Miguel Rodríguez Pulido

Attenuated Foot-and-Mouth Disease Virus RNA Carrying a Deletion in the 3′ Noncoding Region Can Elicit Immunity in Swine

ABSTRACT We constructed foot-and-mouth disease virus (FMDV) mutants bearing independent deletions of the two stem-loop structures predicted in the 3′ noncoding region of viral RNA, SL1 and SL2, respectively. Deletion of SL2 was lethal for viral infectivity in cultured cells, while deletion of SL1 resulted in viruses with slower growth kinetics and downregulated replication associated with impaired negative-strand RNA synthesis. With the aim of exploring the potential of an RNA-based vaccine against foot-and-mouth disease using attenuated viral genomes, full-length chimeric O1K/C-S8 RNAs were first inoculated into pigs. Our results show that FMDV viral transcripts could generate infectious virus and induce disease in swine. In contrast, RNAs carrying the ΔSL1 mutation on an FMDV O1K genome were innocuous for pigs but elicited a specific immune response including both humoral and cellular responses. A single inoculation with 500 μg of RNA was able to induce a neutralizing antibody response. This response could be further boosted by a second RNA injection. The presence of the ΔSL1 mutation was confirmed in viruses isolated from serum samples of RNA-inoculated pigs or after transfection and five passages in cell culture. These findings suggest that deletion of SL1 might contribute to FMDV attenuation in swine and support the potential of RNA technology for the design of new FMDV vaccines.

Tolerance to mutations in the foot-and-mouth disease virus integrin-binding RGD region is different in cultured cells and in vivo and depends on the capsid sequence context.

Engineered RNAs carrying substitutions in the integrin receptor-binding Arg-Gly-Asp (RGD) region of foot-and-mouth disease virus (FMDV) were constructed (aa 141-147 of VP1 capsid protein) and their infectivity was assayed in cultured cells and suckling mice. The effect of these changes was studied in the capsid proteins of two FMDVs, C-S8c1, which enters cells through integrins, and 213hs(-), a derivative highly adapted to cell culture whose ability to infect cells using the glycosaminoglycan heparan sulfate (HS) as receptor, acquired by multiple passage on BHK-21 cells, has been abolished. The capsid sequence context determined infectivity in cultured cells and directed the selection of additional replacements in structural proteins. Interestingly, a viral population derived from a C-S8c1/L144A mutant, carrying only three substitutions in the capsid, was able to expand tropism to wild-type (wt) and mutant (mt) glycosaminoglycan-deficient CHO cells. In contrast, the 213hs(-) capsid tolerated all substitutions analysed with no additional mutations, and the viruses recovered maintained the ability of the 213hs(-) parental virus to infect wt and mt CHO cells. Viruses derived from C-S8c1 with atypical RGD regions were virulent and transmissible for mice with no other changes in the capsid. Substitution of Asp143 for Ala in the C-S8c1 capsid eliminated infectivity in cultured cells and mice. Co-inoculation with a neutralizing monoclonal antibody directed against the type C FMDV RGD region abolished infectivity of C-S8c1 virus on suckling mice, suggesting that FMDV can infect mice using integrins. Sequence requirements imposed for viral entry in vitro and in vivo are discussed.

Combined administration of synthetic RNA and a conventional vaccine improves immune responses and protection against foot-and-mouth disease virus in swine

Abstract Foot‐and‐mouth disease virus (FMDV) is the causative agent of a highly contagious disease and a major concern in animal health worldwide. We have previously reported the use of RNA transcripts mimicking structural domains in the non‐coding regions of the FMDV RNA as potent type‐I interferon (IFN) inducers showing antiviral effect in vivo, as well as their immunomodulatory properties in combination with an FMD vaccine in mice. Here, we describe the enhancing effect of RNA delivery on the immunogenicity and protection induced by a suboptimal dose of a conventional FMD vaccine in pigs. Animals receiving the RNA developed earlier and higher levels of neutralizing antibodies against homologous and heterologous isolates, compared to those immunized with the vaccine alone, and had higher anti‐FMDV titers at late times post‐vaccination. RNA delivery also induced higher specific T‐cell response and protection levels against FMDV challenge. Peripheral blood mononuclear cells from pigs inoculated with RNA and the vaccine had a higher IFN‐&ggr; specific response than those from pigs receiving the vaccine alone. When challenged with FMDV, all three animals immunized with the conventional vaccine developed antibodies to the non‐structural viral proteins 3ABC and two of them developed severe signs of disease. In the group receiving the vaccine together with the RNA, two pigs were fully protected while one showed delayed and mild signs of disease. Our results support the immunomodulatory effect of these RNA molecules in natural hosts and suggest their potential use for improvement of FMD vaccines strategies. HighlightsSynthetic RNA is biologically active in swine at a single dose.Combinatorial RNA‐vaccine treatment induced higher B‐ and T‐cell specific responses.FMDV IRES enhanced wider antigenic spectrum neutralizing responses.RNA delivery increased protection induced by suboptimal vaccination against viral challenge.

