Simon P Graham
email: s.graham@vla.defra.gsi.gov.uk

Helen E Everett

Helen L Johns

Felicity J Haines

S Anna

La Rocca

Meenakshi Khatri

Ian K Wright

Trevor Drew

Helen R Crooke

Characterisation of virus-specific peripheral blood cell cytokine responses following

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

A c c e p t e d M a n u s c r i p t 2 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 proinflammatory 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. 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 [START_REF] Vandeputte | Classical swine fever: the European experience and a guide for infected areas[END_REF][START_REF] Van Oirschot | Vaccinology of classical swine fever: from lab to field[END_REF]. 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 [START_REF] Van Oirschot | Vaccinology of classical swine fever: from lab to field[END_REF].

Keywords

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 [START_REF] Suradhat | The correlation of virus-specific interferon-gamma production and protection against classical swine fever virus infection[END_REF]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 Cstrain 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 A c c e p t e d M a n u s c r i p t 4 demonstrated that the NS3 specific cytotoxic T lymphocytes also secrete IFN-γ [START_REF] Rau | Immunological properties of recombinant classical swine fever virus NS3 protein in vitro and in vivo[END_REF]. While IFN-γ appears to serve as a good marker for anti-CSFV cellmediated responses, its role in the primary response to virulent CSFV is less well studied. [START_REF] Piriou | Humoral and cell-mediated immune responses of d/d histocompatible pigs against classical swine fever (CSF) virus[END_REF] infected pigs with a sub-acute dose of CSFV that resulted in the induction of a strong IFN-γ response. [START_REF] Suradhat | The correlation of virus-specific interferon-gamma production and protection against classical swine fever virus infection[END_REF] 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 [START_REF] Bensaude | Classical swine fever virus induces proinflammatory cytokines and tissue factor expression and inhibits apoptosis and interferon synthesis during the establishment of long-term infection of porcine vascular endothelial cells[END_REF]. 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 [START_REF] Jamin | Classical swine fever virus induces activation of plasmacytoid and conventional dendritic cells in tonsil, blood, and spleen of infected pigs[END_REF]. In addition immunohistochemical studies have demonstrated macrophages expressing proinflammatory cytokines in both the spleen and liver of CSFV infected pigs (Sanchez-Cordon et al., 2005, Nunez 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 A c c e p t e d M a n u s c r i p t 5 virulent isolate from the UK CSF outbreak in 2000 [START_REF] Sandvik | CSF virus in East Anglia: where from?[END_REF]Everett et al., this issue).

A c c e p t e d M a n u s c r i p t 6

Materials and methods

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 CSFV isolate from the disease outbreak in 2000 (UK2000/7.1 [START_REF] Sandvik | CSF virus in East Anglia: where from?[END_REF] was obtained from the Mammalian Virus Investigation Unit (VLA-Weybridge, UK) (Everett et al., this issue). Additional virus stocks of both UK2000/7.1 and C-strain were prepared for in vitro stimulation following inoculation of sub-confluent PK-15 cell monolayers and virus titres determined as previously described [START_REF] Drew | Classical swine fever (hog cholera). In: OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (mammals, birds and bees)[END_REF].

Vaccination and infection with CSFV

In two independent experiments, pigs were inoculated intra-nasally with 10 5.3 TCID 50 (2ml divided equally between each nostril) of the UK2000/7.1 CSFV isolate (n=6) (Expt. 1) or intramuscularly with 100PD 50 (1ml; <100 TCID 50 ) of the attenuated C-strain of CSFV (n=4) (Expt. 2). For each experiment, two uninfected control animals were housed in a separate pen from the vaccinated/infected animals. In a third experiment, pigs were vaccinated with the C-strain virus as described above (n=6) and then challenged with the UK2000/7.1 CSFV isolate after 5 days, a group of unvaccinated pigs (n=4) were infected in parallel (Expt. 3). All work was carried out

Clinical, haematological and virological monitoring

All animals were monitored daily from pre-vaccination/infection through to day 15-21 post-vaccination/infection. Animals were euthanized by lethal injection of anaesthetic when clinical scores reached or approached 15. Clinical scores were recorded twice daily by animal technicians and a veterinarian with 10 clinical parameters scored on a scale of 0-3 (Everett et al., this issue). Leukopenia was monitored every 2-3 days by flow cytometry staining. EDTA blood (50µl) was stained with 10µl of FITC conjugated anti-porcine CD45 (AbD Serotec, Kidlington, UK) for 15 min at room temperature. Erythrocytes were lysed and leukocytes fixed by addition of 940µl of FACS Lysing Solution (BD Biosciences, Oxford, UK) and incubation for 10 min at room temperature. An aliquot of cell suspension was analyzed on a MACSQuant flow cytometer (Miltenyi Biotec, Gergisch Gladbach, Germany) and leukocyte counts obtained by gating on FITC positive events. Viral RNA was detected in EDTA blood samples collected every 2-3 days; total RNA was extracted from 140µl of blood using QIAmp Virus RNA Mini Extraction kits as described by the manufacturer (Qiagen, Crawley, UK) and CSFV viral copy numbers assessed by quantitative RT-PCR (Hoffman et al., 2005. Everett et al., this issue).

In vitro stimulation of peripheral blood and lymphoid cells

Heparinised blood was collected from pigs every 2-3 days post vaccination/infection. For Experiment 1, peripheral blood mononuclear cells (PBMC) were prepared by layering heparinised blood diluted 1:1 in HBSS over an equal A c c e p t e d M a n u s c r i p t 8 volume of Biocoll Separating Solution (Autogen Bioclear, Caine, UK) and centrifuged at 2500rpm for 30 min at RT. PBMC were aspirated from the plasma-Biocoll interface and washed three times in HBSS (Invitrogen, Paisley, UK) by centrifugation at 1500 rpm for 5 min at RT. For Experiments 1 and 3, buffy coat cells were prepared using a standard protocol. In brief, blood was centrifuged at 2500rpm for 10 min and visible buffy coat material aspirated. Contaminating erythrocytes were lysed by addition of 10ml of Pharmlyse Buffer (BD Biosciences) and incubation for 10 min at RT before being washed three times in HBSS.

At the termination of the Expt. 2, necropsies were carried out on the vaccinated pigs and samples of the following lymphoid associated tissues were obtained: bone marrow, spleen, tonsil, mandibular lymph node, inguinal lymph node, mesenteric lymph node and thymus. The tissues were chopped and teased in PBS in a petri-dish to liberate cells and a single cell suspension was obtained by passing the suspension through a 70um cell sieve (BD Biosciences). Contaminating erythrocytes were lysed and cells washed as described for the buffy coat cells. Isolated buffy coat, PBMC and lymphoid tissue cells were finally resuspended in RPMI-1640 medium supplemented with 10% FCS and antibiotics (Invitrogen).

Cell densities were determined by analysing 50µl of cell suspension using a MACSQuant flow cytometer and gating on events with typical SSC and FSC properties for buffy coat and PBMC. Cell densities were adjusted to 5x10 6 cells/ml and 100µl or 1ml transferred to wells of a 96 well round-bottom or 24 well plate, respectively. Cells were stimulated by the addition of an equal volume of medium containing C-strain or UK2000/7.1 CSFV at a multiplicity of infection (MOI) of 1 or 0.1. To serve as a negative control, a mock inoculum prepared from uninfected PK-15 A c c e p t e d M a n u s c r i p t 9 cells was added in an equivalent volume to the viruses. Plates were then incubated for 24-72 hours at 37 o C in a humidified 5% CO 2 atmosphere.

Measurement of cytokines in culture supernatants

After incubation, the contents of wells were mixed by repeated pipetting and then centrifuged at 1500rpm for 5min and cell free supernatants removed and immediately stored at -80 o C until analysed. IFN-γ was measured in the culture supernatants diluted 1:2 in Standard Diluent Buffer using a swine IFN-γ ELISA according to the manufacturers instructions (Biosource, Invitrogen) and absorbance at 440nm read using a FLUOstar OPTIMA microplate reader (BMG Labtech, Aylesbury, UK). The presence of other cytokines in culture supernatants was analysed using the Pierce Searchlight Porcine Cytokine Array (Perbio Science UK Ltd. Cramlington, UK), which simultaneously measures porcine IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IFN-γ and TNF-α. Equal volumes of culture supernatants from each group of vaccinated or infected pigs were pooled and diluted 1:2 in sample diluent before addition to the plates. Plates were incubated and developed as described by the manufacturer and analysed using the SearchLight CCD Imaging and Analysis System (Perbio Science).

Statistical analysis

ANOVA was used for the analysis of fixed effects on different traits using GraphPad Prism 5 (Prism 5 for Windows, Version 5.01, GraphPad Software, Inc. La Jolla, USA).

Outcome of vaccination and infection with CSFV viruses

Following infection with the CSFV isolate from a UK outbreak in 2000 (UK2000/7.1) (Expt. 1) or vaccination of pigs with the C-strain virus (Expt. 2), clinical signs were measured using a clinical score system (Fig. 1A). On infection with UK2000/7.1, pigs displayed signs of CSF from 7-14 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 Cstrain virus. The development of leukopenia was monitored by quantitative flow cytometry with mean leukocyte counts shown in Fig. 1B. Infection with the UK2000/7.1 virus produced a leukopenia; leukocyte counts dropped below preinfection levels after 4-8 days post-infection reaching a relatively steady state of 5-10,000 leukocyte/µl for the remaining period of observation. Leukocyte counts were unaffected by immunisation with the C-strain virus. Viral RNA was first detected in the blood of UK2000/7.1 infected pigs at 2-4 days post-infection and increased exponentially, plateauing around day 11 post-infection with viral loads of above 10 6 virus copies/µl blood (Fig. 1C). No viral RNA could be detected in the blood of Cstrain vaccinated animals, but was detected in some tonsil and retropharyngeal lymph node biopsies collected post-mortem (days 23-24 post-vaccination) (data not shown).

