Synthetic dendrimer peptide vaccines against Foot-and-Mouth Disease Virus

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 24-06-202

Negatively charged amino acids at the foot-and-mouth disease virus capsid reduce the virion-destabilizing effect of viral RNA at acidic pH

Elucidation of the molecular basis of the stability of foot-and-mouth disease virus (FMDV) particles is relevant to understand key aspects of the virus cycle. Residue N17D in VP1, located at the capsid inner surface, modulates the resistance of FMDV virion to dissociation and inactivation at acidic pH. Here we have studied whether the virion-stabilizing effect of amino acid substitution VP1 N17D may be mediated by the alteration of electrostatic charge at this position and/or the presence of the viral RNA. Substitutions that either introduced a positive charge (R,K) or preserved neutrality (A) at position VP1 17 led to increased sensitivity of virions to inactivation at acidic pH, while replacement by negatively charged residues (D,E) increased the resistance of virions to acidic pH. The role in virion stability of viral RNA was addressed using FMDV empty capsids that have a virtually unchanged structure compared to the capsid in the RNA-filled virion, but that are considerably more resistant to acidic pH than WT virions, supporting a virion-destabilizing effect of the RNA. Remarkably, no differences were observed in the resistance to dissociation at acidic pH between the WT empty capsids and those harboring replacement N17D. Thus, the virion-destabilizing effect of viral RNA at acidic pH can be partially restored by introducing negatively charged residues at position VP1 N17Work in F.S´s laboratory was funded by grants from MINECO-FEDER EU (AGL2017–84097-C2–1-R), Comunidad de Madrid co-fnanced with ECFEDER funds (P2018/BAA-4370). Work in M.G.M.´s laboratory was funded by grants from MINECO-FEDER EU (BIO2015–69928-R and RTI2018–096635-B-I00). Work by both groups was also funded by an institutional grant from Fundación Ramón Areces M.G.M. is an associate member of the Institute for Biocomputation and Physics of Complex Systems, Zaragoza, Spai

Equine rhinitis A virus mutants with altered acid resistance unveil a key role of VP3 and intrasubunit interactions in the control of the pH stability of the Aphthovirus capsid

Equine rhinitis A virus (ERAV) is a picornavirus associated with respiratory disease in horses and is genetically closely related to foot-and-mouth disease virus (FMDV), the prototype aphthovirus. ERAV has recently gained interest as an FMDV alternative for the study of aphthovirus biology, including cell entry and uncoating or antiviral testing. As described for FMDV, current data support that acidic pH inside cellular endosomes triggers ERAV uncoating. In order to provide further insights into aphthovirus uncoating mechanism, we have isolated a panel of ERAV mutants with altered acid sensitivity and that differed on their degree of sensitivity to the inhibition of endosome acidification. These results provide functional evidence of the involvement of acidic pH on ERAV uncoating within endosomes. Remarkably, all amino acid substitutions found in acid-labile or acid-resistant ERAVs were located in the capsid protein VP3, indicating that this protein plays a pivotal role for the control of pH stability of the ERAV capsid. Moreover, all amino acid substitutions mapped at the intraprotomer interface between VP3 and VP2 or between VP3 and the N terminus of VP1. These results expand our knowledge on the regions that regulate the acid stability of aphthovirus capsid and should be taken into account when using ERAV as a surrogate of FMDV.Spanish grants BIO2011-24351, AGL2014-52395-C2-01, and S2013/ABI-2906-PLATESA (Comunidad Autónoma de Madrid) (to F.S.), and AGL2014-56518-JIN (to M.A.M.-A.). Ministerio de Economia y CompetitividadFundación Ramón ArecesPeer Reviewe

