Veterinary Immunology and Immunopathology 128 (2009) 205210

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Veterinary Immunology and Immunopathology

journal homepage: www.elsevier.com/locate/vetimm

Research paper

Innate immune responses against foot-and-mouth disease virus: Current understanding and future directions
Artur Summereld *, Laurence Guzylack-Piriou, Lisa Harwood, Kenneth C. McCullough
usern, Switzerland Institute of Virology and Immunoprophylaxis, Sensemattstrasse 293, 3147 Mittelha

A R T I C L E I N F O

A B S T R A C T

Keywords: Foot-and-mouth disease virus Innate immunity Dendritic cells Interferon

Foot-and-mouth disease (FMD) represents one of the most economically important diseases of farm animals. The basis for the threat caused by this virus is the high speed of replication, short incubation time, high contagiousness, and high mutation rate resulting in constant antigenic changes. Thus, although protective immune responses against FMD virus (FMDV) can be efcacious, the rapidity of virus replication and spread can outpace immune defence development and overrun the immune system. FMDV can also evade innate immune responses through its ability to shut down cellular protein synthesis, including IFN type I, in susceptible epithelial cells. This is important for virus evolution, as FMDV is quite sensitive to the action of IFN. Despite this, innate immune responses are probably induced in vivo, although detailed studies on this subject are lacking. Accordingly, this interaction of FMDV with cells of the innate immune system is of particular interest. Dendritic cells (DC) can be infected by FMDV and support viral RNA replication, and viral protein synthesis but the latter is inefcient or abortive, leading most often to incomplete replication and progeny virus release. As a result DC can be activated, and particularly in the case of plasmacytoid DC (pDC), this is manifest in terms of IFN-a release. Our current state of knowledge on innate immune responses induced by FMDV is still only at a relatively early stage of understanding. As we progress, the investigations in this area will help to improve the design of current vaccines and the development of novel control strategies against FMD. 2008 Elsevier B.V. All rights reserved.

1. Introduction Foot-and-mouth disease virus (FMDV) represents one of the most devastating viruses affecting cloven-hoofed livestock including cattle, sheep, goats and pigs. The disease is characterized by high fever and the formation of vesiclesin particular on tongue, lips, coronary band, teats and udder, as well as between and above the claws of the feet. However, this depends on the species affected, with ruminants in particular sheep also showing subclinical or asymptomatic infections (Bachrach, 1968; Alexandersen et al., 2001). The threat of this virus is caused by a

* Corresponding author. Tel.: +41 31 848 9 377; fax: +41 31 848 9 222. E-mail address: artur.summereld@ivi.admin.ch (A. Summereld). 0165-2427/$ see front matter 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2008.10.296

combination of virological characteristics including high contagiousness, virus tenacity, rapidity of replication, high level of virus excretion via aerosols, and short incubation times. Other important characteristics contributing to the difculty in combating this disease are high antigenic variation with a high mutation rate and quasispecies nature of viral isolates (reviewed in Domingo et al., 2005a,b). In a population of susceptible animals FMDV can spread like wildre when not fought quickly and efciently. Seven serotypes of FMDV A, O, C, Asia 1 and South African Territories (SAT) 1, 2 and 3 have been identied (Bachrach, 1968). The virus is a single-stranded, plus-sense RNA virus belonging to the Aphthovirus genus of the Picornaviridae family. Its genome of $8500 bases encodes for a single open reading frame giving rise to a polyprotein,