Challenge of C-strain vaccinated pigs 5 days post-vaccination (Expt. 3) did not result in the development of clinical signs and no virus could be detected in peripheral blood. In contrast, all challenge control animals developed severe clinical CSF requiring euthanasia and had high levels of viraemia (data not shown)

A c c e p t e d M a n u s c r i p t 11

Characterisation of virus specific IFN-γ responses by in vitro stimulation of PBL from pigs vaccinated and challenged with CSFV viruses

Following vaccination of pigs with the C-strain virus (Expt. 2), virus specific IFN-γ responses of peripheral blood leukocyte (PBL) were measured by ELISA after in vitro stimulation and compared to responses from unvaccinated pigs (Fig. 2A).

IFN-γ production in response to stimulation with C-strain virus was first detected in vaccinates 9 days post-vaccination (p<0.001). This response declined but appeared sustained over the period of observation ( 18 PBL's isolated 14 days post-challenge were stimulated with either C-strain or UK2000/7.1 viruses. Unvaccinated animals failed to mount an IFN-γ response to either virus while the vaccinated animals showed a weak response to UK2000/7.1 at an MOI=0.1 (p<0.1), which was similar to the response to the equivalent dose of Cstrain and less than the response to the C-strain virus at an MOI of 1 (p<0.01) (Fig. 2C). The presence of C-strain viral RNA in the lymphoid tissues of the nasopharyngeal region and absence in blood observed in Expt. 2 led us to examine the distribution of CSFV-specific IFN-γ secreting cells in lymphoid organs well described as key sites for viral expansion and/or persistence. From day 23-25 post-vaccination,

Assessment of additional cytokine responses by in vitro stimulation of PBL from pigs vaccinated or challenged with CSFV viruses

Further analyses of other cytokine responses revealed highly divergent responses between vaccinated and infected animals (Fig. 3). Stimulation of PBL from vaccinates with the C-strain virus (Expt. 2) induced a cytokine response that was dominated by IFN-γ (Fig. 3A &B). IL-8 production was also detected, however this response appeared non-specific as it was also observed in all cultures analysed (Fig. 3B-D). Of the other cytokines measured, there was a small increase in the secretion of IL-10 and TNF-α from day 12 post-vaccination, which appeared to be virus specific.

Analysis of PBMC culture supernatants from UK2000/7.1 infected pigs (Expt. 1) showed that UK2000/7.1 virus stimulation resulted in TNF-α, IL-6 and IL-1β being detected from day 8 post-infection (Fig. 3C). However, these cytokine responses were also observed, albeit at a slightly lower magnitude, in supernatants from mock infected cultures (Fig. 3D). These responses followed the onset of viraemia as A c c e p t e d M a n u s c r i p t 14

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., this issue). 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 cytokine responses with a view to attributing them to protection or pathology. As had been previously described [START_REF] Suradhat | The correlation of virus-specific interferon-gamma production and protection against classical swine fever virus infection[END_REF][START_REF] Suradhat | Factors critical for successful vaccination against classical swine fever in endemic areas[END_REF], C-strain vaccination induced significant IFN-γ responses from peripheral blood leukocytes following in vitro stimulation with CSFV, which peaked around day 9 and remained elevated until the termination of the experiment on day 21. Challenge of vaccinated animals induced a strong IFN-γ response and these animals completely controlled the challenge infection. In contrast, cells from unvaccinated animals infected with the UK2000/7.1 virus failed to mount a detectable IFN-γ response. It appears unlikely that this was strain related since no response could be detected against either the vaccine or challenge virus while cells from vaccinates responded to both. It therefore also appears that there are significant cross-reactive T cell epitopes between the 2 viruses of different genotypes.

The absence of an IFN-γ response following infection conflicts with the data of [START_REF] Suradhat | The correlation of virus-specific interferon-gamma production and protection against classical swine fever virus infection[END_REF] that showed an IFN-γ response from animals infected with a virulent isolate. However, the present data supports the hypothesis that virus specific IFN-γ responses provide a marker of T cell-mediated mechanisms that control CSFV.

Understanding the mechanisms by which virulent CSFV prevents the induction of this response may be of value in the efforts to develop new vaccines that aim to trigger that this depletion affects T lymphocytes (Graham, S.P., unpublished data, [START_REF] Pauly | Infection with classical swine fever virus: effects on phenotype and immune responsiveness of porcine T lymphocytes[END_REF][START_REF] Summerfield | Depletion of CD4(+) and CD8(high+) T-cells before the onset of viraemia during classical swine fever[END_REF]. The inability to prime IFN-γ secreting virusspecific T cells may be due to the depletion of cells prior to their activation or the recently described phenomenon of apoptotic cell-induced dendritic cell mediated suppression [START_REF] Williams | Apoptotic cells induce dendritic cell-mediated suppression via interferon-gamma-induced IDO[END_REF]. Alternatively, the failure to prime these responses may stem from the dysregulation of cytokine responses from infected dendritic cell populations. It has been shown that plasmacytoid dendritic cells over-express cytokines such as TNF-α and IFN-α in response to CSFV infection [START_REF] Jamin | Classical swine fever virus induces activation of plasmacytoid and conventional dendritic cells in tonsil, blood, and spleen of infected pigs[END_REF] whereas the Type I IFN response from myeloid derived cells is impaired (Carrasco et al., 2004).

The present study showed evidence of dysregulated cytokine responses in infected animals, with secretion of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 from stimulated and unstimulated cultures following the onset of viraemia and coincident with the appearance of clinical signs. It is unclear whether this proinflammatory response is largely T cell independent and simply reflects a proinflammatory cascade initiated in vivo following the dissemination of replicating virus, as has been suggested from investigations during the early stages of CSFV infection in vivo [START_REF] Jamin | Classical swine fever virus induces activation of plasmacytoid and conventional dendritic cells in tonsil, blood, and spleen of infected pigs[END_REF]. However, the pathology associated with acute CSFV infection that is attributed to a pro-inflammatory cytokine storm is often absent from animals born persistently infected due to in utero infection prior to the onset of immunocompetence [START_REF] Van Oirschot | Experimental production of congenital persistent swine fever infections I. Clinical, pathological and virological observations[END_REF]. It has previously been shown in vitro that CSFV infection of myeloid dendritic cells does not affect their T cell stimulatory capacity (Carrasco et al., 2004). It is therefore tempting to speculate that this

:

  Classical swine fever virus; Swine; Vaccination and infection; Interferonγ; Cytokines.

  UK legislation pertaining to care and use of animals under experimentation.

  days-post vaccination). No significant virus-specific IFN-γ responses were detected in non-vaccinated animals (Fig 2A). PBL IFN-γ responses of pigs vaccinated 5 days previously were similarly determined after challenge with the UK2000/7.1 strain and compared to responses from unvaccinated pigs (Fig 2B) (Expt. 3). IFN-γ responses following in vitro stimulation with C-strain virus peaked in vaccinated pigs 6 days post-challenge (11 days postvaccination) and then showed similar kinetics to that observed in the previous vaccination experiment (Fig. 2B) (p<0.001). In contrast, unvaccinated pigs failed to display virus-specific IFN-γ responses following UK2000/7.1 infection. (Fig. 2B).

  bone marrow, spleen, tonsil, blood, thymus and mandibular, inguinal and mesenteric lymph nodes were stimulated with C-strain CSFV and IFN-γ responses measured(Fig 2D). The most significant responses were obtained from blood (p<0.05) and spleen cell cultures (p<0.001). The lack of response by cells from the tonsil and lymph nodes draining the oronasal mucosae, which are likely to be the first sites of replication for an incoming challenge virus, was surprising and may be because the vaccine was administered by intramuscular inoculation rather than by the intranasal route.

  -time qRT-PCR but were coincident with the detection of CSFV infected cells in peripheral blood as assessed by flow cytometry (data not shown).

  . Both earlier studies and the present study have shown that CSFV infection results in the rapid onset of leucopoenia and it has been shown

  an adaptive component and that the delayed kinetics of the response reflects the time required for induction of an adaptive response. IL-17producing CD4 + T cells (Th-17 cells) have recently been identified as a unique subset of Th cells that develop along a pathway that is distinct from the Th1-and Th2-cells, with both IFN-γ and IL-4 negatively regulating the generation of these Th-17 cells[START_REF] Bettelli | Induction and effector functions of T(H)17 cells[END_REF]. Th-17 cells promote inflammatory responses and are implicated in a variety of pathological conditions. It could be speculated that the early cytokine responses induced following CFSV infection favours priming of a Th17 response leading to activation of granulocytes and macrophages and the resultant tissue inflammation observed in clinical CSF. Utilising a commercially available live attenuated CSFV vaccine and a moderately virulent CSFV isolate, the present study established robust models to study vaccine mediated protection and uncontrolled viral infection leading to clinical disease. These models have been exploited to demonstrate that attenuated and virulent viruses induce dramatically different cytokine responses which likely play important roles in dictating whether protective immunity or pathology develops. With a view to enhancing our understanding of the contribution of these responses, current studies are focussing on the identification of the cells secreting these cytokines and extending the panel of cytokines analysed to include cytokines such as IFN-α, TGF-β, IL-17 and IL-22, which may play an important role in the induction of the responses measured in the present study.

Fig. 1 .

 1 Figure Legends

Fig. 2 .

 2 Fig. 2. Characterisation of virus specific IFN-γ responses by in vitro stimulation of PBL from pigs following vaccination with a live attenuated C-strain CSFV and

  FIG. 1

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 review of the manuscript. This study was supported by a grant (SE0778) from the Department for the Environment, Food and Rural affairs of the United Kingdom.

 Microbiol. 92, 187-194. Suradhat, S., Sada, W., Buranapraditkun, S., Damrongwatanapokin, S., 2005. The kinetics of cytokine production and CD25 expression by porcine lymphocyte subpopulations following exposure to classical swine fever virus (CSFV). Vet.