Inhibition of porcine viruses by different cell-targeted antiviral drugs

Antiviral compounds targeting cellular metabolism instead of virus components have become an interesting issue for preventing and controlling the spread of virus infection, either as sole treatment or as a complement of vaccination. Some of these compounds are involved in the control of lipid metabolism and/or membrane rearrangements. Here, we describe the effect of three of these cell-targeting antivirals: lauryl gallate (LG), valproic acid (VPA), and cerulenin (CRL) in the multiplication of viruses causing important porcine diseases. The results confirm the antiviral action in cultured cells of LG against African swine fever virus (ASFV), foot and mouth disease virus (FMDV), vesicular stomatitis virus (VSV), and swine vesicular disease virus (SVDV), as well as the inhibitory effect of VPA and CRL on ASFV infection. Other gallate esters have been also assayed for their inhibition of FMDV growth. The combined action of these antivirals has been also tested in ASFV infections, with some synergistic effects when LG and VPA were co-administered. Regarding the mode of action of the antivirals, experiments on the effect of the time of its addition in infected cell cultures indicated that the inhibition by VPA and CRL occurred at early times after ASFV infection, while LG inhibited a late step in FMDV infection. In all the cases, the presence of the antiviral reduced or abolished the induction of virus-specific proteins. Interestingly, LG also reduced mortality and FMDV load in a mouse model. The possible use of cell-targeted antivirals against porcine diseases is discussed.Spanish Ministerio de Ciencia e Innovación (2011-20E112), Ministerio de Economía y Competitividad (AGL2014-52395-C2-1-R and AGL2017-84097-C2-1-R), the European Community’s Seventh Framework Programme under grant agreement 311931-ASForce, Comunidad de Madrid co-financed with ECFEDER funds (S20149/ABI-2906-PLATESA; P2018/BAA-4370-PLATESA-CM), and by institutional grants from the Fundación Ramón Areces and Banco Santander Universidade

Protection of a single dose west nile virus recombinant subviral particle vaccine against lineage 1 or 2 strains and analysis of the cross-reactivity with usutu virus

© 2014 Merino-Ramos et al. West Nile virus (WNV) is a neurovirulent mosquito-borne flavivirus. High WNV virulence was mainly associated with lineage 1 strains, but recent outbreaks have unveiled circulation of highly virulent lineage 2 strains. Co-expression of flavivirus prM and E glycoproteins drives the assembly of recombinant subviral particles (RSPs) that share antigenic features with virions. Mouse immunization with lineage 1 WNV RSPs induced a potent humoral response against WNV with production of neutralizing antibodies. A single inoculation of RSPs formulated with Al(OH)3as adjuvant protected mice against a lethal challenge with WNV strains from lineage 1 or 2. The cross-reactivity of the response elicited by these RSPs was analyzed against the related flavivirus Usutu virus (USUV), which shares multiple ecological and antigenic features with WNV. Immunization with WNV-RSPs increased specific, although low, antibody titers found upon subsequent USUV infection. Copyright:Fundación Ramón ArecesPeer Reviewe

Uso de ésteres derivados del ácido gálico como antivirales

La invención hace referencia a un nuevo uso de ésteres derivados del ácido gálico, en particular, galato de laurilo, sus derivados, tautómeros y/o solvatos farmacéuticamente aceptables, como agentes antivirales en el tratamiento y/o prevención de infecciones por el virus de la fiebre aftosa (VFA). Además, la presente invención hace referencia a composiciones que comprenden dichos compuestos. [ES]The invention relates to a new use of esters derived from gallic acid, in particular lauryl gallate, its pharmaceutically acceptable solvates, derivatives and/or tautomers, as antiviral agents in the treatment and/or prevention of infections caused by the foot-and-mouth disease virus (FMDV). In addition, the invention relates to compositions containing said compounds. [EN]Peer reviewedConsejo Superior de Investigaciones Científicas (España)A1 Solicitud de patente con informe sobre el estado de la técnic

Adaptive value of foot-and-mouth disease virus capsid substitutions with opposite effects on particle acid stability

