The antiviral response triggered by the cGAS/STING pathway is subverted by the foot-and-mouth disease virus proteases.

Propagation of viruses requires interaction with host factors in infected cells and repression of innate immune responses triggered by the host viral sensors. Cytosolic DNA sensing pathway of cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) is a major component of the antiviral response to DNA viruses, also known to play a relevant role in response to infection by RNA viruses, including foot-and-mouth disease virus (FMDV). Here, we provide supporting evidence of cGAS degradation in swine cells during FMDV infection and show that the two virally encoded proteases, Leader (Lpro) and 3Cpro, target cGAS for cleavage to dampen the cGAS/STING-dependent antiviral response. The specific target sequence sites on swine cGAS were identified as Q140/T141 for the FMDV 3Cpro and the KVKNNLKRQ motif at residues 322-330 for Lpro. Treatment of swine cells with inhibitors of the cGAS/STING pathway or depletion of cGAS promoted viral infection, while overexpression of a mutant cGAS defective for cGAMP synthesis, unlike wild type cGAS, failed to reduce FMDV replication. Our findings reveal a new mechanism of RNA viral antagonism of the cGAS-STING innate immune sensing pathway, based on the redundant degradation of cGAS through the concomitant proteolytic activities of two proteases encoded by an RNA virus, further proving the key role of cGAS in restricting FMDV infection.

Non-coding RNAs derived from the foot-and-mouth disease virus genome trigger broad antiviral activity against coronaviruses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of a potentially severe respiratory disease, the coronavirus disease 2019 (COVID-19), an ongoing pandemic with limited therapeutic options. Here, we assessed the anti-coronavirus activity of synthetic RNAs mimicking specific domains in the non-coding regions of the foot-and-mouth disease virus (FMDV) genome (ncRNAs). These molecules are known to exert broad-spectrum antiviral activity in cell culture, mice and pigs effectively triggering the host innate immune response. The ncRNAs showed potent antiviral activity against SARS-CoV-2 after transfection in human intestinal Caco-2 and lung epithelium Calu-3 2B4 cells. When the in vivo efficacy of the FMDV ncRNAs was assessed in K18-hACE2 mice, administration of naked ncRNA before intranasal SARS-CoV-2 infection significantly decreased the viral load and the levels of pro-inflammatory cytokines in the lungs compared with untreated infected mice. The ncRNAs were also highly efficacious when assayed against common human HCoV-229E and porcine transmissible gastroenteritis virus (TGEV) in hepatocyte-derived Huh-7 and swine testis ST cells, respectively. These results are a proof of concept of the pan-coronavirus antiviral activity of the FMDV ncRNAs including human and animal divergent coronaviruses and potentially enhance our ability to fight future emerging variants.

Use of Foot-and-Mouth Disease Virus Non-coding Synthetic RNAs as Vaccine Adjuvants.

The ncRNAs are short RNA transcripts with sequence and structure resembling that of specific domains in the non-coding regions of the foot-and-mouth disease (FMD) virus (FMDV ) genome. These synthetic molecules induce a robust antiviral response and have been shown to enhance the immune response and protection induced by an FMD inactivated vaccine in pigs. Here, we describe the method for ncRNAs synthesis, formulation, and delivery into mice and pigs for studies focused on testing the adjuvant effect of RNA-based strategies in combination with veterinarian vaccines.

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.

A Wide-Ranging Antiviral Response in Wild Boar Cells Is Triggered by Non-coding Synthetic RNAs From the Foot-and-Mouth Disease Virus Genome.

Foot-and-mouth disease virus (FMDV) is the causative agent of a highly contagious viral disease that affects multiple cloven-hooved hosts including important livestock (pigs, cattle, sheep and goats) as well as several wild animal species. Crossover of FMDV between domestic and wildlife populations may prolong virus circulation during outbreaks. The wild boar (Sus scrofa) is considered a reservoir of various pathogens that can infect other wildlife, domestic animals, and humans. As wild boar and domestic pigs are susceptible to the same pathogens and can infect each other, infected wild boar populations may represent a threat to the pig industry and to international trade. The ncRNAs are synthetic non-coding RNA transcripts, mimicking structural domains in the FMDV genome, known to exert a broad-spectrum antiviral and immunomodulatory effect in swine, bovine and mice cells. Here, we show the type I interferon-dependent, robust and broad range antiviral activity induced by the ncRNAs in a cell line derived from wild boar lung cells (WSL). Transfection of WSL cells with the ncRNAs exerted a protective effect against infection with FMDV, vesicular stomatitis virus (VSV), swine vesicular disease virus (SVDV) and African swine fever virus (ASFV). Our results prove the biological activity of the ncRNAs in cells of an FMDV wild animal host species against a variety of viruses affecting pigs, including relevant viral pathogens of epizootic risk.