Ilona Reimann

Klaus Depner

Katrin Utke

Immanuel Leifer

Elke Lange

Dr Martin Beer
email: martin.beer@fli.bund.de

Characterisation of a new chimeric marker vaccine candidate with a mutated antigenic E2-epitope

A c c e p t e d M a n u s c r i p t 2 Abstract A new chimeric pestivirus "CP7_E1E2alf_TLA", based on the infectious cDNA of bovine viral diarrhea virus (BVDV) strain CP7, was constructed. The substitution of BVDV E1 and E2 with the respective proteins of classical swine fever (CSF) strain Alfort 187 allows an optimal heterodimerization of E1 and E2 in the chimeric virus, which is beneficial for efficient and authentic virus assembly and growth. In addition, for implementation of E2based marker diagnostics, the previously described antigenic CSFV-specific TAVSPTTLRepitope was exchanged with the corresponding E2-epitope of BVDV strain CP7.

Recombinant virus CP7_E1E2alf_TLA displayed a growth defect, and was not reacting with monoclonal antibodies used in commercial E2 antibody blocking ELISAs. Therefore, efficacy as well as marker properties of CP7_E1E2alf_TLA were investigated in an animal experiment with both a high dose and a low dose vaccine preparation. All CP7_E1E2alf_TLA-vaccinated animals seroconverted until day 14 post challenge infection with neutralizing antibodies. Furthermore, at the day of challenge infection CP7_E1E2alf_TLA-immunised animals showed distinct lower ELISA-values in a commercial CSFV-E2-antibody test in comparison to the C-strain vaccinated controls. However, E2-ELISA reactivity as well as neutralizing titres were directly connected to the dosage used for vaccination, and only the low dose group had E2-ELISA values below threshold until challenge infection. Following challenge infection with highly virulent CSFV-strain Koslov, all vaccinees were protected, however, short-term fever episodes and very limited CSFV-genome detection with very low copy numbers could be observed. In conclusion, manipulation of the TAVSPTTLR-epitope within the tested chimeric virus resulted in an slightly reduced efficacy, but the E2-marker properties unexpectedly did not allow a clear differentiation of infected from vaccinated animals in some cases.

Keywords: Pestivirus, classical swine fever, marker vaccine, DIVA, chimeric construct Large-scale CSF outbreaks have a disastrous economic effect in countries with an industrial pig production and are associated with mass culling of animals. In addition, CSF outbreaks in domestic pigs implicate trade restrictions of pigs and pig products [START_REF] Artois | Classical swine fever (hog cholera) in wild boar in Europe[END_REF]Edwards et al., 2000). Currently a non-vaccination policy is implemented in the EU. But due to ethical, welfare and economical aspects the development of emergency vaccines, allowing the differentiation of vaccinated from infected animals, is indispensable. Furthermore, due to the risk of CSFV transmission from wild boar to domestic pigs [START_REF] Artois | Classical swine fever (hog cholera) in wild boar in Europe[END_REF], a modified live vaccine should be available which also could be used for oral vaccination of wild boar. At the moment, no licensed CSFV live vaccine features "marker properties", allowing a differentiation of vaccinated and infected animals (Differentiating Infected from Vaccinated Animals " DIVA") [START_REF] Beer | Novel marker vaccines against classical swine fever virus[END_REF]. Until now, only CSF subunit marker vaccines based on recombinant, bacculovirus-expressed envelope protein E2 are registered [START_REF] Bouma | Efficacy and stability of a subunit vaccine based on glycoprotein E2 of classical swine fever virus[END_REF]. However, the immune response after vaccination with the E2-subunit vaccine is delayed, and pigs have to be vaccinated twice for a sufficient protection (Depner et al., 2001;de Smit et al., 2001a). Facilitated by the establishment of infectious cDNA clones of classical swine fever virus (CSFV) and bovine viral diarrhea virus (BVDV) (Meyers, et al., 1996;Moorman et al., 1996;Rüggli et al., 1996;Vassilev et al., 1997) trans-complemented deletion mutants (Wjdjoatmodjo et al., 2000;Maurer et al., 2005;[START_REF] Beer | Novel marker vaccines against classical swine fever virus[END_REF] and chimeric pestiviruses [START_REF] Widjojoatmodj | Classical swine fever virus E(rns) deletion mutants: trans-complementation and potential use as nontransmissible, modified, live-attenuated marker vaccines[END_REF]Reimann et al., 2004, König et al.,2007a[START_REF] Wehrle | Chimeric pestiviruses: candidates for live-attenuated classical swine fever marker vaccines[END_REF], Rasmussen et al., 2007) are the most promising candidates for the development of new, attenuated DIVA vaccines.

Serological diagnostics of pestivirus infections are often based on the detection of antibodies directed against the non-structural protein NS3 or the envelope proteins E2 and E rns . Since A c c e p t e d M a n u s c r i p t 4 NS3-specific antibodies are pan-pestivirus specific, only E2 and E rns specific antibodies can be used for serological differentiation of different pestivirus species [START_REF] Wensvoort | Antigenic differentiation of pestivirus strains with monoclonal antibodies against hog cholera virus[END_REF][START_REF] Paton | Pestivirus diversity[END_REF], Beer et al., 2007). Due to the high sensitivity and specificity, CSFV E2-ELISAs would be the best choice for CSF DIVA diagnostics. However, the available vaccines (E2-Subunit, C-strain) are based on unchanged CSFV E2 proteins which do not enable E2-based DIVA diagnostics. Therefore, chimeric pestiviruses had been generated on the basis of CSFV and BVDV backbones, respectively. CSFV backbone chimeras are based on vaccine strain C.

Substitution of CSFV E2 and E rns with the analogous proteins of a BVDV type 2 strain, respectively, resulted in viable chimeric viruses, which protected pigs from a lethal CSFV infection and allowed either an E2 or E rns based marker diagnostics [START_REF] Widjojoatmodj | Classical swine fever virus E(rns) deletion mutants: trans-complementation and potential use as nontransmissible, modified, live-attenuated marker vaccines[END_REF], de Smit et al., 2001b). The BVDV-based chimeric pestivirus CP7_E2alf had been constructed on the basis of BVDV strain CP7 by replacing the original E2 protein with the E2 protein of CSFV Alfort 187 (Reimann et al., 2004). CP7_E2alf virus was proven to be completely avirulent and efficacious after intramuscular (i.m.) and oral immunisation of domestic pigs and wild boar (Reimann et al., 2004;König et al., 2007aKönig et al., , 2007b)). The BVDV background of CP7_E2alf allowed an E rns based serological marker diagnostics as well as a genetic DIVA diagnostics (Reimann et al., 2004;Hoffmann at al., 2005Hoffmann at al., , 2006)).

In this study, we describe a newly developed, BVDV-based chimera, expressing both CSFV E1 and E2. In order to allow E2-based in addition to E rns -based serological marker diagnostics, CSFV E2 was modified by substitution of the antigenic TAVSPTTLR-epitope (Lin et al., 2000;Liu et al., 2006) with the corresponding epitope of BVDV strain CP7. The chimeric virus was characterized in vitro, and vaccine efficacy and marker properties were investigated in an immunization and challenge experiment. Hannover, Germany) and an Alexa Fluor®488-conjugated F(ab') 2 fragment of goat antimouse IgG (Molecular Probes). For detection of the chimeric E2-protein, the monoclonal antibodies CA34, HC34 and HC/TC16 were used (kindly provided by Irene Greiser-WIlke; Institute of Virology, TiHo Hannover, Germany). Standard immunofluorescence analyses using a fluorescence microscope (IX51, Olympus) were performed as previously described [START_REF] Beer | Cytotoxic T-lymphocyte responses in cattle infected with bovine viral diarrhea virus[END_REF].

Material and Methods

Cell cultures and viruses

Polymerase chain reaction and sequencing

For PCR, a PTC-200 thermal cycler (MJ Research, Inc.) was used. DNA based amplification was done with the Expand High Fidelity PCR System (Roche Molecular Biochemicals) according to the supplier´s protocol. For RT-PCR, total RNA of virus-infected cells was extracted using the TRIZOL reagent (Invitrogen). cDNA was produced from RNA by using the OneStep RT-PCR Kit (Qiagen) with sequence-specific primers. The PCR products were directly sequenced using appropriate primers. Sequencing was carried out using a Big Dye® Terminator v1.1 Cycle sequencing Kit (Applied Biosystems). Nucleotide sequences were read with an automatic sequencer (3130 Genetic Analyzer, Applied

Plasmid construction

The chimeric pestivirus clones pA/CP7_E1E2alf and pA/CP7_E1E2alf_TLA, depicted in Fig. 1, were constructed by using the full-length cDNA clone pA/BVDV (Meyers et al., 1996). For the generation of pA/CP7_E1E2alf, genomic regions encoding BVDV E1 and E2 were deleted (nucleotides 1860-3566) and a PCR fragment comprising CSFV E1 and E2 was inserted. In addition, by site-directed mutagenesis amino acid (aa) 479 within BVDV E rns was mutated from glycine to argenine, and restriction sites created during the cloning procedure were deleted resulting in plasmid pA/CP7_E1E2alf. The plasmid with a chimeric E2 gene, pA/CP7_E1E2alf_TLA, was constructed by site-directed mutagenesis of the antigenic CSFVspecific TAVSPTTLR epitope within E2 to TLANKDTLA, the corresponding BVDV CP7 epitope. The specified nucleotide positions (nt) correspond to the BVDV CP7 genome (Accession No. U63479), and the CSFV Alfort 187 sequence (Accession No. X87939).

Detailed information concerning the cloning steps is available on request.

Rescue of recombinant virus

For virus recovery, in vitro-transcribed RNA of plasmids pA/CP7_E1E2alf and pA/CP7_E1E2alf_TLA (1 to 5 µg) were transfected into SK6 cells by electroporation. In vitro transcription, transfection of SK-6 cells, virus rescue and multi-step growth kinetics were performed as described previously (Reimann et al., 2004). Virus stocks were prepared by passaging the virus three times on SK-6 cells, containing a titre of 10 5 -10 7 TCID 50 /ml. The identity of the recombinant viruses was confirmed by RT-PCR and subsequent sequencing of the PCR products.