Foot-and-mouth disease virus (FMDV) is a picornavirus that exhibits an extremely acid sensitive capsid. This acid lability is directly related to its mechanism of uncoating triggered by acidification inside cellular endosomes. Using a collection of FMDV mutants we have systematically analyzed the relationship between acid stability and the requirement for acidic endosomes using ammonium chloride (NH4Cl), an inhibitor of endosome acidification. A FMDV mutant carrying two substitutions with opposite effects on acid-stability (VP3 A116V that reduces acid stability, and VP1 N17D that increases acid stability) displayed a rapid shift towards acid lability that resulted in increased resistance to NH4Cl as well as to concanamicyn A, a different lysosomotropic agent. This resistance could be explained by a higher ability of the mutant populations to produce NH4Cl-resistant variants, as supported by their tendency to accumulate mutations related to NH4Cl-resistance that was higher than that of the WT populations. Competition experiments also indicated that the combination of both amino acid substitutions promoted an increase of viral fitness that likely contributed to NH4Cl resistance. This study provides novel evidences supporting that the combination of mutations in a viral capsid can result in compensatory effects that lead to fitness gain, and facilitate space to an inhibitor of acid-dependent uncoating. Thus, although drug-resistant variants usually exhibit a reduction in viral fitness, our results indicate that compensatory mutations that restore this reduction in fitness can promote emergence of resistance mutantsComunidad de Madrid co-financed with ECFEDER funds (P2018/BAA-4370). Work in F.S.’s laboratory was also funded by Grants from MINECO/MICINN-FEDER EU (AGL2017-84097-C2-1-R; PID2020-113184RB-C21), and through the Spanish National Reserarch Council (CSIC), grant CONEXIONES-Vida/202120E47. Work in E.D.´s laboratory was also funded by MICIN/-FEDE

A single dose of dendrimer B2T peptide vaccine partially protects pigs against foot-and-mouth disease virus infection

Foot-and-mouth disease virus (FMDV) causes a highly contagious disease of cloven-hoofed animals whose control relies on efficient vaccination. We have reported that dendrimer peptide B2T, with two copies of FMDV B-cell epitope VP1 (136-154) linked through maleimide units to T-cell epitope 3A (21-35)], elicits potent B- and T-cell specific responses and confers solid protection in pigs to type-O FMDV challenge after two doses of peptide. Herein we now show that B2T evokes specific protective immune responses after administration of a single dose of either 2 or 0.5 mg of peptide. High titers of ELISA and neutralizing antibodies against FMDV were detectable at day 15 post-immunization. Likewise, activated T cells and induced IFN-γ response to in vitro recall with FMDV peptides were also detected by the same day. Further, in 70% of B2T-vaccinated pigs, full protection-no clinical signs of disease-was observed upon virus challenge at day 25 post-immunization. These results strengthen the potential of B2T as a safe, cost-effective candidate vaccine conferring adequate protection against FMDV with a single dose. The finding is particularly relevant to emergency scenarios permitting only a single shot immunization.This work was supported by the Spanish Ministry of Science, Innovation and Universities (grant AGL2017-89097-C2 to FS and DA; AGL2016-76445-R to EB), Comunidad de Madrid co-financed with ECFEDER funds (S2013/ABI-2906-PLATESA and P2018/BAA-4370 to FS and EB) and Generalitat de Catalunya (2009SGR492 to DA). Work at Centro de Biología Molecular “Severo Ochoa” and at UPF was supported by Fundación Ramón Areces and by the Maria de Maeztu Program of the Spanish Ministry of Science, Innovation and Universities, respectively. R.C.-A. and M.F. were holders of a PhD fellowship from the Spanish Ministry of Science, Innovation and University (FPI programme). Article processing free of charge

Immunogenicity of foot-and-mouth disease virus dendrimer peptides: need for a T-cell epitope and ability to elicit heterotypic responses