Designing Functionally Versatile, Highly Immunogenic Peptide-Based Multiepitopic Vaccines against Foot-and-Mouth Disease Virus.

A broadly protective and biosafe vaccine against foot-and-mouth disease virus (FMDV) remains an unmet need in the animal health sector. We have previously reported solid protection against serotype O FMDV afforded by dendrimeric peptide structures harboring virus-specific B- and T-cell epitopes, and also shown such type of multivalent presentations to be advantageous over simple B-T-epitope linear juxtaposition. Chemically, our vaccine platforms are modular constructions readily made from specified B- and T-cell epitope precursor peptides that are conjugated in solution. With the aim of developing an improved version of our formulations to be used for on-demand vaccine applications, we evaluate in this study a novel design for epitope presentation to the immune system based on a multiple antigen peptide (MAP) containing six immunologically relevant motifs arranged in dendrimeric fashion (named B2T-TB2). Interestingly, two B2T units fused tail-to-tail into a single homodimer platform elicited higher B- and T-cell specific responses than former candidates, with immunization scores remaining stable even after 4 months. Moreover, this macromolecular assembly shows consistent immune response in swine, the natural FMDV host, at reduced dose. Thus, our versatile, immunogenic prototype can find application in the development of peptide-based vaccine candidates for various therapeutic uses using safer and more efficacious vaccination regimens.

A bivalent B-cell epitope dendrimer peptide can confer long-lasting immunity in swine against foot-and-mouth disease.

Foot-and-mouth disease virus (FMDV) causes a widely extended contagious disease of livestock. We have previously reported that a synthetic dendrimeric peptide, termed B2 T(mal), consisting of two copies of a B-cell epitope [VP1(140-158)] linked through maleimide groups to a T-cell epitope [3A(21-35)] of FMDV, elicits potent B- and T-cell-specific responses and confers solid protection in pigs to type O FMDV challenge. Longer duration of the protective response and the possibility of inducing protection after a single dose are important requirements for an efficient FMD vaccine. Herein, we show that administration of two doses of B2 T(mal) elicited high levels of specific total IgGs and neutralizing antibodies that lasted 4-5 months after the peptide boost. Additionally, concomitant levels of IFN-γ-producing specific T cells were observed. Immunization with two doses of B2 T(mal) conferred a long-lasting reduced susceptibility to FMDV infection, up to 136 days (19/20 weeks) post-boost. Remarkably, a similar duration of the protective response was achieved by a single dose of B2 T(mal). The effect on the B2 T(mal) vaccine of RNA transcripts derived from non-coding regions in the FMDV genome, known to enhance the immune response and protection induced by a conventional inactivated vaccine, was also analysed. The contribution of our results to the development of FMD dendrimeric vaccines is discussed.

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-ß 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.

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-ß 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.

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.

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

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-γ 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.

Synthetic RNAs Mimicking Structural Domains in the Foot-and-Mouth Disease Virus Genome Elicit a Broad Innate Immune Response in Porcine Cells Triggered by RIG-I and TLR Activation.

The innate immune system is the first line of defense against viral infections. Exploiting innate responses for antiviral, therapeutic and vaccine adjuvation strategies is being extensively explored. We have previously described, the ability of small in vitro RNA transcripts, mimicking the sequence and structure of different domains in the non-coding regions of the foot-and-mouth disease virus (FMDV) genome (ncRNAs), to trigger a potent and rapid innate immune response. These synthetic non-infectious molecules have proved to have a broad-range antiviral activity and to enhance the immunogenicity of an FMD inactivated vaccine in mice. Here, we have studied the involvement of pattern-recognition receptors (PRRs) in the ncRNA-induced innate response and analyzed the antiviral and cytokine profiles elicited in swine cultured cells, as well as peripheral blood mononuclear cells (PBMCs).

Protection against Rift Valley fever virus infection in mice upon administration of interferon-inducing RNA transcripts from the FMDV genome.