Immunization and challenge experiment

In two animal experiments, 5-to 7-week old crossbred weanling pigs, free of pestiviruses and neutralizing pestivirus antibodies, were randomly divided into vaccine groups of 5 (chimeric constructs) or 3 (C-strain) pigs. The animals of the vaccine group were immunized with CP7_E1E2alf_TLA derived from infectious chimeric cDNA clone pA/CP7_E1E2alf_TLA. All animals were inoculated intramuscularly (i.m.) with 2 ml of cell culture supernatant containing 10 4,75 TCID 50 (low dose [ld] group; n=5) and 10 6,5 TCID 50 , (high dose [hd] group; n=5), respectively. The third group (n=3) was vaccinated with 2 ml [>10 2 50% protective doses (PD 50 ) corresponding to 10 4.1 50% tissue culture-infective doses (TCID 50 )] of a commercial available C-strain vaccine (Riemser Arzneimittel AG) according to the manufacturer's instructions. All vaccinated animals and naïve controls were challenged 28 days later oronasally with highly virulent CSFV strain Koslov (2 x 10 6,0 TCID 50 /animal).

During the course of the experiment, pigs were monitored daily for clinical signs of CSF.

Nasal and oral swabs as well as blood samples were collected for virological and serological examinations in intervals of 2 to 7 days. Blood samples were also analysed for differential leukocyte counts by using an automatic cell counter (Cell-Dyn R 3700, Abbott).

Virus was isolated from blood samples by using co-cultivation of 1 x 10 6 blood leucocytes with SK-6 cells in duplicate. After an incubation period of 5 to 6 days, the cells were analyzed for NS3-expression by immunofluorescence. Viral RNA was detected by realtime RT-PCR with a real-time PCR-Cycler MX3005pro (Stratagene) in serum, nasal swabs and leucocytes as described by Hoffmann et al. (2005).

Neutralizing titres were determined as neutralizing dosage 50% using the CSF virus strain Alfort 187 according to the EU Diagnostic Manual. In addition, serum or plasma samples were tested for the presence of antibodies against CSFV E2 and E rns with the commercialized ELISA assays HerdCheck CSFV Ab, (IDEXX) and Ceditest CSFV-E RNS Ab (Cedi-Diagnostics/Prionics).

A c c e p t e d M a n u s c r i p t 8

Results and discussion

Construction and in vitro-characterization of the recovered BVDV/CSFV chimeras

Aim of this study was to generate a BVDV-based chimeric pestivirus as a potential CSFV vaccine candidate allowing additionally to the E RNS -based marker diagnostics an E2based differentiation of infected from vaccinated animals. Possible E2 markers can rely on the modification of the antigenic regions "BC" or "A" within CSFV E2 [START_REF] Van Rijn | Antigenic structure of envelope glycoprotein E1 of hog cholera virus[END_REF].

However, CSF E2 antibody ELISAs commercially available in Europe are using monoclonal antibodies directed against the antigenic "TAVSPTTLR-epitope" within the antigenic region A. Therefore, one favourite option for E2-DIVA diagnostics could be the use of vaccine viruses with a modified TAVSPTTLR-epitope in combination with the commercial E2 antibody ELISAs as accompanying test systems. Here, in a first step a chimeric BVDV/CSFV full-length clone, pA/CP7_E1E2alf was constructed, in which the E1 and E2 encoding regions of cytopathogenic BVDV strain CP7 were replaced by the corresponding regions of CSFV strain Alfort 187, and amino acid G 479 within the genomic region encoding for BVDV E rns was mutated to R for an optimized virus growth (Hulst et al., 2000;Reimann et al., 2004). Furthermore, based on plasmid pA/CP7_E1E2alf, the cDNA construct pA/CP7_E1E2alf_TLA was generated by substitution of the antigenic TAVSPTTLR-epitope (Lin et al., 2000) within the CSFV E2 with the corresponding BVDV CP7 epitope (TLANKDTLA) resulting in a total of six exchanged amino acids (Fig. 1).

In vitro-transcribed RNA of both cDNA constructs pA/CP7_E1E2alf and pA/CP7_E1E2alf_TLA was able to replicate after transfection into SK-6 cells (data not shown). At 24 h after transfection, recombinant virus progeny CP7_E1E2alf and CP7_E1E2alf_TLA could be isolated from transfection supernatants by inoculation of SK-6 cells. Cells infected with the recombinant viruses could be stained (Fig. 2A) by using a panpesti-specific mab (anti-Pesti, NS3-specific, C16). The modified chimeric Growth kinetics of the chimeric viruses CP7_E1E2alf and CP7_E1E2alf_TLA were performed in porcine SK-6 cells. Surprisingly, the multistep growth curve of CP7_E1E2alf_TLA showed a decreased replication efficiency resulting in about tenfold reduced virus titers in comparison to parental CP7_E1E2alf (Fig. 2B). A possible explanation could be that the modified CSFV E2 protein has an altered structure inducing a reduced virus replication due to changes in the efficacy of virus assembly, release and/or attachment.

However, the role of a change of tropism should be low since virus growth of CP7_E1E2alf_TLA was similarly decreased in bovine KOP-R cells as it was for CP7_E1E2alf or CP7_E2alf (data not shown; Reimann et al., 2004).

Immunization and challenge infection of pigs

The marker and vaccine properties of CP7_E1E2alf_TLA were investigated in immunization and challenge experiments in comparison with the C-strain vaccine (Riemser Arzneimittel AG) as "gold standard". After immunization of pigs with the C-strain vaccine and CP7_E1E2alf_TLA, respectively, the general condition of the animals was good and no clinical symptoms could be observed. CP7_E1E2alf_TLA was completely avirulent, and no vaccine virus transmission was observed (data not shown).

After oro-nasal challenge infection with the highly virulent CSFV strain Koslov at day 28 post vaccination (dpv; day 0 post challenge infection [dpc]), all pigs vaccinated with A c c e p t e d M a n u s c r i p t 11 comparison to the C-strain-vaccinated animals before challenge infection, and animals of the low dose group scored negative until 28 dpv. Nevertheless, 3 out of 5 animals of the high dose group reached ELISA titers above the threshold, and scored positive before challenge infection (Fig. 3). Following challenge infection with CSFV Koslov, all vaccinated animals scored clearly positive in all CSFV-specific antibody ELISAs. Therefore, despite a marked difference in the E2-specific antibody reactivity of the E2-manipulated recombinants, a clear differentiation of vaccinated from infected pigs was not possible with the commercial E2antibody ELISAs in some cases, especially when a high titer preparation induced higher immune responses. Obviously, the exchange of several amino acids within the TAVSPTTLRepitope could not completely block the induction of E2-antibodies with a certain blocking efficacy against the A-epitope specific mabs of the commercial ELISAs. This is in contrast to previously published data using a C-strain-based chimera with "BC" and "A" epitopes exchanged with the corresponding sequences of a BVDV type 2 strain [START_REF] Widjojoatmodj | Classical swine fever virus E(rns) deletion mutants: trans-complementation and potential use as nontransmissible, modified, live-attenuated marker vaccines[END_REF]. However, further studies are needed to evaluate the data of the C-strain chimera using high titer preparations or multiple vaccinations. In addition, further studies have to test the suitability of changed cut-off values of the E2-ELISAs allowing e.g. the differentiation with an acceptable loss of sensitivity. Nevertheless, DIVA diagnostics was possible on the basis of the tested E rns -antibody ELISA, but the sensitivity of this assay for the detection of wild type CSFV infections is lower than that of the widely used CSFV E2-antibody ELISAs.

We conclude from the data, that the TAVSPTTLR-mutant CP7_E1E2alf_TLA induced an only slightly reduced protection compared to the C-strain vaccine, but did not achieve the expected additional DIVA properties concerning E2-based serology.

Nevertheless, the new marker vaccine candidate allowed E rns -based serological marker diagnostics as well as genetic DIVA, and could be a good basis for further adaptations to optimize the DIVA properties. However, "fine tuning" of BVDV backbone constructs for improved DIVA properties includes the risk of reduced efficacy due to e.g. E2 manipulation.

A c c e p t e d M a n u s c r i p t 12 Therefore, the ideal marker vaccine will be a balance between efficacy and safety on one site, and DIVA properties on the other site. Further studies will focus on the definition of antigenic regions/epitopes with low influence on the protective efficacy and a potential for DIVA diagnostics.

A c c e p t e d M a n u s c r i p t 13 Depner, K.R., Bouma, A., Koenen, F., Klinkenberg, D., Lange, E., de Smit, H., Vanderhallen, H., 2001 Table 1:

Results of the immunization and challenge experiments C-strain vaccine CP7_E1E2alf_TLA

Group "low dose" (10 4,75 TCID 50 )

CP7_E1E2alf_TLA

Group "high dose" (10 6,5 TCID50)

Clinical symptoms

No fever

  Classical swine fever (CSF) is one of the most important infectious diseases of pigs.

  kidney cells (SK-6; CCLV RIE262) were obtained from the Collection of Cell Lines in Veterinary Medicine, FLI, Insel Riems, Germany (CCLV). Cells were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% BVDV free fetal calf serum at 37°C in a humidified atmosphere containing 5% CO2. CSFV strains Alfort 187 and Koslov were obtained from the National Reference Laboratory of CSF (Friedrich-Loeffler-Institut Insel Riems, Germany). Virus titers were determined by end point titrations. Cells seeded in microtitration plates were infected with 10fold serial dilutions of clarified supernatants. The titres expressed in TCID 50 per milliliter were obtained by immunofluorescence (IF) staining of the SK-6 cells with the monoclonal antibody (mab) C16 directed against the pestiviral protein NS3 (Institute of Virology, TiHo

  A c c e p t e d M a n u s c r i p t 6 Biosystems) and analyzed using the Genetics Computer Group software version 11.1 (Accelrys Inc., San Diego, USA).

  be differentiated from parental CP7_E1E2alf virus by IF staining with CSFV E2-specific mabs directed against the TAVSPTTLR-epitope (e.g. anti-CSFV E2, TAV, HC34). In contrast to a non-TAVSPTTLR-specific mab (anti-CSFV E2, none TAV, HC/TC16), the TAVSPTTLR-specific mab detected only CP7_E1E2alf-infected SK-6 cells. In addition, due to the substitution of BVDV E2 with CSFV E2, SK-6 cells infected with either CP7_E1E2alf or CP7_E1E2alf_TLA could not be detected with a BVDV E2-specific mab (anti-BVDV E2, CA34).