An approach based on a dendrimer display of B- and T-cell epitopes relevant for antibody induction has been shown to be effective as a foot-and-mouth disease (FMD) vaccine. B2T dendrimers combining two copies of the major FMD virus (FMDV) type O B-cell epitope (capsid proteinVP1 (140-158)) covalently linked to a heterotypic T-cell epitope from non-structural protein 3A (21-35), henceforth B2T-3A, has previously been shown to elicit high neutralizing antibody (nAb) titers and IFN-γ-producing cells in both mice and pigs. Here, we provide evidence that the B- and T-cell epitopes need to be tethered to a single molecular platform for successful T-cell help, leading to efficient nAb induction in mice. In addition, mice immunized with a non-covalent mixture of B2T-3A dendrimers containing the B-cell epitopes of FMDV types O and C induced similarly high nAb levels against both serotypes, opening the way for a multivalent vaccine platform against a variety of serologically different FMDVs. These findings are relevant for the design of vaccine strategies based on B- and T-cell epitope combinations.This research was funded by the Spanish Ministry of Science, Innovation and Universities (grants AGL2014-48923-C2 and AGL2017-84097-C2-2-R (to D.A. and F.S), and PID2019-107145RB-100 (to E.B.)), as well as by Comunidad de Madrid co-financed ECFEDER funds (S2013/ABI-350 2906-PLATESA and P2018/BAA-4370 to F.S. and E.B.), and by Generalitat de Catalunya (2009SGR492 to D.A.). Work at Centro de Biología Molecular “Severo Ochoa” and at UPF was supported by the Fundación Ramón Areces and by the Maria de Maeztu Program of the Spanish Ministry of Science, Innovation and Universities, respectively. M.F. and R.C.-A. were holders of a PhD fellowship from the Spanish Ministry of Science, Innovation and Universities

Peptide-based vaccines: foot-and-mouth disease virus, a paradigm in animal health

Vaccines are considered one of the greatest global health achievements, improving the welfare of society by saving lives and substantially reducing the burden of infectious diseases. However, few vaccines are fully effective, for reasons ranging from intrinsic limitations to more contingent shortcomings related, e.g., to cold chain transport, handling and storage. In this context, subunit vaccines where the essential antigenic traits (but not the entire pathogen) are presented in rationally designed fashion have emerged as an attractive alternative to conventional ones. In particular, this includes the option of fully synthetic peptide vaccines able to mimic well-defined B- and T-cell epitopes from the infectious agent and to induce protection against it. Although, in general, linear peptides have been associated to low immunogenicity and partial protection, there are several strategies to address such issues. In this review, we report the progress towards the development of peptide-based vaccines against foot-and-mouth disease (FMD) a highly transmissible, economically devastating animal disease. Starting from preliminary experiments using single linear B-cell epitopes, recent research has led to more complex and successful second-generation vaccines featuring peptide dendrimers containing multiple copies of B- and T-cell epitopes against FMD virus or classical swine fever virus (CSFV). The usefulness of this strategy to prevent other animal and human diseases is discussed.This research was funded by the European Commission (project H2020-FETOPEN-2018-2019-2020-01 n° 828774), by the Spanish Ministry of Science, Innovation and Universities, grants AGL2014-48923-C2 and AGL2017-84097-C2-2-R (to D.A. and F.S.), and AGL2016-349 76445-R (to E.B.), as well as by Comunidad de Madrid co-financed ECFEDER funds (S2013/ABI-350 2906-PLATESA and P2018/BAA-4370 to F.S. and E.B.), and by Generalitat de Catalunya (2009SGR492 to D.A.). Work at Centro de Biología Molecular “Severo Ochoa” and at UPF was supported by Fundación Ramón Areces and by the Maria de Maeztu Program of the Spanish Ministry of Science, Innovation and Universities, respectively. M.F. and R.C.-A. were holders of a PhD fellowship from the Spanish Ministry of Science, Innovation and Universities