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co- and post-translationally processed into in individual viral proteins. The virion has an icosahedral capsid, 28 30 nm in diameter, comprising 60 copies each of four structural virus proteins VP1, VP2, VP3 and VP4. In addition to these structural proteins, the viral genome encodes seven non-structural proteins. FMDV infects and replicates efciently in epithelial cells. Infection occurs via av integrins, in particular avb3 and avb6 interacting with a highly conserved Arg-Gly-Asp (RGD) sequence found on the VP1 protein (Pfaff et al., 1988; Leippert et al., 1997). Viral uptake depends on clathrin-mediated endocytosis followed by rapid acidication resulting in capsid disassembly and release of the viral genome into the cytoplasm through an unknown process (Berryman et al., 2005; ODonnell et al., 2005; MartinAcebes et al., 2007). Replication of the genome and virion assembly occur in the infected cell cytoplasm, and release of progeny virus is believed to occur solely by cell lysis. The details of the molecular and cell biological characteristics of this replication are reviewed elsewhere (Mason et al., 2003; Grubman and Baxt, 2004). With respect to innate immune responses it is important to stress the activity of the viral leader protein Lpro, which efciently shuts down host protein synthesis through its capacity to cleave the eIF4G, a factor essential for CAP-dependent translation. As a result of this, infected epithelial cells will have difculties to mount antiviral responses based on de novo protein synthesis. FMDV infection elicits a rapid humoral response accompanied by clearance of virusantibody complexes through phagocytic cells (McCullough et al., 1988). Protection against FMDV correlates with the induction of high levels of neutralising antibody in serum, rst detectable as early as 34 days following infection (Doel, 2005), although protection can be witnessed in certain cases when the levels of such antibodies are low or undetectable (Barnett and Carabin, 2002). In contrast to adaptive immunity, relatively little is known about the contribution of innate immune responses in controlling FMDV replication. Only recently have such studies become available, in particular in the light of developing new control strategies for emergency scenarios. Consequently this review will focus on the interaction of FMDV with the innate immune system. The aim is not only to review the current knowledge, but also to address controversial areas and gaps in the eld. 2. Virus interaction with cells of the innate immune system The data presented in this section is summarized in Table 1. 2.1. Macrophages (MF) It has been reported that FMDV infection of porcine MF is abortive and has little or no inuence on cell viability (Rigden et al., 2002). Virus-binding to the MF is less efcient than when virus is opsonised with antibody. Internalization of non-opsonised virus is also relatively slow compared to uptake of antibodyvirus complexes,

and is probably mediated through macropinocytosis (Rigden et al., 2002). Interestingly, these studies demonstrated the release of small quantities of infectious virus, but only during the rst 10 h post-infection. It is not absolutely clear whether this virus was exocytosed uptake virus or would represent progeny virus released before the abortion of virus replication. Certainly, it is possible to nd non-structural FMDV proteins and a reduction of cellular protein synthesis in infected macrophages, the latter suggesting de novo synthesis of Lpro (Rigden et al., 2002). In an earlier study, similar observations including RNA replication in macrophages were only found with antibody-opsonised FMDV (Baxt and Mason, 1995). However it is now clear that the virus can enter MF without aid from antibody, although the antibody will certainly increase the efciency and rate of uptake, through the involvement of the cellular Fc receptors (McCullough et al., 1988). 2.2. Dendritic cells (DC) Based on the central role played by DC in the induction of innate and adaptive immune responses, the importance of studying the interaction of FMDV with DC has already been addressed recently (Bayry and Tough, 2006). Despite this, there are conicting results with respect to the interaction of FMDV with DC, and all published studies are restricted to porcine and murine DC. The rst report on DC infection by FMDV was in 1995 (Gregg et al., 1995), in which cytopathic effects of the virus after infection of porcine skin DC were reported. Ten years later, Bautista et al. (2005) demonstrated that such skin DC are refractory to infection by FMDV, and that this was possibly resulting from constitutive expression of IFN type I in response to the FMDV infection (Bautista et al., 2005). In our laboratory, various subsets of porcine DC including plasmacytoid DC (pDC), monocyte-derived DC, and bone marrow-derived DC were shown to be susceptible to FMDV infection (Guzylack-Piriou et al., 2006; Harwood et al., 2008). Following infection, it was possible to detect structural and non-structural viral proteins as well as double-stranded RNA up to 24 h postinfection. In monocyte-derived DC, it was possible to detect small quantities of virus released between 2 and 8 h post-infection, only occurring in the absence of cycloheximide implying a full replicative cycle (Harwood et al., 2008). However, the characteristics of infection were not typical of those seen in susceptible cells such as epithelial cellsthe virus did not induce detectable cytopathic effects in the DC, and the infection was nally abortive in terms of both RNA and infectious progeny production. Nevertheless, the infection was not inapparent, because the porcine DC did respond in terms of phenotypic maturationincreased expression of MHC class II and CD86 (Guzylack-Piriou et al., unpublished data). A recent study demonstrated reduced IFN-alpha responses in monocyte-derived DC and skin DC generated or isolated from FMDV-infected pigs (Nfon et al., 2008). These DC were not infected indicating an indirect bystander functional modulation responsible for the suppressed responses.