(Fig 1 :

 1 Figure legends:

Figure 2 :

 2 Figure 2: (A) IF analysis of SK6 cells infected with CP7_E1E2alf and CP7_E1E2alf_TLA,

Figure 3 :

 3 Figure 3: CSFV E2-specific ELISA antibodies after immunization with the C-strain vaccine

  Figures

  . Classical swine fever (CSF) marker vaccine Trial II: Challenge study in pregnant sows. Vet. Microbiol. 83, 107-120.

	de Smit, A.J., Bouma, A., de Kluijver, E.P., Terpstra, C., Moormann, R.J., 2001a. Duration of
	the protection of an E2 subunit marker vaccine against classical swine fever after a
	single vaccination. Vet. Microbiol. 78,307-317.
	de Smit, A.J., Bouma, A., van Gennip, H.G., de Kluijver, E,P., Moormann, R.J., 2001b.
	Chimeric (marker) C-strain viruses induce clinical protection against virulent classical
	swine fever virus (CSFV) and reduce transmission of CSFV between vaccinated pigs.
	Vaccine 19,1467-1476.
	Edwards, S., Fukusho, A., Lefevre, P.C., Lipowski, A., Pejsak, Z., Roehe, P., Westergaard, J.,
	2000. Classical swine fever: the global situation. Vet. Microbiol. 73, 103-119.
	Hoffmann, B., Beer, M., Schelp, C., Schirrmeier, H., Depner, K., 2005. Validation of a real-
	time RT-PCR assay for sensitive and specific detection of classical swine fever. J.
	Virol. Methods 130, 36-44.
	Hoffmann, B., Depner, K., Schirrmeier, H., Beer, M., 2006. A universal heterologous internal
	control system for duplex real-time RT-PCR assays used in a detection system for
	pestiviruses. J. Virol. Methods 136, 200-209.
	Hulst, M.M., van Gennip, H.G., Moormann, R.J., 2000. Passage of classical swine fever virus
	in cultured swine kidney cells selects virus variants that bind to heparan sulfate due
	to a single amino acid change in envelope protein E(rns). J. Virol. 74, 9553-9561.
	König, P., Hoffmann, B., Depner, K.R., Reimann, I., Teifke, J.P., Beer, M. 2007a. Detection
	of classical swine fever vaccine virus in blood and tissue samples of pigs vaccinated
	either with a conventional C-strain vaccine or a modified live marker vaccine.
	Vet. Microbiol. 120, 343-51.

Acknowledgments

We thank Gabriela Adam, Doreen Reichelt and Ulrike Polenz for excellent technical assistance, Gregor Meyers for providing the infectious BVDV clone pA/BVDV, and Irene Greiser-Wilke (TiHo, Hannover, Germany) for providing NS3-, E rns -and E2-specific antibodies. The study was supported by the EU project "CSF Vaccine and Wild Boar" (SSPE- CT-2003-501559).

A c c e p t e d M a n u s c r i p t 10 CP7_E1E2alf_TLA showed a short increase of body temperatures (>40°C) between 3 and 5 dpc (Table 1). At the day of challenge infection, mean homologous antibody titers of 1:15 (low dosis group = ld group) and 1:74 (high dosis group = hd group) could be detected in the CP7_E1E2alf_TLA-immunized animals with a CSFV Alfort 187 specific neutralization assay. In comparison, a mean titer of CSFV Alfort 187 neutralizing antibodies of 1:33 was determined for the C-strain-immunized group. No challenge virus could be re-isolated from blood leucocytes of any of the immunized animals between 0 and 28 dpc, suggesting that no virus shedding occurred. However, low levels of CSFV genome could be detected in serum samples of animals of both groups immunized with CP7_E1E2alf_TLA between 3 to 6 dpc with cycle of threshold (ct) values of about 35 (less than 100 genome copies). In detail, CSFV genome was detected in 5/5 vaccinated pigs (low dose) at 4 dpc, and in 1/5 pigs at 6 dpc (Table 1). In serum samples of animals, which were immunized with a high dose of CP7_E1E2alf_TLA, CSFV genomes were detected at 3 dpc (4/5 animals), and 5 dpc (2/5 animals, Table 1). In summary, CP7_E1E2alf_TLA showed a slightly reduced protective efficacy in comparison to the C-strain vaccine. In addition, the newly constructed mutants were also less efficacious than the prototype BVDV-based chimera CP7_E2alf (Reimann et al., 2004, König et al., 2007b).

Differentiating infected from immunized animals

Following immunization and challenge infection, "marker or DIVA serology" was determined using selected CSFV-specific E RNS and E2 antibody blocking ELISAs. Pigs immunized with CP7_E1E2alf_TLA scored negative until day 28 p.v. by using the E RNSantibody ELISA (data not shown), a result which is comparable to the data obtained with the chimeric vaccine candidate CP7_E2alf (Reimann et al., 2004), and which allowed a differentiation of vaccinated from infected animals. Furthermore, in the more sensitive CSFV E2 antibody ELISA, CP7_E1E2alf_TLA-immunized animals showed distinct lower titers in

Conflict of interest statement None


J Tarradas

J M Argilaguet

R Rosell

M Nofrarías

E Crisci

L Córdoba

E Pérez

I Díaz

F Rodríguez

M Domingo

M Montoya

L Ganges

Interferon gamma induction correlates with protection by DNA vaccine expressing E2 glycoprotein against classical swine fever virus infection in domestic pigs

Keywords: Classical swine fever virus, DNA vaccines, interferon gamma, protection

A c c e p t e d M a n u s c r i p t 2 Abstract Classical swine fever is a highly contagious viral infection affecting domestic and wild pigs. For CSFV, immunization with plasmids expressing different versions of glycoprotein E2 has proven an effective way to induce protection. Previously, we have also shown that immunization with DNA vaccine expressing glycoprotein E2 (DNA-E2) induced specific T helper cell responses in the absence of neutralizing antibodies. However, the role of T-cell responses in protection against CSFV is largely unknown.

Here we have extended these studies to deeply characterize the role of T-cell responses by a DNA-E2 and their correlation with protection against CSFV infection. Thus, pigs vaccinated with the DNA vaccine induced a strong cellular immune response, characterized by the specific induction IFN-gamma expressing T cells after vaccination without any detectable levels of CSFV neutralizing antibodies.

Constant levels of CSFV-specific IFN gamma producing cells observed from the beginning of the infection until 7 days after challenge in vaccinated animals might contribute to early control of CSFV replication, at least until neutralizing antibodies are developed.

Severe clinical signs of the disease, including high titters of viremia, pyrexia and virus spread to different organs, were recorded in the non vaccinated challenge animals, in comparison to the vaccinated animals where only one animal showed mild clinical signs and a short pick of viremia. Lack of complete protection in this animal correlated with a delay on the induction of neutralizing antibodies, detectable only from day 11 post-CSFV challenge. Conversely, the rest of the pigs within the group developed neutralizing antibodies as early as at day two post-challenge, correlating with sterile protection. Finally, an inverse correlation seemed to exist between early induction of IFN-alpha and the protection observed, while IL-10 seemed to be differentially regulated in vaccinated and non-vaccinated animals.

Our results support the relevance of the induction of a strong T cellular response to confer a solid protection upon DNA vaccination against CSFV. Further experiments are needed to be done in order to clarify the key cytokines playing a role in CSFV-protection and to obtain emergency vaccines capable to confer robust and fast protection.

Introduction

Classical swine fever (CSF), a highly contagious viral infection of domestic and wild pigs, is one of the more devastating porcine diseases worldwide [START_REF] Moennig | Clinical Signs and Epidemiology of Classical Swine Fever: A Review of New Knowledge[END_REF]. The disease is endemic in Asia and prevails in many Central and South American countries, as well as in Eastern Europe. Despite the stringent controls adopted in the EU from the early 1990s, CSFV has been periodically reintroduced in the EU, either via wild pigs or through external imports, with outbreaks in the 1990s in Belgium, Germany, Holland, Spain and Italy, and in the 2000s in UK, Spain and Germany [START_REF] Dong | Candidate Peptide-Vaccines Induced Immunity Against CSFV and Identified Sequential Neutralizing Determinants in Antigenic Domain A of Glycoprotein E2[END_REF]).

The etiological agent, classical swine fever virus (CSFV), is an icosahedral RNA virus with (+) polarity and a lipid envelope that integrates the genera Pestivirus along with bovine diarrhea virus (BVDV) and border disease virus (BDV), all closely related at both genomic and antigenic levels.

Clinical presentation and severity of CSF are diverse, depending largely on the virulence of the strain and the immunological status of the host. In its natural host, CSFV infection results in haemorrhage, leukocytopenia and immunosupression [START_REF] Susa | Pathogenesis of Classical Swine Fever: B-Lymphocyte Deficiency Caused by Hog Cholera Virus[END_REF]. The virus displays tropism towards immune cells such as dendritic and monocyte/macrophage lineage cells [START_REF] Carrasco | Interaction of Classical Swine Fever Virus with Dendritic Cells[END_REF], and infection of such cells is assumed to play a key role in immunosuppression, dissemination and/or viral persistence, through hitherto unknown mechanisms. Thus, reductions in circulating CD4 + and CD8 + T cells subset and granulocytes have been reported, the latter being replaced by immature precursors. The immunomodulation induced by a highly virulent strain is detected in the first day post-infection, much earlier than viremia, clinical signs or specific antibodies appear [START_REF] Torlone | Circulating Interferon Production in Pigs Infected with Hog Cholera Virus[END_REF][START_REF] Summerfield | Lymphocyte Apoptosis during Classical Swine Fever: Implication of Activation-Induced Cell Death[END_REF]. Thus, besides its basic interest, the study of the early immunomodulation induced by CSFV can lead to the future development of early diagnostic and preventive strategies [START_REF] Ganges | Recent Advances in the Development of Recombinant Vaccines Against Classical Swine Fever Virus: Cellular Responses also Play a Role in Protection[END_REF].