Molecular Mechanisms of Foot-and-Mouth Disease Virus Targeting the Host Antiviral Response

Foot-and-mouth disease virus (FMDV) is the causative agent of an acute vesicular disease affecting pigs, cattle and other domestic, and wild animals worldwide. The aim of the host interferon (IFN) response is to limit viral replication and spread. Detection of the viral genome and products by specialized cellular sensors initiates a signaling cascade that leads to a rapid antiviral response involving the secretion of type I- and type III-IFNs and other antiviral cytokines with antiproliferative and immunomodulatory functions. During co-evolution with their hosts, viruses have acquired strategies to actively counteract host antiviral responses and the balance between innate response and viral antagonism may determine the outcome of disease and pathogenesis. FMDV proteases Lpro and 3C have been found to antagonize the host IFN response by a repertoire of mechanisms. Moreover, the putative role of other viral proteins in IFN antagonism is being recently unveiled, uncovering sophisticated immune evasion strategies different to those reported to date for other members of the Picornaviridae family. Here, we review the interplay between antiviral responses induced by FMDV infection and viral countermeasures to block them. Research on strategies used by viruses to modulate immunity will provide insights into the function of host pathways involved in defense against pathogens and will also lead to development of new therapeutic strategies to fight virus infections.

RNA immunization can protect mice against foot-and-mouth disease virus.

In previous work we have reported the immunization of swine using in vitro-transcribed foot-and-mouth disease virus (FMDV) RNA. With the aim of testing whether RNA-induced immunization can mediate protection against viral infection, a group of Swiss adult mice was inoculated with FMDV infectious transcripts. In most inoculated animals viral RNA was detected in serum at 48-72h postinoculation. A group of the RNA-inoculated mice (11 out of 19) developed significant titers of neutralizing antibodies against FMDV. Among those animals that were successfully challenged with infectious virus (15 out of 19), three out of the eight animals immunized upon RNA inoculation were protected, as infectious virus could not be isolated from sera but specific anti-FMDV antibodies could be readily detected. These results suggest the potential of the inoculation of genetically engineered FMDV RNA for virulence and protection assays in the murine model and allow to explore the suitability of RNA-based FMDV vaccination in natural host animals.

Innate immune sensor LGP2 is cleaved by the Leader protease of foot-and-mouth disease virus

The RNA helicase LGP2 (Laboratory of Genetics and Physiology 2) is a non-signaling member of the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), whose pivotal role on innate immune responses against RNA viruses is being increasingly uncovered. LGP2 is known to work in synergy with melanoma differentiation-associated gene 5 (MDA5) to promote the antiviral response induced by picornavirus infection. Here, we describe the activity of the foot-and-mouth disease virus (FMDV) Leader protease (Lpro) targeting LGP2 for cleavage. When LGP2 and Lpro were co-expressed, cleavage products were observed in an Lpro dose-dependent manner while co-expression with a catalytically inactive Lpro mutant had no effect on LGP2 levels or pattern. We further show that Lpro localizes and immunoprecipitates with LGP2 in transfected cells supporting their interaction within the cytoplasm. Evidence of LGP2 proteolysis was also detected during FMDV infection. Moreover, the inhibitory effect of LGP2 overexpression on FMDV growth observed was reverted when Lpro was co-expressed, concomitant with lower levels of IFN-β mRNA and antiviral activity in those cells. The Lpro target site in LGP2 was identified as an RGRAR sequence in a conserved helicase motif whose replacement to EGEAE abrogated LGP2 cleavage by Lpro. Taken together, these data suggest that LGP2 cleavage by the Leader protease of aphthoviruses may represent a novel antagonistic mechanism for immune evasion.

Use of RNA Domains in the Viral Genome as Innate Immunity Inducers for Antiviral Strategies and Vaccine Improvement

This chapter will focus on the role of innate immunity induction on antiviral responses with an emphasis on nucleic acids as type-I interferon (IFN) inducers and their use as antiviral compounds and vaccine adjuvants. A general and up-to-date view of the different mechanisms operating in the host cell for sensing viral genomes will be given, as well as viral strategies counteracting this response through immune evasion or specifically targeted antagonism. Our own recent data describing the ability to induce IFN and mediate protection against viral infection in vivo of synthetic RNA transcripts enclosing structural domains present in the 5 ́and 3 ́-terminal regions of the foot-and-mouth disease virus (FMDV) genome will be sum‐ marized and discussed in this context. New vaccine formulations including innate immunity inducers are being developed for improvement of current vaccines. The potential of exogenous nucleic acids as modulators of immune response outcomes and vaccine adjuvants will be reviewed and discussed. A schematic summary of the interrelated topics addressed in this chapter is shown in Figure 1. Additionally, a glossary of all the acronyms and abbreviations used in the text and figures is shown in Table 1.

Synthetic RNA derived from the foot-and-mouth disease virus genome elicits antiviral responses in bovine and porcine cells through IRF3 activation

Foot-and-mouth disease virus (FMDV) is the causative agent of a highly transmissible disease affecting wild and domestic animals including pigs, cattle and sheep. The ability of synthetic RNA transcripts mimicking distinct domains in the non-coding regions of the FMDV genome (ncRNAs) to induce a potent innate immune response in swine cultured cells and mice has been previously described, as well as their enhancing effect on conventional inactivated FMD vaccines. Here, we provide evidence of the activation of interferon regulatory factor 3 (IRF3), a key transcriptional regulator of type I interferon (IFN)-dependent immune responses after transfection of swine and bovine cells with transcripts corresponding to the FMDV 3´ non-coding region (3´NCR). Induction of IFN-β and Mx1expression, concomitantly with antiviral activity and IRF3 activation was observed in bovine MDBK cells transfected with the 3´NCR. Our results link the stimulation of the innate immune response observed in 3´NCR-transfected cells to the intracellular type I IFN signaling pathway and suggest the potential use of these molecules for antiviral strategies in cattle.