Biology subjects

Calvo-Pinilla, E.

Publications and source records attributed to Calvo-Pinilla, E..

4 recordsLinked to original sources

bioRxiv2026

A bivalent VP2 based vaccine approach reveals co expression driven interference between VP2 antigens impairing BTV neutralizing antibody induction

Bluetongue virus (BTV) is an important arboviral pathogen of ruminants that causes a significant impact on the primary sector. More than 29 different BTV serotypes exist, hindering control and prevention of Bluetongue (BT) disease. Conventional vaccines possess safety and efficacy limitation and do not permit to confer multiserotype protection and differentiate between vaccinated and infected animals (DIVA strategy). Here, we developed recombinant Modified Vaccinia Ankara (rMVA) viral vectors individually expressing or co-expressing the neutralizing antibody (nAb) target proteins VP2 of BTV serotypes 4 and 8. We evaluated their immunogenicity and protective efficacy in IFNAR(-/-) mice and sheep. Immunization of IFNAR(-/-) mice with the rMVA co-expressing of BTV-4 VP2 and BTV-8 VP2 led to abolished nAb induction while eliciting non neutralizing antibodies against both VP2 proteins, suggesting a previously unrecognized interference between VP2 antigens. Combined immunization of IFNAR(-/-) mice with rMVAs expressing either BTV-4 VP2 or BTV-8 VP2 elicited a strong neutralizing immune response and complete protection against lethal challenge with BTV-4 or BTV-8. In sheep, combinatorial prime-boost immunization with these monovalent rMVAs induced nAbs against both homologous BTV serotypes. Immunized sheep were fully protected against BTV-4, as no fever or viral replication were detected after challenge. Similarly, immunized sheep were also protected against BTV-8 although detectable but mild RNAemia and slight increase in rectal temperatures were observed after challenge. Overall, we identified a relevant constraint for multivalent VP2 based vaccine design and present a DIVA vaccination strategy that offers bivalent protection against BTV in a natural host of the disease.

microbiology↗

bioRxiv2025

Generation and characterization of an ovine cell line derived from peripheral blood and its potential use to study livestock and zoonotic viral infections

BackgroundMesenchymal stromal cells (MSCs) are a population of undifferentiated non-hematopoietic fibroblast-like cells isolated from several tissues with multipotent differentiation capacity in vitro. This study focused on the establishment and characterization of a mesenchymal stromal cell line derived from ovine peripheral blood mononuclear cells (PBMCs). MethodsMSC were isolated from ovine blood and used to develop a cell line. The characterization of the cell line to confirm its mesenchymal origin was carried out by different assays. First at all, cells were characterized by flow cytometry (FACS) using monoclonal antibodies specific for MSC and hematopoietic non-stromal cell surface antigens. Real-time quantitative PCR (RT-qPCR) was performed using gene primer pairs specific for hematopoietic and mesenchymal cell surface markers. In addition, we assayed the potential of the cell line to differentiate in vitro to other lineages such as osteoblasts and neurons and confirmed that by specific staining with Alizarin Red (AR) and RT-qPCR and immunofluorescence (IFA). Finally, we assayed the susceptibility of the cell line to various livestock, animal and human viruses. ResultsFACS and RT-qPCR analyses revealed that the ovine cell line expressed mesenchymal markers and was negative for hematopoietic markers assayed. MSC were able to differentiate to osteoblasts and neurons, these results were confirmed by quantitative analyses of AR staining and RT-qPCR and IFA assays. The cell line showed susceptibility to infection with all viruses assayed confirmed by IFA asssays. ConclusionsIn this work, we used isolated MSCs from peripheral blood to establish a new ovine cell line. The characterization and the osteogenic and neuronal differentiation carried out confirmed that the cell line had a mesenchymal stromal origin. In fact, the new established ovine cell line was permissive to all viruses studied in this work. The new sheep cell line could be a useful tool for isolating and characterizing viruses and studying virus-host interactions.

cell biology↗

bioRxiv2025

The hyper-attenuated RVFV 40Fp8 strain can be safely administered to pregnant ewes to protect them from a virulent challenge

In the present study, we evaluated the immunogenicity, safety and protective efficacy of the attenuated RVFV-40Fp8 strain in natural hosts (non-pregnant ewes) and in a highly susceptible host infection model such as pregnant ewes in the first third of pregnancy. Our results confirm the immunogenicity of 40Fp8 administration in non-pregnant and pregnant ewes, as well as the absence of foetal damage even after a high-dose vaccination regime in pregnant ewes. In addition, the ewes and their foetuses were protected against a virulent RVFV-56/74 strain challenge, as shown by comparative histopathological evaluation of tissue samples from vaccinated and non-vaccinated pregnant ewes. These results confirm the potential use of 40Fp8 as a RVF live-attenuated vaccine candidate and pave the way for further clinical developments.

microbiology↗

bioRxiv2022

Pharmacological elevation of cellular dihydrosphingomyelin provides a novel antiviral strategy against West Nile virus infection

Flavivirus life cycle is strictly dependent on cellular lipid metabolism. Polyphenols like gallic acid and its derivatives are promising lead compounds for new therapeutic agents as they can exert multiple pharmacological activities, including the alteration of lipid metabolism. The evaluation of our own collection of polyphenols against West Nile virus, a representative medically relevant flavivirus, led to the identification of N,N'-(dodecane-1,12-diyl)bis(3,4,5-trihydroxybenzamide) and its 2,3,4-trihydroxybenzamide regioisomer as selective antivirals with low cytotoxicity and high antiviral activity (EC50 of 2.2 and 0.24 M, respectively in Vero cells; EC50 of 2.2 and 1.9 M, respectively in SH-SY5Y cells). These polyphenols also inhibited the multiplication of other flaviviruses, namely Usutu, dengue, and Zika viruses, exhibiting lower antiviral or negligible antiviral activity against other RNA viruses. The mechanism underlying their antiviral activity against WNV involved the alteration of sphingolipid metabolism. These compounds inhibited ceramide desaturase (Des1) promoting the accumulation of dihydrosphingomyelin (dhSM), a minor component of cellular sphingolipids with important roles on membrane properties. Addition of exogenous dhSM, or Des1 blockage by using the reference inhibitor GT-11, confirmed the involvement of this pathway in WNV infection. These results unveil the potential of novel antiviral strategies based on the modulation of the cellular levels of dhSM and Des1 activity for the control of flavivirus infection.

microbiology↗