Glycoprotein E2 is regarded as the most immunogenic of CSFV proteins. It is mainly responsible for the induction of neutralizing antibodies and it is the only viral protein that can elicit them and it can confer protection when given alone [START_REF] Van Zijl | Live Attenuated Pseudorabies Virus Expressing Envelope Glycoprotein E1 of Hog Cholera Virus Protects Swine Against both Pseudorabies and Hog Cholera[END_REF][START_REF] Hulst | Glycoprotein E1 of Hog Cholera Virus Expressed in Insect Cells Protects Swine from Hog Cholera[END_REF][START_REF] Konig | Classical Swine Fever Virus: Independent Induction of Protective Immunity by Two Structural Glycoproteins[END_REF]. For these reasons glycoprotein E2 is an excellent antigen and potentially an ideal candidate which is using currently for the development of a different strategies of recombinant vaccines against CSFV (Reviewed in [START_REF] Dong | Marker Vaccine Strategies and Candidate CSFV Marker Vaccines[END_REF][START_REF] Ganges | Recent Advances in the Development of Recombinant Vaccines Against Classical Swine Fever Virus: Cellular Responses also Play a Role in Protection[END_REF]. Although considerable efforts towards the development of a marker vaccine, new strategies and better vaccines against CSFV are urgently needed that can confer protection at very early time after single administration, even when no neutralizing antibodies are detected. That prevent and fulfil safety requirement (Reviewed in [START_REF] Dong | Marker Vaccine Strategies and Candidate CSFV Marker Vaccines[END_REF][START_REF] Ganges | Recent Advances in the Development of Recombinant Vaccines Against Classical Swine Fever Virus: Cellular Responses also Play a Role in Protection[END_REF].

Despite the correlation between the induction of neutralizing antibodies by vaccination and protection against CSFV [START_REF] Terpstra | The Protective Value of Vaccine-Induced Neutralising Antibody Titres in Swine Fever[END_REF][START_REF] Ganges | Recent Advances in the Development of Recombinant Vaccines Against Classical Swine Fever Virus: Cellular Responses also Play a Role in Protection[END_REF], there are occasions in which this protection was conferred in the absence of detectable anti-CSFV antibodies, suggesting that other immune mechanisms, such as cellular responses against CSF antigens, might be involved in the protection observed [START_REF] Rumenapf | Structural Proteins of Hog Cholera Virus Expressed by Vaccinia Virus: Further Characterization and Induction of Protective Immunity[END_REF][START_REF] Hulst | Glycoprotein E1 of Hog Cholera Virus Expressed in Insect Cells Protects Swine from Hog Cholera[END_REF][START_REF] Suradhat | The Correlation of Virus-Specific Interferon-Gamma Production and Protection Against Classical Swine Fever Virus Infection[END_REF][START_REF] Ganges | Recent Advances in the Development of Recombinant Vaccines Against Classical Swine Fever Virus: Cellular Responses also Play a Role in Protection[END_REF].

In spite of the potential involvement of T cellular response in protection against CSFV in absence of neutralizing antibodies, its role in CSFV protection is not well defined. In particular, the role of different cytoquine such as interferon (IFN) gamma (pro-inflammatory) and in elimination and pathogenesis of the virus is poorly understood.

Previously, we have also shown that immunization with DNA-E2 induced specific T helper cell responses in the absence of neutralizing antibodies [START_REF] Ganges | Recent Advances in the Development of Recombinant Vaccines Against Classical Swine Fever Virus: Cellular Responses also Play a Role in Protection[END_REF]. Interestingly, T cell response elicited can prime an efficient B cell response, since immunized animals developed an accelerated neutralizing antibody response immediately after challenge and they were fully protected upon a severe viral challenge against CSFV [START_REF] Ganges | Recent Advances in the Development of Recombinant Vaccines Against Classical Swine Fever Virus: Cellular Responses also Play a Role in Protection[END_REF].

Our aim is to unravel the possible immunological mechanisms involved in DNA-E2 protection but not the efficacy of our experimental DNA vaccine, as it has been previously proven [START_REF] Ganges | Recent Advances in the Development of Recombinant Vaccines Against Classical Swine Fever Virus: Cellular Responses also Play a Role in Protection[END_REF]. The ultimate objective of this work was to use a DNA -E2 [START_REF] Ganges | Recent Advances in the Development of Recombinant Vaccines Against Classical Swine Fever Virus: Cellular Responses also Play a Role in Protection[END_REF] as a model to study the cellular immune response focusing on the effective role of different cytoquine as interferon (IFN) gamma (pro-inflammatory) and IL-10 (anti-inflammatory) associated with protection and CSFV infection.

A c c e p t e d M a n u s c r i p t 5

Materials and Methods

Cells and Viruses

PK-15 cell line was cultured in complete DMEM media supplemented with 10% fetal bovine serum (FBS) at 37ºC in 5% CO 2 . Cells were infected with 0.1 TCID 50 /cell in 2% FBS and virus was harvested 48 h later. Viral stocks were titrated by using peroxidase-linked assay (PLA) [START_REF] Wensvoort | Production of Monoclonal Antibodies Against Swine Fever Virus and their use in Laboratory Diagnosis[END_REF], following the statistical method described by [START_REF] Reed | A Simple Method of Estimating Fifty Percent Endpoints[END_REF]. The virulent strain Margarita used in this study was isolated in Havana in 1958, and has been used since 1965 for vaccine potency tests in Cuba [START_REF] Ganges | Antigenic, Biological and Molecular Characterization of the Cuban CSFV Isolates "Margarita[END_REF].

DNA immunization of pigs

To evaluate immune response induced by DNA-E2 [START_REF] Ganges | Recent Advances in the Development of Recombinant Vaccines Against Classical Swine Fever Virus: Cellular Responses also Play a Role in Protection[END_REF], 5 pigs (Landrace x Large white, 8 weeks old; numbered from 1 to 5) were used. As control, 5 additional pigs were inoculated with pcDNA3.1+ (numbered 6 to 10). Three doses of 400 μg of DNA were administered at 14-day intervals. In all cases, one third of the total amount of DNA was intramuscularly (i.m) injected in the femoral quadriceps, one third in the neck muscle and the last third was subcutaneously injected in the ear.

CSFV challenge

45 days after the first injection all pigs were challenged with 10 5 TCID 50 of CSFV (strain Margarita) by i.m injection in the neck. This viral dose often causes the death of injected animals after 10 to 15 days [START_REF] Ganges | Recent Advances in the Development of Recombinant Vaccines Against Classical Swine Fever Virus: Cellular Responses also Play a Role in Protection[END_REF][START_REF] Ganges | Antigenic, Biological and Molecular Characterization of the Cuban CSFV Isolates "Margarita[END_REF]. Both rectal temperature and external clinical signs of disease were scored daily (1 point: pyrexia; 2 point: pyrexia + mild clinical signs; 3 point: severe clinical signs and 4: death). After euthanized, animals were subjected to an exhaustive necropsy in which the presence of pathological signs in different organs and tissues was evaluated.

Isolation of porcine PBMC and Elispot assay for detection CSFV-specific IFN gamma and IL-10 producing cells

Pigs were bled 45 days after the first DNA immunization (Pre-challenge) and later at 7 and 14 days post challenge (p.c.). Blood was collected with 5mM EDTA and used to obtain periphery blood Mercaptoethanol (Sigma), 50 000 UI penicillin 1 -1 (Invitrogen), 50 mg streptomycin 1 -1 (Invitrogen) and50 mg gentamicin 1 -1 (Sigma). Elispot assay for detection CSFV-specific IFN gamma and IL-10 producing cells was performed as described [START_REF] Diaz | Use of ELISPOT and ELISA to Evaluate IFN-Gamma, IL-10 and IL-4 Responses in Conventional Pigs[END_REF]. Briefly, for IFN gamma ELISA plates (Costar 3590, Corning) were coated overnight with 5 μg / ml of capture antibody (P2G10, Pharmigen). For detecting we use biotinylated detection antibody (P2C11, Pharmigen). In both cases (for IL-10 and IFN gamma producing cells), 5 x10 5 live PBMC/well was plated in triplicates at 0.02 multiplicity of infection of CSFV Margarita strain. As controls, triplicate of cells were incubated in absence of virus (negative control), or with phytohaemagglutinin (PHA) (10 μg/ml). To calculate the antigen-specific frequencies of IFN gamma and IL-10 producing cells, count of spots in the media for non stimulated wells were subtracted from counts of the media for CSFV stimulates wells. Frequencies of cytokine-producing cells were expressed as responding cells in 5x10 5 PBMC.

Antibody detection

Pigs were bled weekly to follow CSFV specific neutralizing antibody induction. Serum samples were tested by neutralization peroxidase-linked assay (NPLA) [START_REF] Terpstra | The Neutralizing Peroxidase-Linked Assay for Detection of Antibody Against Swine Fever Virus[END_REF] and titers were expressed as the reciprocal dilution of serum that neutralized 100 TCID 50 of strain Margarita in 50% of the culture replicates. E2 specific antibodies were detected using an ELISA (CEDITEST; Lelystad), following manufacturer recommendations.

RT-PCR and Virus isolation

The presence of CSFV RNA in different tissues and organs was analyzed using the RT-PCR assay previously described [START_REF] Diaz De Arce | An RT-PCR Assay for the Specific Detection of Classical Swine Fever Virus in Clinical Samples[END_REF], which allows detection of 2 TCID 50 in samples from CSFV infected animals. Serum samples, nasal swabs collected at 2 and 7 days post challenge (p.c) and samples (1g) from different organs collected at necropsy were used to isolate virus after three consecutive passages in susceptible PK-15 cells [START_REF] Ganges | Recent Advances in the Development of Recombinant Vaccines Against Classical Swine Fever Virus: Cellular Responses also Play a Role in Protection[END_REF]. Anti-IFN alpha monoclonal antibodies (K9 and K17) and an IFN alpha recombinant protein (PBL Biomedical laboratories, Piscataway, New Jersey) were used in ELISA as described [START_REF] Guzylack-Piriou | Type-A CpG Oligonucleotides Activate Exclusively Porcine Natural Interferon-Producing Cells to Secrete Interferon-Alpha, Tumour Necrosis Factor-Alpha and Interleukin-12[END_REF]. TMB (3,3',5,5'tetramethylbenzidine) was from Sigma.