In this work we have addressed the effect of synthetic, non-infectious, RNA transcripts, mimicking structural domains of the non-coding regions (NCRs) of the foot-and-mouth disease virus (FMDV) genome on the infection of mice with Rift Valley fever virus (RVFV). Groups of 5 mice were inoculated intraperitoneally (i.p.) with 200 µg of synthetic RNA resembling the 5'-terminal S region, the internal ribosome entry site (IRES) or the 3'-NCR of the FMDV genome. RNA inoculation was performed 24h before (-24 h), 24 h after (+24 h) or simultaneously to the challenge with a lethal dose of RVFV. Administration of the IRES RNA afforded higher survival rates than administration of S or 3'NCR transcripts either at -24h or +24h after challenge. In contrast, when RNA inoculation and viral challenge were performed simultaneously, all mice survived in both IRES- and 3'NCR-inoculated groups, with an 80% survival in mice receiving the S RNA. Among survivors, a complete correlation between significant anti-RVFV circulating antibody titers and resistance to a second lethal challenge with the virus was observed, supporting a limited viral replication in the RNA-inoculated animals upon the first challenge. All three RNA transcripts were able to induce the production of systemic antiviral and pro-inflammatory cytokines. These data show that triggering of intracellular pathogen sensing pathways constitutes a promising approach towards development of novel RVF preventive or therapeutic strategies.

Delivery of synthetic RNA can enhance the immunogenicity of vaccines against foot-and-mouth disease virus (FMDV) in mice.

We have recently described the antiviral effect in mice of in vitro-transcribed RNAs mimicking structural domains in the non-coding regions of the foot-and-mouth disease virus (FMDV) genome RNA. These small, synthetic and non-infectious RNA molecules (ncRNAs) are potent type-I interferon (IFN) inducers in vivo. In this work, the immunomodulatory effect of the ncRNA corresponding to the internal ribosome entry site (IRES) on immunization with two different FMD vaccine formulations, both based on inactivated virus, including or not a commercial adjuvant, was analyzed in the mice model. The effect of the time interval between RNA inoculation and immunization was also studied. RNA delivery consistently increased the titers of specific anti-FMDV antibodies, including neutralizing antibodies, elicited after vaccination. Moreover, at day 2 after immunization, significant differences in mean antibody titers could be detected between the groups of mice receiving either vaccine co-administered with the RNA and the control group, unlike those immunized with the vaccine alone. When vaccinated mice were challenged with FMDV, the mean values of viral load were lower in the groups receiving the RNA together with the vaccine. Our results show the enhancing effect of the IRES RNA on the immune response elicited after vaccination and suggest the potential of this molecule as an adjuvant for new FMD vaccine design.

Protection against West Nile virus infection in mice after inoculation with type I interferon-inducing RNA transcripts.

West Nile virus (WNV) is a neurovirulent single stranded RNA mosquito-borne flavivirus, whose main natural hosts are birds, but it also infects humans and horses. Nowadays, no human vaccine is commercially available and clinical treatment is only supportive. Recently, it has been shown that RNA transcripts, mimicking structural domains in the non-coding regions (NCRs) of the foot-and mouth disease virus (FMDV) induce a potent IFN response and antiviral activity in transfected cultured cells, and also reduced mice susceptibility to FMDV. By using different transcripts combinations, administration schedules, and infecting routes and doses, we have demonstrated that these FMDV RNA transcripts protect suckling and adult mice against lethal challenge with WNV. The protective activity induced by the transcripts was systemic and dependent on the infection route and dose. These results confirm the antiviral potential of these synthetic RNAs for fighting viruses of different families relevant for human and animal health.

Modulation of foot-and-mouth disease virus pH threshold for uncoating correlates with differential sensitivity to inhibition of cellular Rab GTPases and decreases infectivity in vivo.

The role of cellular Rab GTPases that govern traffic between different endosome populations was analysed on foot-and-mouth disease virus (FMDV) infection. Changes of viral receptor specificity did not alter Rab5 requirement for infection. However, a correlation between uncoating pH and requirement of Rab5 for infection was observed. A mutant FMDV with less acidic uncoating pH threshold was less sensitive to inhibition of Rab5, whereas another mutant with more acidic requirements was more sensitive to inhibition of Rab5. On the contrary, opposed correlations between uncoating pH and dependence of Rab function were observed upon expression of dominant-negative forms of Rab7 or 11. Modulation of uncoating pH also reduced FMDV virulence in suckling mice. These results are consistent with FMDV uncoating inside early endosomes and indicate that displacements from optimum pH for uncoating reduce viral fitness in vivo.