A. Summereld et al. / Veterinary Immunology and Immunopathology 128 (2009) 205210 Table 1 Summary of current knowledge on the interaction of live FMDV with cells of the innate immune system in vitro. Replication CPE IFN type I Epithelial cells Porcine macrophages Porcine MoDC Porcine dermal DC Porcine Flt3L-DC Murine DC Porcine pDC NK/LAK
a b c d e f g

207

Activation/modulation
a

References Sanz-Parra et al. (1998), de Los Santos et al. (2006,2007) Mason et al. (1993), Baxt and Mason (1995), Rigden et al. (2002) Harwood et al. (2008), Nfon et al. (2008) Bautista et al. (2005) Unpublished results Ostrowski et al. (2005) Guzylack-Piriou et al. (2006) Amadori et al. (1992)

+++ Abortiveb Abortive Abortived Abortive Abortive Abortive ?

+++ /+ ?

Only mRNA Protein synthesis## ; MHCI# Protein synthesis# + MHCII/CD86"c + MHCII/CD86le ++ MHCII/CD86" ? IL-10"; MHCII/CD40#f MHCII/CD86" +++g ? Kill infected cells

Viral Lpro-mediated host protein shutoff and NFkB degradation in epithelial cells prevents translation of IFN and other genes. Fc receptor mediated enhancement if FDMV infection. Expression upregulated. Expression downregulated. Expression unchanged. No expression of viral proteins. Immune complex mediated.

Attempts to infect mouse DC with FMDV have also resulted in an abortive infection. In contrast to infection of porcine cells, the virus induced a modulation of the murine DC towards immunosuppressive DC with a downregulation of MHC class II and CD40 as well as IL-10 secretion (Ostrowski et al., 2005). Inactivated FMDV was also shown to inuence murine DC, inducing apoptosis (Jin et al., 2007). Again, this is in contrast to the porcine model in which inactivated FMDV-loaded DC remains both viable and efcient antigen presenting cells (APC) (Carrasco et al., 2004; Vincent et al., 2005; Saurer et al., 2007). Considering that pigs are a natural host of FMDV, the studies with the murine DC may be reecting a scenario peculiar to the mouse and therefore not necessarily relevant for FMDV and its natural hosts. It is possible that strain differences as well as differences with respect to receptor usage could inuence FMDVDC interaction. For example, cell culture-adapted FMDV uses heparan sulphate in preference to integrins as receptor. We have recently compared a pair of FMDV viruses differing in this respect and found that cell cultureadapted virus was more efciently taken up by the DC when compared to integrin-binding virus (Harwood et al., 2008). Other DC receptors can also play a role, particularly when specic antibody reactions come into play. Fc receptors efciently promote uptake of immune-complexed FMDV by MF (McCullough et al., 1988; Baxt and Mason, 1995; Rigden et al., 2002), as well as monocytes and pDC (Guzylack-Piriou et al., 2006) and enhance virus uptake by cell lines transfected to express Fc receptors (Mason et al., 1993). The virus is not only efciently taken up and internalized, but replication processes are also initiated, although this remains an abortive replication in MF and DC. Importantly, the consequence of this live FMDV uptake by pDC through the FcRIIg was induction of high levels of IFN-a production by the pDC (GuzylackPiriou et al., 2006). 2.3. Lymphocytes In addition to DC and MF, there are a number of other cells, which could play an important role in the control of early phases of FMD infections. Amongst them are NK or

