Characterisation of virus-specific peripheral blood cell cytokine responses following vaccination or infection with classical swine fever viruses

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Abstract

Existing live attenuated classical swine fever virus (CSFV) vaccines provide a rapid onset of complete protection but pose problems in discriminating infected amongst vaccinated animals. With a view to providing additional information on the cellular mechanisms that may contribute to protection, which in turn may aid the development of the next generation of CSFV vaccines, we explored the kinetics of the cytokine responses from peripheral blood cells of pigs vaccinated with an attenuated C-strain vaccine strain and/or infected with a recent CSFV isolate. Peripheral blood cells were isolated over the course of vaccination/infection and stimulated in vitro with C-strain or UK2000/7.1 viruses. Virus-specific responses of peripheral blood cells isolated from C-strain vaccinated pigs were dominated by the production of IFN-γ. IFN-γ production in response to the C-strain virus was first detected in vaccinates 9 days post-vaccination and was sustained over the period of observation. In contrast, cells from challenge control animals did not secrete IFN-γ in response to stimulation with C-strain or UK2000/7.1 viruses. Supernatants from UK2000/7.1 infected animals contained significant levels of pro-inflammatory cytokines from day 8 post-infection and these cytokines were present in both virus and mock stimulated cultures. The results suggest that the C-strain virus is a potent inducer of a type-1 T cell response, which may play a role in the protection afforded by such vaccines, whereas the pro-inflammatory cytokine responses observed in cultures from infected pigs may reflect a pathological pro-inflammatory cascade initiated in vivo following the replication and spread of CSFV.

Introduction

Classical swine fever (CSF) is a devastating disease that poses one of the greatest risks to the swine industry worldwide. CSF is caused by the classical swine fever virus (CSFV), a highly contagious, small enveloped, single-stranded RNA virus that belongs to the family Flaviviridae. CSF has, since 1990, been controlled in the EU through a ‘stamping-out’ slaughter policy but the presence of a CSFV reservoir in European wild-boar populations, together with increasing public opposition against stamping-out policies, has now led to an increased likelihood that vaccination may be deployed as a last resort component of a control policy (Vandeputte and Chappuis, 1999, van Oirschot, 2003). Existing live attenuated CSFV vaccines, such as those based on lapinised or culture attenuated C-strain viruses, provide a rapid onset of complete protection but pose problems in discriminating infected amongst vaccinated animals whereas questions remain about the efficacy of available marker sub-unit vaccines for use under emergency outbreak conditions (van Oirschot, 2003).

The immunological mechanisms that underlie the rapid protection afforded by live attenuated C-strain vaccines are not well defined, however protection may precede the appearance of neutralising antibody but not IFN-γ secreting cells in peripheral blood (Suradhat et al., 2001, Suradhat and Damrongwatanapokin, 2003), suggesting that cellular immunity is responsible. A number of investigations have attempted to characterise these cellular mechanisms; both virus-specific CD4+ and CD8+ T cell IFN-γ responses occur following vaccination, with different studies suggesting prominent roles for either population (Suradhat et al., 2005). Following C-strain vaccination and challenge, CD6+CD8+ MHC class I restricted cytotoxic T lymphocyte responses, directed against the non-structural viral protein NS3, were evoked that could lyse virus-infected cells (Pauly et al., 1995). It was later demonstrated that the NS3 specific cytotoxic T lymphocytes also secrete IFN-γ (Rau et al., 2006). While IFN-γ appears to serve as a good marker for anti-CSFV cell-mediated responses, its role in the primary response to virulent CSFV is less well studied. Piriou et al. (2003) infected pigs with a sub-acute dose of CSFV that resulted in the induction of a strong IFN-γ response. Suradhat et al. (2001) reported a virus-specific IFN-γ response in pigs 8 days after a virulent CSFV infection and yet all animals died within 14 days.

Other cytokine responses induced by CSFV have been studied in vitro and in vivo but the mechanisms underlying these responses and their contribution to immunity/disease remain to be defined. In vitro infection of primary endothelial cells with virulent CSFV induces an up-regulation in the transcription of pro-inflammatory cytokines (Bensaude et al., 2004). Analyses of early cytokine responses following CSFV infection have shown that different dendritic cell types express different cytokine profiles. The dominant effect appeared to be highly elevated secretion of TNF-α and IFN-α, which could also be detected in sera, and may play a role in the disruption of immune responses (Jamin et al., 2008). In addition immunohistochemical studies have demonstrated macrophages expressing proinflammatory cytokines in both the spleen and liver of CSFV infected pigs (Sánchez-Cordón et al., 2005, Núñez et al., 2005).

With a view to providing additional information concerning the cytokine mediated mechanisms that may contribute to protection or pathology, and therefore aid the development of the next generation of CSFV vaccines, we explored the kinetics of the cytokine response from peripheral blood cells of pigs vaccinated with a highly efficacious attenuated C-strain vaccine and/or infected with a moderately virulent isolate from the UK CSF outbreak in 2000 (Sandvik et al., 2000, Everett et al., 2009).

Section snippets

Animals and viruses

‘High-health’ status Large White/Landrace cross male pigs, 8–10 weeks of age were purchased from a local commercial source. Lyophilized live attenuated Riemser C-strain CSFV (AC Reimser Schweinepestvakzine, Reimser Arzneimittel AG, Germany) was provided by the European Commission Vaccine Bank. For inoculation of pigs, the virus was reconstituted in vaccine diluent as described by the manufacturer. A UK CSFV isolate from the disease outbreak in 2000 (UK2000/7.1 Sandvik et al., 2000) was obtained

Outcome of vaccination and infection with CSFV viruses

Following infection with the CSFV isolate from a UK outbreak in 2000 (UK2000/7.1) (Experiment 1) or vaccination of pigs with the C-strain virus (Experiment 2), clinical signs were measured using a clinical score system (Fig. 1A). On infection with UK2000/7.1, pigs displayed signs of CSF from 7 to 14 days post-infection and progressed to reach clinical scores between 10 and 15 by 13–19 days post-infection. In contrast, no clinical signs were observed in any of the pigs vaccinated with the

Discussion

Experimental infection of pigs with UK2000/7.1 demonstrated this isolate to be of moderate virulence, causing high viraemia and clinical symptoms, and appears to be representative of CSFV strains causing recent outbreaks (Floegel-Niesmann et al., 2003, Everett et al., 2009). Vaccination of pigs with the C-strain virus induced no clinical signs and provided solid protection against the UK2000/7.1 virus after only 5 days. Thus, these viruses offered excellent model systems with which to analyse

Conflict of interest statement

The authors declare no conflict of interests.

Acknowledgments

We would like to thank Nicole Piontkowski, Reimser Arzneimittel AG, Germany and the European Commission for supplying the C-strain CSFV vaccine; Derek Clifford and colleagues at the Veterinary Laboratories Agency (VLA) Animal Services Unit for animal husbandry and provision of samples; Alex Nunez and Javier Salguero of the VLA Pathology Department for post-mortem dissection of lymphoid tissues; Joseph Newman and Falko Steinbach, VLA Virology Department, for statistical advice and critical

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