DNA immunization of pigs induces IFN gamma producing cells in absence of detectable anti-CSFV antibodies.

Previously, we have shown that immunization with DNA-E2 induced specific helper T cell responses. In order to investigate whether immunization also induced IFN gamma production, an Elispot assay was performed to examine number of IFN gamma producing cells after three doses of the same vaccination design (45 days p.i). Increased number of specific CSFV IFN gamma producing cells was observed in animals vaccinated with DNA-E2, whereas control non vaccinated animals (6 to 10) showed no responses to the virus (Fig. 1). Similar to our previous work, no detectable levels of neutralizing antibodies were found in sera collected prior to the second DNA boost (day 28 p.i) and at subsequent time intervals (days 35 and 45 p.i). The absence of antibodies was further confirmed using an ELISA specific for E2 protein (data not shown).

DNA vaccine confers protection against lethal challenge with CSFV

All vaccinated and non-vaccinated animals were challenged with a lethal dose (10 5 TCID 50 ) of Margarita strain 15 days after the third dose of plasmid DNA-E2 (45 days p.i). As expected, control non vaccinated animals (pigs 6 to 10) showed pyrexia (rectal temperature above 40ºC) that appeared, in average, at day 5 p.c, with peaks reaching above 41ºC. Additional clinical signs of the disease were developed from day 5 p.c in control non vaccinated pigs, such as anorexia, conjunctivitis, constipation, fibrin accumulation in feces, abdominal petechiae, nervous disturbers and prostration (Fig. 2). Control non vaccinated animals were euthanized at day 14 p.c to avoid suffering. Post-mortem analysis of these pigs showed pathological lesions typical of CSFV infection such as: marginal spleen infarcts, hemorrhagic mesenteric and mediastinic lymph nodes, enteritis, kidney petechiae, pulmonary edema and hydrothorax (data not shown).

As we have previously shown, none of the vaccinated animal (pigs 1 to 5) developed any of the clinical signs observed in control non vaccinated pigs. These animals were euthanized at day 17 p.c and no pathological changes were observed upon post-mortem analysis. A mild clinical symptom was observed only in one of the vaccinated animals (pig 3) with transient pyrexia (with a peak of 41ºC) at days 5 to 6 p.c. This animal (pig 3) was euthanized at day 14 p.c for analysis revealing a mild congestion in mesenteric lymph nodes (data not shown).

A c c e p t e d M a n u s c r i p t 9

Protection against CSFV is associated with induction of cellular and humoral responses upon challenge

To further understand the mechanisms underlying protection against CSFV viral challenge elicited after vaccination with DNA-E2, CSFV neutralizing antibody titers were determined for both groups of animals at different days p.c. As shown in Figure 3, no specific antibodies were detected in control non vaccinated animals (pigs 6 to 10). Conversely, elevated levels of E2 specifics neutralizing antibodies were determined at 7 days post infection in vaccinated animals, reaching the highest values at day 17 p.c. One animal (pig 3) which showed transient pyrexia upon challenge, elicited low titers of neutralizing antibodies, with a peak of 1:80 at day 14 p.c (Fig. 3).

After CSFV challenge, numbers of specific CSFV IFN gamma producing cells were analyzed. Levels of IFN gamma producing cells in vaccinated animals (Fig. 4) remained in the same range than unchallenged vaccinated animals (Fig. 1). On the other hand, control non vaccinated animals showed no significant numbers of IFN gamma producing cells in response to virus challenge (Fig. 4).

Absence of CSFV in animals protected by DNA vaccine

To correlate the lack of clinical and pathological symptoms after viral challenge with virus clearance RT-PCR was used, in serum, nasal swabs and organs. CSFV RNA was amplified from serum and nasal swabs samples of 5 non vaccinated control pigs at day 7 p.c. (Table 1). Viral nucleic acid was detected in non vaccinated control animals when RNA extracted at necropsy from diverse organs such as spleen, lymph nodes and kidney were used as template for the assay (data not shown).

Consistent with the protection of pigs immunized with DNA-E2, negative results were obtained in the attempts to amplify viral RNA from serum of these animals at different days p.c (Table 1). Lack of amplification was also observed in samples from organs collected at day 17 p.c, when vaccinated pigs were euthanized. Pig 3 showed positive amplification at day 7 p.c; however no CSFV RNA was amplified from any of the organ samples collected at day 14 p.c, when this animal was euthanized (Table 1). Detection of infectious CSFV by viral isolation in tissue culture in samples collected after animal necropsy confirmed the results obtained by RT-PCR. CSFV was isolated in all organ samples from nonimmunized control pigs after a single passage in PK-15 cells. In contrast, no virus was recovered from immunized pigs even after three serial passages in this cell line (data not shown).

A c c e p t e d M a n u s c r i p t 10

Augmented levels of IFN alpha associated with CSFV infection

Presence of elevated levels of IFN alpha in serum have been reported as a response of natural interferon producing cells (NIPC) to the presence of virus [START_REF] Summerfield | High IFN-Alpha Responses Associated with Depletion of Lymphocytes and Natural IFN-Producing Cells during Classical Swine Fever[END_REF]. Systemic replication of virulent CSFV in vivo during the acute phase of infection induces type I IFN [START_REF] Torlone | Circulating Interferon Production in Pigs Infected with Hog Cholera Virus[END_REF][START_REF] Summerfield | High IFN-Alpha Responses Associated with Depletion of Lymphocytes and Natural IFN-Producing Cells during Classical Swine Fever[END_REF]. Therefore, levels of IFN alpha in serum samples obtained from vaccinated and non vaccinated animals at 2 and 7 days post challenge were measured. Only infected non vaccinated animals (6 to 10) show high amount of IFN alpha as early as day 2 until day 7 post infection (Fig. 5). In the vaccinated group, only one animal (pig number 3) showed high amounts of IFN alpha at 7 days post challenge. This response correlated very well with viral detection in serum at 7 days post infection and the mild clinical signs observed in this pig (Table 1).

Increased numbers of IL-10 producing cells after CSFV challenge

Finding high amounts of gamma IFN producing cells (pro-inflammatory cytokine) in protecting animals lead us to investigate whether the levels of other important anti-inflammatory cytokine, IL-10, were altered. After vaccination (45 p.i.), both groups (vaccinated and non vaccinated infected animals) showed similar levels of IL-10 producing cells. Surprisingly, higher amount of IL-10 producing cells specific to CSFV was observed in both groups at 7 days post infection (Fig. 6). However, vaccinated animals showed almost double levels of IL-10 producing cells specific to CSFV when compared to levels in non vaccinated infected animals, suggesting that vaccination with our DNA-E2 construct was able to modulate immune responses in those animals after viral infection. Moreover, IL-10 producing cells specific to CSFV disappeared at 10 days post infection from non-vaccinated infected animals whereas vaccinated animals recovered pre-challenge levels (fig. 6).

A c c e p t e d M a n u s c r i p t 11

Discussion

DNA immunization is a relatively new method of vaccination with promising future and several advantages over more conventional vaccines [START_REF] Rajcani | Current Developments in Viral DNA Vaccines: Shall they Solve the Unsolved?[END_REF]. During the last years, this methodology has shown successful results against various pathogens and tumor models, and many strategies have been developed to enhance its immunogenicity (Rodríguez et al. 2000;[START_REF] Belakova | DNA Vaccines: Are they Still just a Powerful Tool for the Future?[END_REF]. Nowadays, there are several DNA vaccines included in clinical trials, and four have already been licensed for animal use [START_REF] Kutzler | DNA Vaccines: Ready for Prime Time?[END_REF]. For CSFV, immunization with plasmids expressing different versions of glycoprotein E2 has proven an effective way to induce protection [START_REF] Andrew | Protection of Pigs Against Classical Swine Fever with DNA-Delivered gp55[END_REF][START_REF] Ganges | Recent Advances in the Development of Recombinant Vaccines Against Classical Swine Fever Virus: Cellular Responses also Play a Role in Protection[END_REF].

We have reported that immunization of domestic pigs with a DNA-E2 of CSFV induced specific CD4 + T cell responses against CSFV without any detectable antibodies to CSFV [START_REF] Ganges | Recent Advances in the Development of Recombinant Vaccines Against Classical Swine Fever Virus: Cellular Responses also Play a Role in Protection[END_REF]. The elicited T cell response after DNA vaccination seemed to efficiently prime B cell response, since immunized animals developed significant titers of neutralizing antibodies and they were fully protected upon a severe viral challenge [START_REF] Ganges | Recent Advances in the Development of Recombinant Vaccines Against Classical Swine Fever Virus: Cellular Responses also Play a Role in Protection[END_REF]. Thus, our previous data paved the way for further understanding the involvement in protection of the T cell responses elicited after administration of our DNA-E2. In this report, we analyzed the Th1 and Th2 cytokine profile induced by our vaccine, in particular IFN gamma and IL-10. Additionally, the induction of a key component of the innate immunity, IFN alpha, was also followed after vaccination and viral CSFV infection.

Our results clearly showed that the DNA vaccine triggered the induction of CSFV -specific IFN gamma producing cells in vaccinated animals prior to challenge with CSFV, consistently detected also after CSFV infection. In concordance with our previous results using same DNA vaccine prototype [START_REF] Ganges | Recent Advances in the Development of Recombinant Vaccines Against Classical Swine Fever Virus: Cellular Responses also Play a Role in Protection[END_REF], no neutralizing antibodies were detectable prior to challenge. Early upon challenge with a severe viral dose in the range indicated by the OIE Manual, vaccinated animals induced detectable neutralizing antibody levels and were fully protected from the clinical signs of CSFV infection. Despite being clinically protected, one vaccinated challenged animal (pig 3 immunized with DNA vaccine) developed a mild and transient peak of pyrexia (Fig. 2). Although transient viremia was detected in serum collected at day 7 p.c from this animal (Table 1) neither signs of disease were observed nor was virus recovered from the tissue samples analyzed upon necropsy.