Mutations that hamper dimerization of foot-and-mouth disease virus 3A protein are detrimental for infectivity.

Foot-and-mouth disease virus (FMDV) nonstructural protein 3A plays important roles in virus replication, virulence, and host range. In other picornaviruses, homodimerization of 3A has been shown to be relevant for its biological activity. In this work, FMDV 3A homodimerization was evidenced by an in situ protein fluorescent ligation assay. A molecular model of the FMDV 3A protein, derived from the nuclear magnetic resonance (NMR) structure of the poliovirus 3A protein, predicted a hydrophobic interface spanning residues 25 to 44 as the main determinant for 3A dimerization. Replacements L38E and L41E, involving charge acquisition at residues predicted to contribute to the hydrophobic interface, reduced the dimerization signal in the protein ligation assay and prevented the detection of dimer/multimer species in both transiently expressed 3A proteins and in synthetic peptides reproducing the N terminus of 3A. These replacements also led to production of infective viruses that replaced the acidic residues introduced (E) by nonpolar amino acids, indicating that preservation of the hydrophobic interface is essential for virus replication. Replacements that favored (Q44R) or impaired (Q44D) the polar interactions predicted between residues Q44 and D32 did not abolish dimer formation of transiently expressed 3A, indicating that these interactions are not critical for 3A dimerization. Nevertheless, while Q44R led to recovery of viruses that maintained the mutation, Q44D resulted in selection of infective viruses with substitution D44E with acidic charge but with structural features similar to those of the parental virus, suggesting that Q44 is involved in functions other than 3A dimerization.

Inoculation of newborn mice with non-coding regions of foot-and-mouth disease virus RNA can induce a rapid, solid and wide-range protection against viral infection.

We have recently described the ability of in vitro-transcribed RNAs, mimicking structural domains in the 5' and 3' non-coding regions (NCRs) of the foot-and-mouth disease virus (FMDV) genome, to trigger the innate immune response in porcine cultured cells and mice. In this work, the antiviral effect exerted in vivo by these small synthetic non-infectious RNA molecules was analyzed extensively. The susceptibility of transfected newborn Swiss mice to FMDV challenge was tested using a wide range of viral doses. The level of protection depended on the specific RNA inoculated and was dose-dependent. The RNA giving the best protection was the internal ribosome entry site (IRES), followed by the transcripts corresponding to the S fragment. The time course of resistance to FMDV of the RNA-transfected mice was studied. Our results show the efficacy of these RNAs to prevent viral infection as well as to contain ongoing FMDV infection in certain time intervals. Protection proved to be independent of the serotype of FMDV used for challenge. These results support the potential use of the FMDV NCR transcripts as both prophylactic and therapeutic molecules for new FMDV control strategies.

RNA structural domains in noncoding regions of the foot-and-mouth disease virus genome trigger innate immunity in porcine cells and mice.

The induction of type I interferons (alpha/beta interferon [IFN-α/ß]) in response to viral infection is a crucial step leading to the antiviral state in the host. Viruses produce double-stranded RNA (dsDNA) during their replication cycle that is sensed as nonself by host cells through different receptors. A signaling cascade then is activated to block viral replication and spread. Foot-and-mouth disease virus (FMDV) is a picornavirus that is highly sensitive to IFN, and it causes one of the world's most important animal diseases. In this study, we showed the ability of structural domains predicted to enclose stable dsRNA regions in the 5'- and 3'-noncoding regions (NCRs) of the FMDV genome to trigger an IFN-α/ß response in porcine kidney cultured cells and newborn mice. These RNAs, generated by in vitro transcription, were able to stimulate IFN-ß transcription and induce an antiviral state in SK-6 cells. The induction levels elicited by the different NCR RNAs were compared. Among them, the 3'NCR was identified as a potent IFN activator, and the features in this region involved in signaling have been analyzed. To address whether the FMDV NCR transcripts were able to trigger the innate immune response in vivo, Swiss suckling mice were inoculated intraperitoneally with the RNAs. All transcripts induced the innate response in transfected animals, measured as IFN-α/ß protein levels, antiviral activity in sera, and reduced susceptibility to FMDV infection. Our work provides new insight into innate responses against FMDV and identifies these small noninfectious RNA molecules as potential adjuvants for vaccine improvement and antiviral strategies against picornaviruses.

Attenuated foot-and-mouth disease virus RNA carrying a deletion in the 3' noncoding region can elicit immunity in swine.

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 DeltaSL1 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 microg 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 DeltaSL1 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.