LAK cells, which have been obtained from FMDV restimulated bovine PBMC of vaccinated cattle, and shown to have cytotoxic activity against FMDV-infected target cells (Amadori et al., 1992). In this respect the observed rapid loss of MHC class I on infected epithelial cells (Sanz-Parra et al., 1998) could represent a important trigger of NK cellmediated cytolysis. On the other hand, it is evident that this effect of the virus could also contribute to viral evasion from MHC class I-restricted cytotoxic T-lymphocytes responses. Another potentially relevant cell type is the gd T-cell, shown to proliferate and produce cytokines in response to FMDV antigen (Takamatsu et al., 2006). Innatelike CD9+ splenic B cells may also have a role to play. In the mouse model at least, these cells are responsible for an FMDV-specic thymus-independent neutralising IgM response (Ostrowski et al., 2007). However, it is unknown whether this response exists in any natural host for FMDV. While T- and B-lymphocytes are best known for their roles in adaptive immune defences against FMDV, it is likely that T-lymphocytes and NK cells can contribute to innate immune responses. This would be through the secretion of IFN-g, shown to mediate antiviral effects against FMDV (Zhang et al., 2002), acting synergistically with type I IFNs (Moraes et al., 2007). 3. Innate responses in non-immunological cells Many non-immunological cells such as epithelial cells and endothelial cells contribute to innate immune defences through the production of IFNs, chemokines and other cytokines. For example, keratinocytes produce TNF-a after FMDV infection of pigs (Ku et al., 2005). Laser microdissection in combination with quantitative RT-PCR has detected IL-1a, TNF-a, and IFN-a/b/g mRNA in epithelial cells of the bovine tongue, coronary band and dorsal soft palate after infection with FMDV (Zhang et al., 2007). Interestingly, the quantity of these mRNA levels correlated with viral RNA. Unfortunately, it was not investigated whether these cytokine mRNAs were translated into proteins, although work from our own laboratory has shown that in vitro infection of the porcine PK-15 cell line at low multiplicity of infection can result in the release of IL-6 protein (unpublished data).

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Recently, it was demonstrated that the Lpro of FMDV can also interfere with NFkB signalling and suppress the levels of cytokine and chemokine mRNA in infected epithelial cell lines (de Los Santos et al., 2007). Although FMDV induced NFkB translocation at early time points post-infection, a decrease in the levels of the p65/RelA subunit of NFkB was observed later during infection. 4. Induction of interferon type I FMDV is highly sensitive to the action of IFN type I in vitro (Ahl and Rump, 1976; Chinsangaram et al., 2001) and in vivo (Chinsangaram et al., 2003). Consequently, the IFN response is of particular relevance for innate immunity against FMDV. Most if not all viruses probably possess mechanisms for interference with the induction and/or signalling pathways of the IFN system (Weber et al., 2004). As mentioned above, the Lpro of FMDV is of central importance due to its capacity to shut down CAPdependent host protein synthesis, and thereby prevent IFN type I release (Grubman and Baxt, 2004). While both wildtype FMDV and Lpro deleted mutants induce IFN-a and IFN-b mRNA, only the Lpro deleted FMDV induces IFN proteins in infected epithelial cells (de Los Santos et al., 2006). However, the overall picture cannot be so simple there are older reports demonstrating that IFN secretion can be induced following FMDV infection of epithelial cells (Dinter and Philipson, 1962). This is particularly the case with attenuated strains of FMDV (Sellers, 1963, 1964). While shut down of host protein synthesis may be responsible for the lack of type I IFN found in infected MF, the situation in DC cannot be explained so easily. Skin DC can produce IFN type I in response to FMDV infection, albeit at low levels (Bautista et al., 2005). As mentioned above, pDC can also produce IFN in response to FMDV, in this case in large quantities (Guzylack-Piriou et al., 2006). Current investigations in our laboratory have also found that in vitro Flt3-ligand generated bone marrow-derived DC also respond to FMDV by the production of large quantities of INF-a (Guzylack-Piriou et al., in preparation). Altogether, these studies indicate that DC play a major role in the innate immune defence against FMDV, and that type I IFN is an important component therein. Moreover, this is not simply an in vitro phenomenon. IFNa and/or IFN-b mRNA has been found after experimental infection in vivo. This has been reported with mononuclear cells of the lung (Brown et al., 2000), nasalassociated lymphoid tissue (Zhang et al., 2006), and epithelial cells of the bovine tongue, coronary band and dorsal soft palate (Zhang et al., 2007). Nevertheless, it remains unclear whether this message was also translated into protein. 5. Innate immune response and emergency vaccination The current conventional high-potency vaccines can induce early protection 45 days post-vaccination (Barnett and Carabin, 2002). This has been attributed to induction of a combination of innate and early adaptive immune responses (Barnett et al., 2002; Rigden et al., 2003; Barnard

et al., 2005). The innate responses induced by the vaccine in the peripheral blood compartment included proinammatory cytokines and increased migratory and chemotactic activity, although no direct antiviral activity was found. The latter was achieved when the vaccine included an Adenovirus-based vector for expression of IFN-aunder these conditions protection against challenge infection was demonstrated as early as 1 day postvaccination (Grubman, 2005). In addition, providing IFN-g to the immune system has been shown to be benecial (Moraes et al., 2007). This cytokine has not only antiviral activity against FMDV but also promotes NK and MF activation, which are likely to contribute to control FMDV replication and spread within the host. Taken together, these studies demonstrate the importance of innate immune defences for the control of FMDV, particularly early during infection. Moreover, the results uphold the principle that targeting innate immune responses represents a promising strategy to develop improved emergency vaccines capable of rapidly establishing a protected status. Based on such ideas, IFNinducers have been tested for their capacity to promote innate protection against FMDV. Such an approach found some success in mouse models using polyinosinic:polycytidylic acid (Richmond and Hamilton, 1969) and CpG (Kamstrup et al., 2006). Unfortunately, in natural hosts of FMDV neither polyinosinic:polycytidylic acid (McVicar et al., 1973; Cunliffe et al., 1977) nor CpG (Alves et al., in preparation) application has proven successful. These disappointing experiences highlight the limited value of mouse model for FMDV vaccination/challenge studies, and demand a more elaborate study on how modulators of innate defences could be employed for delivery to and activation of the innate immune system. 6. Conclusions and open questions 6.1. Does FMDV induce potent innate immune defences in vivo? While it is evident that innate immune defences in particular the IFN system can control FMDV replication, it is still unclear to which extent such responses are induced in the course of a natural infection. The relative rapid control of viraemia during FMD and the early induction of adaptive immune responses would indicate that innate immune responses are active. However, more detailed future studies are required to characterize such responses, both in sites of FMDV replication and in immunological organs and tissue. It will be important to analyse cytokine responses particularly that of the type I IFNs at the protein level. 6.2. Which cells participate in innate immune defences against FMDV? We are only beginning to understand the contribution of individual cell types in innate immune defences, although both MF and DC, and likely NK cells, are involved. Characteristics of the various roles played by these cells, especially the different DC subsets, requires further study. Of particular importance is the additional

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complexity that will come from the interactions amongst these cell types in orchestrating the immune response. 6.3. How does FMDV interact with the ruminant immune system? Many studies on interaction of FMDV with cells of the innate immune system have been performed in porcine or murine models, and it will be important to verify these nding with ruminant cells. 6.4. What are the cell biological characteristics in innate immunity against FMDV? A better denition of the basic virological characteristics, including aspects of receptor usage, entry, cytoplasmic trafcking and localization, viral RNA replication and protein expression as well as cytopathic effects in cells of the innate immune system is required. Understanding each of these processes will be of particular importance for DC, considering the important function of these cells in innate immunity, viral clearance and antigen presentation. 6.5. What are the cellular receptors involved in sensing FMDV? Recently many new immune-sensing receptors for sensing RNA viruses have been identied, including the helicase and TLR families (Kawai and Akira, 2006). In nonimmunological cells, a candidate for sensing FMDV is the ubiquitously expressed helicase mda-5, a known sensor of another picornavirus encephaomyocarditis virus (Kato et al., 2006). For cells of the immune system in particular DC TLR3 and TLR7/8 are likely to play important roles. Both represent endosomal sensors of RNA, with TLR3 being triggered by double-stranded RNA while TLR7/8 recognizing single-stranded RNA (Kawai and Akira, 2006). 6.6. Translate the knowledge on innate immune responses into better vaccines for controlling FMD in endemic and epidemic settings It can be anticipated that detailed knowledge on FMDVinduced innate immune response will help to develop new intervention strategies. In particular, more knowledge on the contribution of interferon, cytokines, chemokines as well as cellular elements of innate antiviral responses will enable the design of improved vaccines, including those used in emergency scenarios as well as those triggering potent memory immune responses. The latter is based on the known importance of the innate responses for the development of adaptive immunity. Conict of interest None. Acknowledgements This work was supported by the State Secretariat for Education and Research grant #03.0519 linked to EU

project FP6 503603 and grant #02.003 linked to EU project QLTR-2001-00825.

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