knockout mice were inoculated with either MVA-VP2 or baculovirus-expressed VP2 vaccines using one dose or the standard two-dose vaccination regime. After vaccination, all four vaccinated groups were challenged with AHSV and clinical responses, lethality and viraemia compared between the groups. Our results show that complete clinical protection was achieved after a single vaccination with either MVA-VP2 or baculovirus sub-unit VP2 vaccines.

Research paper thumbnail of Microspheres-prime/rMVA-boost vaccination enhances humoral and cellular immune response in IFNAR(-/-) mice conferring protection against serotypes 1 and 4 of bluetongue virus

Antiviral research, Jun 1, 2017

Bluetongue virus (BTV) is the causative agent of bluetongue disease (BT), which affects domestic ... more Bluetongue virus (BTV) is the causative agent of bluetongue disease (BT), which affects domestic and wild ruminants. At the present, 27 different serotypes have been documented. Vaccination has been demonstrated as one of the most effective methods to avoid viral dissemination. To overcome the drawbacks associated with the use of inactivated and attenuated vaccines we engineered a new recombinant BTV vaccine candidate based on proteins VP2, VP7, and NS1 of BTV-4 that were incorporated into avian reovirus muNS-Mi microspheres (MS-VP2/VP7/NS1) and recombinant modified vaccinia virus Ankara (rMVA). The combination of these two antigen delivery systems in a heterologous prime-boost vaccination strategy generated significant levels of neutralizing antibodies in IFNAR(-/-) mice. Furthermore, this immunization strategy increased the ratio of IgG2a/IgG1 in sera, indicating an induction of a Th1 response, and elicited a CD8 T cell response. Immunized mice were protected against lethal challe...

Research paper thumbnail of Pathological Characterization Of IFNAR(-/-) Mice Infected With Bluetongue Virus Serotype 4

International Journal of Biological Sciences, 2016

Bluetongue virus (BTV) replicates in lymphoid tissues where infected mononuclear leukocytes secre... more Bluetongue virus (BTV) replicates in lymphoid tissues where infected mononuclear leukocytes secrete proinflammatory and vasoactive mediators that can contribute to bluetongue (BT) pathogenesis. Using the well-characterized IFNAR(-/-) mice animal model, we have now studied the histopathology and dynamics of leukocyte populations in different target tissues (spleen, thymus, and lung) during BTV-4 infection by histological and immunohistochemical techniques. The spleen and thymus of BTV-4 infected mice showed severe lymphoid depletion on H&E stained sections. This finding was confirmed by IHC, showing moderate decreased immunopositivity against CD3 in the thymus, and scarce immunoreactivity against CD3 and CD79 in the rest of the white pulp in the spleen, together with an increase in MAC387 immunostaining. BTV-4 infection also induced the expression of active caspase-3 in the spleen, where apoptotic debris was observed by H&E. A dramatic increase in iNOS immunoreactivity associated to necrotic areas of the white pulp was observed, being less noticeable in the thymus and the lung. The induction of pro-inflammatory cytokines in tissues where BTV replicates was evaluated by measuring transcript levels by RT-qPCR. BTV-4 infection led to enhance transcription of IFN-γ, TNF, IL-6, IL-12-p40, and IL-1β mRNA in the thymus, spleen and lung, correlating with the level of virus replication in these tissues. Disease progression and pathogenesis in IFNAR(-/-) mice closely mimics hallmarks of bluetongue disease in ruminants. IFNAR(-/-) mice are a good choice to facilitate a faster advance in the field of orbiviruses.

Research paper thumbnail of Transmissible gastroenteritis virus (TGEV)-based vectors with engineered murine tropism express the rotavirus VP7 protein and immunize mice against rotavirus

A coronavirus vector based on the genome of the porcine transmissible gastroenteritis virus (TGEV... more A coronavirus vector based on the genome of the porcine transmissible gastroenteritis virus (TGEV) expressing the rotavirus VP7 protein was constructed to immunize and protect against rotavirus infections in a murine model. The tropism of this TGEV-derived vector was modified by replacing the spike S protein with the homologous protein from mouse hepatitis virus (MHV). The rotavirus gene encoding the VP7 protein was cloned into the coronavirus cDNA. BALB/c and STAT1-deficient mice were inoculated with the recombinant viral vector rTGEV S-MHV-VP7, which replicates in the intestine and spreads to other organs such as liver, spleen and lungs. TGEV-specific antibodies were detected in all the inoculated BALB/c mice, while rotavirusspecific antibodies were found only after immunization by the intraperitoneal route. Partial protection against rotavirus-induced diarrhea was achieved in suckling BALB/c mice born to dams immunized with the recombinant virus expressing VP7 when they were orally challenged with the homotypic rotavirus strain.

Research paper thumbnail of Antiserum from mice vaccinated with modified vaccinia Ankara virus expressing African horse sickness virus (AHSV) VP2 provides protection when it is administered 48h before, or 48h after challenge

Antiviral Research, 2015

Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on ... more Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre-including this research content-immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.