These results pointed towards the specific IFN gamma producing T-cells, playing a role in the elicited protection against CSFV. Our findings is in agreement with previous reports showing full protection and elevated number of IFN gamma producing cells as early as 6 days after vaccination with A c c e p t e d M a n u s c r i p t 12 life CSFV vaccines [START_REF] Suradhat | The Correlation of Virus-Specific Interferon-Gamma Production and Protection Against Classical Swine Fever Virus Infection[END_REF][START_REF] Suradhat | The Kinetics of Cytokine Production and CD25 Expression by Porcine Lymphocyte Subpopulations Following Exposure to Classical Swine Fever Virus (CSFV)[END_REF]. The presence of constant levels of CSFV-specific IFN gamma producing cells from the beginning of the infection until 7 days after challenge in vaccinated animals, might contribute to the early control of CSFV replication, at least until neutralizing antibodies are developed, as it has been previously shown for other viral infectious [START_REF] Neveu | Selection of High-Avidity CD8 T Cells Correlates with Control of Hepatitis C Virus Infection[END_REF][START_REF] Savarin | Memory CD4+ T-Cell-Mediated Protection from Lethal Coronavirus Encephalomyelitis[END_REF]. Conversely, CSFV-specific IFN gamma producing cells in non vaccinated animals was below the detection level after CSFV infection, (almost zero) consistent with the fact that these animals were unprotected and so, immune-suppressed (see Table 1 and Fig. 2).

Besides IFN gamma, our results also depicted IFN alpha involvement in the resistance against CSFV. Lower level of IFN alpha was detected in the serum of DNA vaccinated challenged animals at 2 and 7 days after challenge compared to non-vaccinated challenged animals. The DNA-E2 was efficient in limiting the viremia as no virus (or viral RNA) could be detected in clinical and tissue samples obtained from protected animals. Therefore, elevated levels of serum IFN alpha in infected pigs might correlate with leucopenia caused by CSFV infection and is suggestive of a certain level of virus replication or persistence (Rau et al., 2006;[START_REF] Summerfield | High IFN-Alpha Responses Associated with Depletion of Lymphocytes and Natural IFN-Producing Cells during Classical Swine Fever[END_REF]. As consequence of virus replication detected in all non vaccinated infected animals, higher levels of IFN alpha was detecting in the serum of these animals at 2 and 7 days after challenge.

The lack of complete inhibition of virus spread in vaccinated challenged animal (pig 3 immunized with DNA-E2) correlated with high levels of IFN alpha detection at 7 days after challenge.

Anyhow, finding elevated levels of serum IFN alpha can be a good marker of the disease progression.

Our observation also supports the relevant role that neutralizing antibodies play in protection against CSFV (Tepstra and Wensvoort, 1988;van Rijn et al., 1996;Bouma et al., 1999;Moormann et al., 2000), since the only pig vaccinated with the DNA-E2 that did not result fully protected (pig 3), showed a clear delay in the induction of neutralizing antibodies, (see Fig 2 andFig 3).

The fact that both IFN gamma and IL-10 are concomitantly expressed upon CSFV challenge in protected animals suggests a possible modulation in the immune response balance (Th1 vs. Th2) might be relevant to obtain protection against CSFV. It has been suggested that emergency vaccination for foot and mouth disease virus with classical vaccines induces protecting immune responses with early expression of simultaneous presence of Th1 and Th2 like-cytokines, including IFN gamma and IL-10 ( [START_REF] Barnard | Immune Response Characteristics Following Emergency Vaccination of Pigs Against Foot-and-Mouth Disease[END_REF][START_REF] Eble | Comparison of Immune Responses After Intra-Typic Heterologous and Homologous Vaccination Against Footand-Mouth Disease Virus Infection in Pigs[END_REF]. IL-10 is a pleiotropic cytokine involved in many different events having a complex role leading to different functions in the immune system [START_REF] Li | Contextual Regulation of Inflammation: A Duet by Transforming Growth Factor-Beta and Interleukin-10[END_REF], including 2008). Taking into account IL-10 function on B cell survival, it is plausible to think that this cytokine could be involved in keeping B cell homeostasis after vaccination, by comparison with CSFV infected animals suffering from leucopenia [START_REF] Susa | Pathogenesis of Classical Swine Fever: B-Lymphocyte Deficiency Caused by Hog Cholera Virus[END_REF][START_REF] Summerfield | Pathogenesis of Granulocytopenia and Bone Marrow Atrophy during Classical Swine Fever Involves Apoptosis and Necrosis of Uninfected Cells[END_REF]. However, in CSF unvaccinated infected animals, we have described and increased number of IL-10 producing cells after challenge (Figure 6), suggesting a major role for this cytokine in CSFV infection. Additionally, it has been reported that CSFV modulates T cells for cytokines secretion, such as IL-10, which is probably a key cytokine in the immunosuppresion observed after CSFV infection [START_REF] Suradhat | The Kinetics of Cytokine Production and CD25 Expression by Porcine Lymphocyte Subpopulations Following Exposure to Classical Swine Fever Virus (CSFV)[END_REF]. Thus, we assume that the amount of IL-10 produced in the animals regulates the fine tuned balance between the protection induced and the IL-10 low secretion pattern in non-vaccinated challenged animals. IL-10 involvement in CSFV infection will be the topic of further experiments. Table 1:

Conclusion

Conflict of interest

None

  virus spreading, permit differentiation of infected from vaccinated animals (DIVA)

  PBMC) by density-gradient centrifugation with Histopaque 1077 (Sigma). The total number of live PBMC recovered was estimated by trypan-blue staining. PBMC were cultivated in RPMI-10% FBS, 1 mM non-essencial amino acids (Invitrogen), 1 mM sodium pyruvate (Invitrogen), 5 mM 2-

  ELISA for IFN alpha detection from serum samples

  cell survival, proliferation and antibody production, (Reviewed inMosser and Zhang

  DNA immunization with a plasmid encoding full-length E2 induced a strong cellular immune response characterized by specific induction of CSFV-specific IFN gamma expressing T cells. Constant levels of CSFV-specific IFN gamma producing cells observed from the beginning of the infection until 7 days after challenge in vaccinated animals might contribute to early control of CSFV replication, at least until neutralizing antibodies are developed. The immunological profile observed in protecting animals against CSFV also was associated with high levels of specific CSFV IL-10 producing cells and low levels of IFN alpha in serum at 2 and 7 days post challenge.Further studies are necessary for continuing with the characterization of cytokine profile after different strategies of vaccination and after infection with CSFV. Understanding the immune mechanisms operating during CSFV infection and the key components mediating the immunoprotection conferred by ours experimental vaccines can be useful to develop new marker vaccines and more importantly innovative new diagnostic tools against CSFV in the future.

  We are grateful to Dr. Ayub Darji for critically reviewing the manuscript and to David Solanes, Iván Cordon and Juan Carlos Prieto for their support and help in the biosafety facility and animal handling. Work at CReSA was supported by the following projects: AGL2004-07857-C03-01, partially AGL2006-13809-C03-01, and Consolider-Ingenio 2010 from the Spanish Government. LG was supported by Juan de la Cierva program and FR from the Ramon y Cajal program, all sponsored by the MCyT Spanish Government.

  Rapid Antibody Production and Confer Total Protection upon Viral Challenge.

Figure 1 :

 1 Figure 1: Elispot of specific CSFV IFN gamma producing cells after 3 dose of DNA vaccine (Prechallenge). Pigs numbered 1 to 5 were immunized three times with DNA-E2 (black bars). Pigs numbered 6 to 10 were inoculated three times with an empty plasmid, pCDNA3.1+ (grey bars). Specific CSFV IFN gamma producing cells were expressed as spot number per 5x10 5 PBMC.

Figure 2 :

 2 Figure 2: Clinical signs associated with CSFV. Both rectal temperature and external clinical signs of disease were scored daily (1 point: pyrexia; 2 point: pyrexia + mild clinical signs; 3 point: severe clinical signs and 4: death). Pigs numbered 1 to 5 were immunized DNA-E2 (black bars). Pigs numbered 6 to 10 were inoculated three times with an empty plasmid, pCDNA3.1+ (grey bars). Both groups were later challenged with virulent CSFV Margarita strain.

Figure 3 :

 3 Figure 3: Induction of neutralized antibodies after challenge with virulent Margarita strain of CSFV. Pigs numbered 1 to 5 were immunized DNA-E2. Pigs numbered 6 to 10 were inoculated three times with an empty plasmid, pCDNA3.1+. Both groups were later challenged with virulent Margarita strain of CSFV. Blood sampling were collected at 2, 7, 11, 14 and 17 days post challenge. Pig number 3 (vaccinated animal) and pig 6-10 were euthanized at 14 days post challenge.

Figure 4 :

 4 Figure 4: Induction of specific CSFV IFN gamma producing cells in vaccinated and non vaccinated animals at 7 days post challenge. Pigs numbered 1 to 5 were immunized DNA-E2 (black bars). Pigs numbered 6 to 10 were inoculated three times with an empty plasmid, pCDNA3.1+ (grey bars). Both groups were later challenged with virulent CSFV Margarita strain. Specific-CSFV IFN gamma producing cells were expressed as spot number per 5x10 5 PBMC.

Figure 5 :Figure 6 :

 56 Figure 5: IFN alpha levels in serum at 2 and 7 days post challenge with CSFV. Pigs numbered 1 to 5 were immunized DNA-E2. Pigs numbered 6 to 10 were inoculated three times with an empty plasmid, pCDNA3.1+. Both groups were later challenged with virulent CSFV Margarita strain. Black bars indicate IFN alpha levels at 2 days after challenge and grey bars indicate levels at day 7 post challenge.

  Figure 2:

  Figure 3: