Veterinary Microbiology 169 (2014) 8–17

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Efficacy of marker vaccine candidate CP7_E2alf against challenge with classical swine fever virus isolates of different genotypes Sandra Blome a,*, Claudia Gabriel a, Stefanie Schmeiser b, Denise Meyer b, Alexandra Meindl-Bo¨hmer b,1, Frank Koenen c, Martin Beer a a

Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Suedufer 10, 17493 Greifswald – Insel Riems, Germany European Union Reference Laboratory for CSF, Institute of Virology, Department of Infectious Diseases, University of Veterinary Medicine Hannover, Buenteweg 17, 30559 Hannover, Germany c CODA-CERVA, Groeselenberg 99, 1180 Ukkel, Belgium b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 1 March 2013 Received in revised form 30 November 2013 Accepted 2 December 2013

Classical swine fever (CSF) is among the most important viral disease of domestic and feral pigs and has a serious impact on animal health and pig industry. In most countries with industrialized pig production, prophylactic vaccination against CSF is banned, and all efforts are directed towards eradication of the disease, e.g. by culling of infected herds and animal movement restrictions. Nevertheless, emergency vaccination remains an option to minimize the socio-economic impact of outbreaks. For this application, potent vaccines are needed that allow differentiation of infected from vaccinated animals. Among the promising candidates for next generation marker vaccines is the chimeric pestivirus CP7_E2alf. Efficacy studies are usually carried out using highly virulent CSFV strains of genotype 1 that do not mirror the current field situation where strains of genotype 2 predominate. To prove that CP7_E2alf also protects against these strains, efficacy was assessed after single oral vaccination of wild boar and single intramuscular vaccination of domestic pigs using challenge models with recent CSFV strains and the highly virulent strain ‘‘Koslov’’ (genotype 1.1). It could be demonstrated that CP7_E2alf pilot vaccine batches for intramuscular and oral use were able to protect pigs from challenge infection with a highly virulent CSFV. Moreover, solid protection was also achieved in case of challenge infection with recent field strains of genotypes 2.1 and 2.3. Thus, broad applicability under field conditions can be assumed. ß 2013 Elsevier B.V. All rights reserved.

Keywords: Classical swine fever Vaccine candidate CP7_E2alf Oral vaccination wild boar Intramuscular vaccination domestic pigs Efficacy against field strains

1. Introduction

* Corresponding author. Tel.: +49 38351 71144; fax: +49 38351 71275. E-mail address: sandra.blome@fli.bund.de (S. Blome). 1 Present address: Lower Saxony State Office for Consumer Protection and Food Safety, Veterinarian Task Force, Eintrachtweg 19, 30173 Hannover, Germany. 0378-1135/$ – see front matter ß 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2013.12.002

Classical swine fever (CSF) is among the most important infectious disease that threaten profitable and sustainable pig production world-wide (Edwards et al., 2000). For this reason CSF outbreaks are notifiable to the World Organization of Animal Health (OIE). It is caused by an enveloped RNA virus of the genus Pestivirus within the Flaviviridae family (Lindenbach et al., 2007).

S. Blome et al. / Veterinary Microbiology 169 (2014) 8–17

Due to trade restrictions that are imposed on vaccinated animals and their products, prophylactic vaccination against CSF is prohibited within the European Union (EU) and in many other countries with industrialized pig production. Here, the ultimate goal is to eradicate the disease through strict control measures, like culling of infected herds, animal movement restrictions, and sanitary measures. Nevertheless, emergency vaccination remains an option to minimize the socio-economic impact of outbreaks and legal provisions are laid, e.g. in community legislation (van Oirschot, 2003). For this particular use, the ideal vaccine should not only confer fast and solid protection, but also offer differentiability of infected from vaccinated animals (DIVA). Following this principle, deviations from the above-mentioned longlasting trade restrictions are feasible. So far, all commercially available vaccines have drawbacks in one or both of these prerequisites that hamper implementation (Beer et al., 2007). As an important epidemiological and complicating fact, wild boar can be affected by the disease and were shown to pose a considerable risk for disease introduction into domestic herds (Fritzemeier et al., 2000). To control the disease in the wildlife host, and to protect the domestic pig population from introduction, oral emergency vaccination of wild boar is nowadays regularly implemented (Rossi et al., 2010). Also in this case, a DIVA vaccine would be of great benefit for strategy design. Only live vaccines are suitable for oral use and at the moment, no modified live marker vaccine is available for this application. So far, in areas where the disease is spread among the wild boar population and vaccination is carried out, animals cannot be identified as infected or vaccinated by serological testing. Among the promising candidates for a next generation of marker vaccines for parenteral and oral vaccination is the chimeric pestivirus CP7_E2alf (Reimann et al., 2004). It consists of a Bovine viral diarrhea virus (BVDV) backbone containing the main immunogen of CSF virus (CSFV), the surface glycoprotein E2. This vaccine candidate has proven safety for target and non-target species as well as high efficacy both after oral and intramuscular vaccination (Blome et al., 2012; Eble et al., 2012; Gabriel et al., 2012; Koenig et al., 2007a, 2007b; Ko¨nig et al., 2011; Leifer et al., 2009; Tignon et al., 2010). Following the guidelines of the European Pharmacopoeia and the World Organization for Animal Health Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, vaccination-challenge-trials are usually carried out using highly virulent strains of CSFV genotype 1.1 that do not necessarily reflect the circulating isolates in the field but match the genotype of the vaccine strains. Thus, efficacy against field strains may often only be assumed. Moreover, efficacy studies in the main target species for oral vaccination, the wild boar are rare. For this reason, the presented studies were conducted to supplement the existing efficacy data. In the first experiment, efficacy of CP7_E2alf against highly virulent challenge with the genotype 1 CSFV strain ‘‘Koslov’’ was assessed after single oral vaccination of wild boar or single intramuscular vaccination of domestic pigs,

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respectively. This approach was undertaken to generate a harmonized comparator for the below-mentioned studies. The main experimental parts aimed at challenge studies with recent field isolates. Here, two field isolates of genotypes 2.1 and 2.3 were used.

2. Materials and methods 2.1. Experimental settings and animals The whole study comprised three animal experiments of which two (trials A and B) were carried out at the Friedrich-Loeffler-Institut (FLI), Isle of Riems, Germany. The third trial (trial C) was carried out at the EU Reference Laboratory (EURL) for CSF in Hannover, Germany. All trials were carried out under appropriate high containment conditions taking into account animal welfare regulations and standards according to EU Directive 2010/63/EU and institutional guidelines. Animals were kept litter-less, were fed a commercial pig feed for their age class, and had access to water ad libitum. In all trial parts, animals were monitored for clinical signs upon vaccination and challenge using the clinical score system according to Mittelholzer et al. (2000) with slight modifications. In addition, rectal body temperatures were assessed and recorded daily for domestic pigs and at the day of blood sampling for wild boar. Fever was defined as a body temperature  40.0 8C for at least two consecutive days. Elevated body temperatures  40.0 8C for just one day were reported as such. A detailed overview of the different experimental setups outlined below is presented in Table 1. 2.1.1. Trial A: efficacy against challenge with CSFV strain ‘‘Koslov’’ Trial A was designed to confirm efficacy of CP7_E2alf pilot vaccines against highly virulent challenge with CSFV ‘‘Koslov’’ 14 days after intramuscular vaccination of domestic pigs, and 21 days after oral vaccination of European wild boar. 2.1.2. Trial B: efficacy against challenge with CSFV strain CSF1045 ‘‘Roesrath’’ To evaluate the efficacy against current field isolates of wild boar origin, domestic pigs and European wild boar were challenged with CSFV ‘‘Roesrath’’ (genotype 2.3, Germany 2009). Oro-nasal challenge was performed 15 days after intramuscular vaccination of domestic pigs and 21 days after oral vaccination of wild boar with a CP7_E2alf. 2.1.3. Trial C: efficacy against challenge with CSFV strain CSF1047 Trial C was carried out with another currently circulating genotype (CSF1047, Israel 2009, genotype 2.1). This virus was used for challenge infection of five domestic weaner pigs 14 days after intramuscular vaccination with CP7_E2alf. All trials included appropriate unvaccinated challenge controls (see Table 1).

S. Blome et al. / Veterinary Microbiology 169 (2014) 8–17

10 Table 1 Experimental design. Trial

Animals

Vaccination

Challenge

Sampling

Trial A (FLI)

5 Domestic weaner pigs (6–8 weeks of age)

CP7_E2alf Batch 280710 104.6 TCID50/ml 1 ml i.m. Challenge controls

CSFV ‘‘Koslov’’ gt 1.1 107.14 TCID50, oro-nasally 14 dpv

Serum at 0 dpv, 0 dpc, 23 dpc; additional blood and tonsil samples at necropsy

CP7_E2alf Batch 081010 106.1 TCID50/ml 1.6 ml orally Challenge controls

CSFV ‘‘Koslov’’ gt 1.1 1.5  107.14 TCID50, oro-nasally 21 dpv

Serum at 0 dpv, 0 dpc, 18 dpc; blood samples 5 dpc; additional blood and tonsil samples at necropsy

CP7_E2alf VMRD 11-008 1:10 103.7 TCID50/ml 1 ml i.m. Challenge controls

CSFV ‘‘Roesrath’’ gt 2.3 2  106 TCID50, oro-nasally 15 dpv

Serum and EDTA samples 0 dpv

CP7_E2alf Batch 081010; 1:3 105.6 TCID50/ml 1.6 ml orally Challenge controls

CSFV ‘‘Roesrath’’ gt 2.3 2  106 TCID50, oro-nasally 21 dpv

Serum and EDTA samples 0 dpv and 0, 4, 7, 10, 14, 21, 28 dpc; organ samples at necropsy

CP7_E2alf Batch 081010 106.1TCID50/ml 1 ml i.m. Challenge controls

CSFV ‘‘CSF1047’’ gt 2.1 3.6  105 TCID50, oro-nasally 14 dpv

0 dpv, 0, 3, 7, 10, 14, 21 dpc (22 and 41 dpc); blood and organ samples at necropsy

2 Domestic weaner pigs (6–8 weeks of age) 5 Wild boar piglets (3–4 month of age) 2 Wild boar piglets (3–4 month of age) Trial B (FLI)

5 Domestic weaner pigs (6–8 weeks of age) 2 Domestic weaner pigs (6–8 weeks of age) 5 Wild boar piglets (3–4 month of age) 2 Wild boar piglets (3–4 month of age)

Trial C (EURL)

5 Domestic weaner pigs (6–8 weeks of age) 4 Domestic weaner pigs (6–8 weeks of age)

and 0, 4, 7, 10, 14, 21, 28 dpc; organ samples at necropsy

i.m., intramuscularly, gt, genotype, dpv, days post vaccination, dpc, days post challenge.

2.2. Vaccine and challenge viruses 2.2.1. Vaccine batches Information on the used vaccine batches, vaccine dilutions, and their respective titres are presented in Table 1; titres obtained in the respective back-titrations can be found in Supplementary Table 1. Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.vetmic.2013.12.002. 2.2.2. Challenge viruses For trial part A, CSFV strain ‘‘Koslov’’ was obtained from the German National Reference Laboratory (NRL) for CSF (FLI, Insel Riems, Germany). This well characterized challenge strain belongs to genotype 1.1 and is highly virulent for all age classes of animals. It causes severe acute disease but rather unspecific clinical signs including central nervous disorders. The ‘‘Koslov’’ virus originated from an animal experiment at the FLI where whole blood from a viraemic animal was collected. This material was subsequently defibrinated, aliquoted, and titrated. For challenge infection, approx. 1 ml was applied to each animal by oro-nasal inoculation using a 2 ml syringe without needle. For challenge infection in trial part B, CSFV strain ‘‘Roesrath’’ (CSF1045) was obtained from the German NRL. This strain shows an age-dependent clinical course and belongs to genotype 2.3. Clinical signs include several unspecific symptoms but also haemorrhagic lesions. It was originally isolated from a German wild boar piglet in 2009 (Leifer et al., 2010). The virus used in this trial was

passaged twice on permanent porcine kidney cells (PK15) and was applied after dilution in cell culture medium. In trial part C, a CSFV isolate was used that originated from an outbreak in Israel that affected a farm of domestic pigs close to the Lebanese border in 2009 (David et al., 2011). This isolate belongs to genotype 2.1 and caused high fever, severe unspecific lesions, neurological symptoms, and abortions under field conditions. The isolate was obtained from the virus collection of the EURL for CSF and passaged four times on PK15 cells prior to inoculation. 2.3. Cells and test viruses Cells for neutralization assays were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% foetal bovine serum (free of pestiviruses and antipestivirus antibodies) at 37 8C in a humidified atmosphere containing 5% CO2. Porcine kidney cell line 15 (PK15/5; CCLV-RIE0005), Madin–Darby bovine kidney cells (MDBK; CCLV-RIE261), and sheep foetal thymus cells (SFT-R; CCLVRIE0043) were obtained from the Collection of Cell Lines in Veterinary Medicine (CCLV, FLI, Insel Riems, Germany). CSFV strain ‘‘Alfort/187’’, BVDV strain ‘‘NADL’’ and Border disease virus (BDV) strain ‘‘Moredun’’ were obtained from the NRL for CSF (FLI, Insel Riems, Germany) for use in neutralization tests. 2.4. Laboratory investigations 2.4.1. Processing of samples EDTA blood as well as serum samples, which were obtained from native blood by centrifugation at 2031  g at

S. Blome et al. / Veterinary Microbiology 169 (2014) 8–17

20 8C for 20 min, were aliquoted and stored at 70 8C. Tissue samples of tonsils and lymphnodes collected at necropsy were stored at 70 8C until further use. For realtime reverse transcription polymerase chain reaction (RTqPCR) and virus isolation, tissue samples were homogenized in 1 ml DMEM using a TissueLyser II (QIAGEN GmbH, Hilden, Germany). All subsequent laboratory protocols were harmonized across the different trials. 2.4.2. Virus detection To obtain the administered titres of vaccines and challenge viruses, titrations were performed by end point dilution on PK15 cells using standard procedures. The titres expressed as tissue culture infectious doses 50% (TCID50) per ml were obtained by indirect immunoperoxidase staining of heat-fixated cells which was performed 72 h post inoculation using a mouse-antiCSFV-E2 monoclonal antibody mix and a polyclonal goat anti-mouse horseradish peroxidase conjugated secondary antibody (Thermo Fisher Scientific, Waltham, USA). Manual extraction of viral RNA from all EDTA blood or serum samples was performed using the QIAamp1 Viral RNA Mini Kit (QIAGEN GmbH, Hilden, Germany) according to manufacturer’s recommendations. From all tissue samples, viral RNA was extracted using the RNeasy Mini Kit (QIAGEN GmbH, Hilden, Germany). In both extraction methods, an internal control RNA (IC2) was added as described previously (Hoffmann et al., 2006). Viral genome was detected by RT-qPCR using either the real-time PCR-cycler MX3005PTM (Stratagene, La Jolla, USA) or the C1000TM Thermal cycler in combination with the CFX96TM Real-Time System (Bio-Rad Laboratories GmbH, Mu¨nchen, Germany). To determine the absence of pestiviral infections in the animals prior to vaccination, all sera were tested in a panpestivirus specific RT-qPCR as published previously (Hoffmann et al., 2006). Upon challenge infection, a CSFV specific RT-qPCR protocol was used for EDTA blood and organ samples (Hoffmann et al., 2005). Results were recorded as quantification cycle (cq) values. Virus isolation was carried out on all RT-qPCR positive blood samples and on all tissue samples according to the standard protocols laid down in the Technical Annex of European Commission Decision 2002/106/EC (EU Diagnostic Manual for CSF). Results were assessed after one virus passage using indirect immuno-peroxidase staining as described above. Additionally, antigen detection was carried out on all sera collected after challenge infection using the HerdChek1 CSFV Ag/Serum ELISA (IDEXX Laboratories, Hoofddorp, The Netherlands) following the standard protocol provided by the manufacturer. 2.4.3. Antibody detection All sera were tested in neutralization peroxidase-linked antibody assays (NPLA) according to the EU Diagnostic Manual and the Technical Annex accompanying it. Neutralizing antibody titres against CSFV ‘‘Alfort/187’’ were determined over the course of the trial on PK15 cells. For the samples taken prior to vaccination, these tests were supplemented with NPLAs utilizing BVDV ‘‘NADL’’ on MDBK cells, and BDV ‘‘Moredun’’ on SFT-R cells to prove

11

freedom from other (ruminant) pestiviral antibodies. Indirect immuno-peroxidase staining was performed as mentioned above. Titres were calculated as neutralization doses 50% (ND50). Furthermore, sera were tested for the presence of CSFV E2-specific antibodies with the HerdChek1 CSFV Ab ELISA (IDEXX Laboratories, Hoofddorp, The Netherlands) and for the detection of CSFV Erns-specific antibodies using the PrioCHECK1 Erns ELISA (Prionics Lelystad BV, Lelystad, The Netherlands) according to the manufacturer’s instructions. 3. Results 3.1. Clinical signs and pathological lesions 3.1.1. Trial A (CSFV 1.1 challenge) All animals remained healthy during the vaccination phase of the experiment (apart from mange in several wild boar that was treated with antiparasitic drugs). Domestic control animals developed fever starting from 3 dpc (see Supplementary Table 2). Along with the fever reaction, these animals developed severe unspecific clinical signs (including severe depression, somnolence, anorexia) from 6 dpc and were euthanized in a moribund state 10 dpc. Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.vetmic.2013.12.002. The intramuscularly vaccinated animals showed a high temperature peak 4 dpc that was not accompanied by any other clinical signs. At this day, rectal body temperatures ranged from 41.6 to 43 8C. Three out of five animals were back to normal one day later. The remaining animals showed transient and intermittent fever up to day 8 dpc (see Supplementary Table 2). Post-mortem examinations revealed CSF specific lesions only in control animals. Wild boar controls developed clinical signs starting 3– 4 dpc. These animals were euthanized moribund at 7 dpc. As also the vaccinees showed slight depression on 4 dpc, animals were blood sampled on that day and their temperature was taken. At this occasion, two out of five vaccinated wild boar had febrile temperatures (40.1 and 40.6 8C). No other clinical signs were observed in vaccinated wild boar. During necropsy, atypical signs including severe mange, verminous pneumonia, and nematode infestation of the gut were observed in control animals. No CSF specific lesions were observed in vaccinees. 3.1.2. Trial B (CSFV 2.3 challenge) In this part, domestic control animals showed first febrile temperatures between 2 and 4 dpc (see Supplementary Table 2). One of the animals showed constant fever from 2 to 16 dpc and was euthanized 16 dpc in a moribund state. This animal showed severe but rather unspecific clinical signs. However, lesion indicative for CSF were observed during post-mortem examination. The second animal showed remittent fever between 4 and 24 dpc. Besides, only moderate depression and conjunctivitis were observed between 9 and 16 dpc. This animal recovered and was slaughtered together with the

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S. Blome et al. / Veterinary Microbiology 169 (2014) 8–17

vaccinees at the end of the trial (28 dpc). At that time point it did not show any pathological lesions indicative for CSF. From the vaccinated animals, four out of five domestic pigs showed slightly elevated temperatures on 5 dpc (all 40.1 8C). Two animals were back to physiological temperatures one day later, whereas two animals showed two or three days with slightly febrile temperatures. During the rest of the trial, sporadic incidents of slightly elevated temperatures were observed, especially at the days of blood sampling (see Supplementary Table 2). Apart from a slight conjunctivitis in two animals, no other signs were observed upon challenge infection. Both control wild boar succumbed to infection. One animal was found dead at 7 dpc after having shown only moderate depression and diarrhoea. Necropsy revealed severe catarrhous enteritis, poor nutritional state, necrotic foci in the tonsils, and focal fibrinous pleuritis. The other control animal developed severe unspecific signs. They were most obvious from 9 to 12 dpc. At 12 dpc, this animal was euthanized. Pathological changes were indicative for CSF (including enlarged and haemorrhagic lymphnodes and necrotic lesions in tonsils and at the ileocaecal valve). At all time-points of temperature measurement (4, 7, 10, 14, and 21 dpc), some vaccinated animals (up to four out of five animals at 4 and 7 dpc, or three animals at 21 dpc) showed febrile temperatures unrelated to any clinical signs. Slight conjunctivitis was seen in two wild boar piglets. No pathological lesions were observed.

(cq ranging from 33 to 37), whereas serum samples taken on 18 dpc were negative in all virological tests. The tonsils of vaccinated wild boar were RT-qPCR positive with cqvalues ranging from 30 to 34. 3.2.2. Trial B Also in this trial, no challenge virus was isolated from samples of vaccinated animals and the antigen ELISA yielded only negative results (see Supplementary Table 3). Nevertheless, two positive reactions in individual domestic animals were observed in RT-qPCR on days 4 dpc and 7 dpc, respectively (cq-values of 40 and 38, see Fig. 1a).

3.1.3. Trial C (CSFV 2.1 challenge) Control animals of trial C showed first obvious signs at 7 dpc. While two animals succumbed to infection between days 14 and 22 post infection, two animals survived. In the animal euthanized at 14 dpc, fever was observed from 4 to 14 dpc (see Supplementary Table 2). The pig euthanized at 22 dpc showed remittent fever from 5 dpc accompanied by unspecific symptoms. The other two animals showed transient fever and clinical signs (7–34 dpc). All vaccinated animals showed elevated temperatures at 5 dpc (40.1 to 40.6 8C). At the following day, only one animal showed still a febrile temperature (40.5 8C). No other clinical signs were observed. The pathological observations mirrored the clinical course. 3.2. Virus detection 3.2.1. Trial A All vaccinated domestic pigs remained negative in virus isolation and antigen ELISA throughout the trial. Likewise, serum samples taken on 23 dpc were all negative in RTqPCR. Yet, all tonsils yielded positive results. For the tonsil samples, cq-values ranged from 29 to 34. In the domestic control group, virus could be isolated from all blood and organ samples taken at necropsy. These samples were also strongly positive in RT-qPCR (data not shown). Sera from the wild boar control group were strong positive in RT-qPCR (cq 18), and in virus isolation at 7 dpc. Likewise, tonsils gave positive results in both RT-qPCR (cq 20) and virus isolation. Serum samples taken from vaccinated wild boar on 5 dpc were positive in RT-qPCR

Fig. 1. RT-qPCR results for trials B and C depicted as 45-cq. (a) Responses of domestic pigs in trial B, (b) wild boar responses in trial B. Pigs of trial C are presented in (c). Vaccinated animal are presented in black lines, controls in grey. dpv, days post vaccination; dpc, days post challenge; ED, end day (slaughter or euthanasia); dp, domestic pig; wb, wild boar.

S. Blome et al. / Veterinary Microbiology 169 (2014) 8–17

Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.vetmic.2013.12.002. In contrast, four out of five orally vaccinated wild boar were positive at 4 dpc with cq-values ranging from 37 to 40 (see Fig. 1b and Supplementary Table 3). On day 7 post challenge (pc), three animals were still positive of which two remained positive also on day 10 pc. All positive results showed very high cq-values (>36). No positive reactions occurred after day 10 pc (see Fig. 1b and Supplementary Table 3). Tonsil samples from all vaccinees were positive in RT-qPCR with cq-values ranging from 30 to 40, but no virus was ever isolated. In lymphnodes, low amounts of viral genome could be detected in four orally and two intramuscularly vaccinated animals (all cq-values >33). EDTA blood samples of domestic control animals became positive in RT-qPCR at 4 dpc (see Fig. 1a). At 7 and 10 dpc, positive RT-qPCR results were accompanied by positive virus isolations and antigen detection in the respective ELISA (see Supplementary Table 3). For the animal euthanized due to the severe symptoms, RT-qPCR and antigen ELISA remained positive until euthanasia on 16 dpc. Virus isolation was negative on 14 dpc but positive again on day 16 dpc. The surviving control pig was positive in RT-qPCR until the end of the trial but negative in antigen ELISA and virus isolation from 14 dpc onwards (see Supplementary Table 3). Tonsil and lymphnode samples from this pig taken at necropsy were still positive in RTqPCR (cq-values > 30) but negative in virus isolation. Wild boar controls were positive in RT-qPCR and virus isolation starting from 4 dpc. Thereafter, these findings were accompanied by positive virus isolations till the day of euthanasia respective death (see Supplementary Table 3). Organ samples derived from animals succumbing to infection showed high genome loads (cq-values ranging from 19 to 26) and were positive in virus isolation. 3.2.3. Trial C In the vaccinated animals neither virus nor antigen was detected throughout the trial (see Supplementary Table 3), but again, viral genome was sporadically detected (see Fig. 1c). In detail, two animals were positive at 7 dpc (cqvalues of 32 and 38, respectively), and three animals were positive at 10 dpc (cq-values of 34–39). One animal remained weak positive also on days 14 (cq-value 35) and 21 pc (cq-value 40). All tonsil samples were positive for viral genome with cq-values ranging from 30 to 35. Virus could not be isolated from sera and tonsils of vaccinated animals. Three out of four control animals showed positive reactions in RT-qPCR from 3 dpc (see Fig. 1c and Supplementary Table 3). At this time point virus was already isolated from one of the control animals. On day 7 and 10 pc all control animals were positive for viral genome, antigen and virus (see Supplementary Table 3). On day 14 pc three animals were still positive in all virological tests, whereas one animal showed a negative result in virus isolation at that time point. All remaining control animals were positive again in virus isolation and RT-qPCR 21 dpc. Two of these animals were also positive in

13

antigen ELISA, the third was just below the cut-off. The same pattern was seen for the latter animal one day later when the animal was euthanized. The other controls remained positive in all virological tests until euthanasia at 41 dpc (see Supplementary Table 3). Tonsil samples of all control animals were strong positive for viral genome (cqvalues < 20). 3.3. Antibody detection 3.3.1. Trial A At the day of challenge, four out of five vaccinated domestic pigs showed low but measurable antibody titres in NPLAs with CSFV ‘‘Alfort/187’’ (see Fig. 2A1). Of these animals, two were positive in the E2 antibody ELISA (43% and 78% inhibition), another two were doubtful (33% and 39% inhibition) and one was just below the cut-off (29.8% inhibition). Of the wild boar, all were positive in ‘‘Alfort/ 187’’ NPLAs (see Fig. 2A1). These values corresponded to 100% positive results in E2 antibody ELISAs (79–88% inhibition, see Fig. 2A2). On 23 dpc and 18 dpc, respectively, all vaccinees had developed high neutralizing antibody titres (all exceeding 480 ND50) accompanied by positive E2 ELISA results with high inhibition percentages (all exceeding 84%). Moreover, all vaccinees had seroconverted to CSFV Erns by the end of the trial, with a first Erns antibody detection as early as 5 dpc for three wild boar (see Fig. 2A3). None of the control animals developed measurable E2 or Erns antibodies (see Fig. 2A2 and A3). 3.3.2. Trial B Four out of five intramuscularly vaccinated animals of trial B showed measurable antibodies in NPLA with ‘‘Alfort/ 187’’ at 15 dpv (see Fig. 2B1 and Supplementary Table 4). The titre range was comparable to trial part A (mean titre of 15 ND50). Here, only one animal showed a doubtful result in the E2 ELISA (36% inhibition), and two other animals were close to the cut-off (27% and 28% inhibition). In this trial part, four out of five orally vaccinated wild boar were positive in E2 antibody ELISA at the day of challenge (21 dpv), another showed a doubtful result (see Fig. 2B2 and Supplementary Table 4). The NPLA utilizing ‘‘Alfort/ 187’’ showed low antibody titres (10–60 ND50) in four out of five animals (see Fig. 2B1). The NPLA result for the animal that was doubtful in ELISA was 10 ND50, whereas another ELISA positive animal (54% inhibition) stayed below the detection limit (640 ND50 (see Fig. 2B1) and inhibition percentages

S. Blome et al. / Veterinary Microbiology 169 (2014) 8–17

Neutralization test

120

(A2)

60 40 20

(B3)

(B2)

60 40 20 (C2)

(C3) 80 60

Experimental time

c

28

dp

c

c dp

dp

21

14

c

c dp

10

dp

14

21

c

c dp

10

dp

7

4

dp

c

c dp 28 c dp c

0

c

20

0

v

20

c

40

dp

Inhibition [%]

40

dp

c

c

dp

dp

21

Experimental time

28

c

c dp

dp

10

14

c

c dp

dp

4

7

0

dp

c

v

0

60

dp

1

80

0

2

100

0

Inhibition [%]

3

dp

40 20

100

0

60

dp

(C1)

80

7

1

80

v

2

100

Inhibition [%]

3

Inhibition [%]

ND50 (log10)

40 20

100

ND50 (log10)

60

c

(B1)

80

dp

1

80

dp

2

(A3)

100

Inhibition [%]

Inhibition [%]

ND50 (log10)

100 3

0

120

(A1)

0

4

CSFV Erns Ab ELISA

CSFV E2 Ab ELISA

4

14

Experimental time

Vaccinated domestic pigs Vaccinated wild boar Unvaccinated domestic pigs Unvaccinated wild boar Fig. 2. Antibody response after oral vaccination of wild boar and intramuscular vaccination of domestic pigs with CP7_E2alf and challenge with CSFV strain CSFV Koslov (A), CSF1045 (B), or CSFV 1047 (C). Results of neutralization tests (NPLA) using CSFV ‘‘Alfort/187’’ (A1–C1) are shown as group mean values, titres are depicted as log 10 neutralization doses 50% (ND50). Neutralization titres < 5 ND50 are given as 1 ND50, neutralization titres exceeding 640 ND50 were set to 641 ND50. Results of the CSFV E2-specific antibody ELISA (A2–C2) and CSFV Erns-specific antibody ELISA (A3–C3) are shown as group reactivity in inhibition percentages. Prior to analysis, all negative inhibition percentages were set to 0. To increase readability of the figure, the following sampling days were reported together: Test results obtained after challenge with CSFV ‘‘Israel’’ at 3 dpc and CSFV ‘‘Koslov’’ at 5 dpc are reported together with findings from 4 dpc after CSFV ‘‘Roesrath’’ challenge. For control animals, values obtained at the day of euthanasia are reported together with the next sampling day of vaccinees, final days of trial A (18/23 dpc) are reported together with 21 dpc of trials B and C. For trial C, final days of control animals are reported under 28 dpc. Dashed lines represent the ELISA cut-off, arrows mark the day of challenge infection. dpv, days post vaccination; dpc, days post challenge.

from 84 to 92 (see Fig. 2B2). For the surviving control pig, neutralizing antibodies were first detected on 21 dpc. In the Erns ELISA, three out of five vaccinated animals were positive by the end of the trial with reactions starting 14 dpc. One vaccinated and one control animal were already positive 10 dpc. Both control pigs were positive for Erns antibodies from 14 dpc onwards (see Fig. 2B3). All orally vaccinated wild boar were positive in ‘‘Alfort/ 187’’ NPLAs and E2 antibody ELISAs from 4 dpc (see Fig. 2B1 and B2 and Supplementary Table 4). Again, titres rose quickly and reached high values and inhibition percentages at the end of the trial. Here, all vaccinated wild boar showed titres exceeding 640 ND50 (see Fig. 2B1) accompanied by E2 ELISA inhibition percentages of 91– 94% (see Fig. 2B2). The control pigs did not show E2 antibodies. In the Erns ELISA, four out of five orally

vaccinated animals were positive from 7 dpc (see Fig. 2B3). From 14 dpc, all oral vaccinees showed positive results till the end of the trial. No Erns antibodies were detected in control wild boar. 3.3.3. Trial C In trial part C, all intramuscularly vaccinated animals showed low antibody titres in NPLA (15–20 ND50, see Fig. 2C1) and positive results in E2 antibody ELISA (65–81% inhibition, see Fig. 2C2) at the day of challenge. Upon challenge infection, titres and inhibition percentages in E2 ELISAs rose for all vaccinated animals (see Fig. 2C1 and C2 and Supplementary Table 4). By the end of the trial, titres in ‘‘Alfort/187’’ NPLAs exceeded 640 ND50, and inhibition percentages ranged from 94% to 95%. Three out of five animals seroconverted for Erns with first positive

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reactions from 7 dpc (see Fig. 2C3). All control animals showed positive reactions in the E2 antibody ELISA at 14 dpc that were not accompanied by positive results in NPLAs (see Supplementary Table 4). At 21 dpc, two out of three remaining controls were positive in E2 ELISA, the other reacted doubtful (see Fig. 2C2). Again, no neutralization titres were obtained (see Fig. 2C1). The two animals euthanized at 41 dpc were negative in E2 ELISA and NPLA at the end of the trial. Erns antibodies were detectable in all controls from day 14 pc (see Supplementary Fig. 4). In all trials, samples of the control animals were found negative in serological tests in the pre-challenge period (see Supplementary Table 4). 4. Discussion Classical swine fever marker vaccine candidate CP7_E2alf has recently proven safety and efficacy against challenge infection with highly virulent CSFV strains of genotype 1 after both intramuscular and oral application (Blome et al., 2012; Eble et al., 2012; Gabriel et al., 2012; Koenig et al., 2007a, 2007b; Ko¨nig et al., 2011; Leifer et al., 2009; Rangelova et al., 2012; Reimann et al., 2004; Tignon et al., 2010). The choice of challenge viruses for previous studies was led by requirements of international guidelines for vaccine testing like the European Pharmacopoeia and the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. In this context, high virulence of challenge strains is mandatory for discrimination of responses. While these strains ensure rather uniform test conditions and reproducible disease courses in control animals, the field situation is not mirrored. CSFV strain ‘‘Koslov’’ and most other highly virulent challenge strains (e.g. ‘‘Margarita’’, ‘‘Washington’’ or ‘‘Quilota’’), belong to genotype 1. Genotype 1 is also found in most vaccine strains and constructs. Also the E2 insert in CP7_E2alf is derived from a genotype 1.1 strain (CSFV ‘‘Alfort/187’’). In contrast, strains of genotype 2, especially 2.3 and 2.1, predominate in several regions of the world including Europe, parts of Africa, and Asia (Biagetti et al., 2001; Blome et al., 2010; David et al., 2011; Jemersic et al., 2003; Jeoung et al., 2013; Jiang et al., 2013; Leifer et al., 2010; Postel et al., 2013; Vlasova et al., 2003; Sandvik et al., 2005). To evaluate the efficacy of CP7_E2alf against these field isolates and to include the target species for oral vaccination, the European wild boar, into the studies, the presented trials were undertaken to supplement the existing data. Challenge times, i.e. 14 days after intramuscular vaccination and 21 days after oral vaccination, were chosen based on the experiences obtained in the comparative trials with CP7_E2alf (Blome et al., 2012; Eble et al., 2012) and on the requirements of the European Pharmacopoeia for intramuscular vaccines. To compare challenge infection with recent field isolates to the established challenge model using highly virulent CSFV strain ‘‘Koslov’’, a first trial (A) was carried out under routine conditions with CSFV strain ‘‘Koslov’’. It could be demonstrated that all vaccinees were protected from lethal challenge, whereas all control animals developed CSF and died. All observations, including reactions of

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wild boar upon oral vaccination, were in line with previous studies in domestic pigs (Blome et al., 2012; Gabriel et al., 2012). In trial parts B and C, protection was demonstrated against challenge with recent field strains of genotypes 2.1 (isolate from Israel) and 2.3 (isolate from Germany). As in previous trials with CSFV ‘‘Koslov’’ challenge, including trial part A, limited replication of challenge viruses occurred accompanied by detectable viral genome and a short fever reaction in most animals. In contrast to all control animals that showed clinical symptoms accompanied by the detection of high viral genome loads, live virus and antigen, none of the vaccinees was found positive in virus isolation (neither in blood nor in organs) or antigen ELISA. For this reason, the impact of transient viral genome detection in terms of virus transmission seems low. This assumption is strengthened by the fact that transmission to vaccinated (Eble et al., 2012) or unvaccinated (Blome et al., 2012) contact controls was not observed in trials with similar challenge schemes (14 days after intramuscular and 21 days after oral vaccination). Among the possible explanations for the low impact of challenge virus detections by real-time RT-PCR is the fact that the limited replication is accompanied by a strong boost of immune responses as evidenced by the antibody titres towards CSFV E2. Neutralizing antibodies are probably able to block further dissemination and shedding of challenge virus. In addition, detection of challenge virus genome seems to be quite restricted to cellular fractions (probably leukocytes) as comparison of EDTA blood and serum led to detection only in EDTA blood (Blome et al., unpublished results). Apart from these biological factors, it has to be kept in mind that high sensitivity of the employed RT-PCR assays nowadays leads to detection of vaccine and challenge virus even with the ‘‘gold standard’’ of CSF vaccines, the C-strain (Blome et al., 2012, 2010). Apart from safety and efficacy aspects, the marker concept is of paramount importance for a potential DIVA vaccine. In all trial parts, the concept worked well after vaccination. None of the animals was detected positive prior to challenge infection. Upon challenge with CSFV ‘‘Roesrath’’, Erns antibodies were developed by all vaccinated and challenged wild boar and by three out of five intramuscularly vaccinated domestic pigs. The same pattern was seen upon challenge with CSF1047. Again, three out of five intramuscularly vaccinated animals seroconverted to CSFV Erns. These findings were also in line with previous observations (Blome et al., 2012; Eble et al., 2012; Gabriel et al., 2012). In both trial parts, several control animals seroconverted very early against Erns (between 10 and 14 dpc), sometimes even earlier than against E2, proving sensitivity of the assay as such. As Erns is secreted by infected cells early in the course of infection and is thus quite easily accessible by the immune system, these responses may not be completely unexpected. This pattern was also observed in other trials by the same group of authors (Petrov and Blome, personal communication). The lack of detectable Erns antibodies in vaccinated pigs could have different explanations. As the animals that stayed negative in the Erns-antibody ELISA had shown none or very low genome loads in RT-qPCR upon challenge, it can be speculated that lacking replication of challenge

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virus, and thus Erns presentation may lead to non-response against Erns. In this case, very solid protection can be assumed and non-responders (false negative animals) are at least not ‘‘dangerous’’ from an animal disease control point of view. The same probably applies for slightly delayed responses against CSFV Erns in vaccinees. In emergency vaccination scenarios, testing will only be done in the context of lifting zones and mostly accompanied by genome detection methods that would rule out CSFV infection prior to onset of clinical symptoms and humoral responses. In general, a ‘‘doubtful’’ range in the ELISA interpretation system might be helpful as the percentage of inhibition for these animals were quite close to the cut-off. Furthermore, confirmatory assays are needed, especially to reduce false positive reactions. Such concepts have been designed (Aebischer et al., 2013) and are under evaluation. In trial part C, interesting findings were observed in the group of control pigs. The parallel existence of positive reactions in antibody ELISA but not in NPLA together with positive results in virology suggest that these control animals were probably chronically infected with the challenge virus. 5. Conclusions Within the framework of the presented studies it could be demonstrated that CP7_E2alf does not only protect domestic pigs and European wild boar against highly virulent challenge with a challenge strain representing the same genotype as the E2 in the chimaera, but also against recent field strains of genotype 2. Thus, field applicability was confirmed. Acknowledgements We would like to thank all animal caretakers and technicians for their excellent work. CP7_E2alf pilot vaccine was kindly provided by Pfizer Olot S.L.U., Vall de Bianya (Girona), Spain. The research leading to these results has received funding from the European Community’s Seventh Framework (FP7/2007–2013) under grant agreement no. 227003 CP-FP (CSFV_goDIVA). References Aebischer, A., Mu¨ller, M., Hofmann, M.A., 2013. Two newly developed E(rns)-based ELISAs allow the differentiation of Classical Swine Fever virus-infected from marker-vaccinated animals and the discrimination of pestivirus antibodies. Vet. Microbiol. 161, 274–285. Beer, M., Reimann, I., Hoffmann, B., Depner, K., 2007. Novel marker vaccines against classical swine fever. Vaccine 25, 5665–5670. Biagetti, M., Greiser-Wilke, I., Rutili, D., 2001. Molecular epidemiology of classical swine fever in Italy. Vet. Microbiol. 83, 205–215. Blome, S., Aebischer, A., Lange, E., Hofmann, M., Leifer, I., Loeffen, W., Koenen, F., Beer, M., 2012. Comparative evaluation of live marker vaccine candidates ‘‘CP7_E2alf’’ and ‘‘flc11’’ along with C-strain ‘‘Riems’’ after oral vaccination. Vet. Microbiol. 158, 42–59. Blome, S., Grotha, I., Moennig, V., Greiser-Wilke, I., 2010. Classical swine fever virus in South-Eastern Europe – retrospective analysis of the disease situation and molecular epidemiology. Vet. Microbiol. 146, 276–284.

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Efficacy of marker vaccine candidate CP7_E2alf against challenge with classical swine fever virus isolates of different genotypes.

Classical swine fever (CSF) is among the most important viral disease of domestic and feral pigs and has a serious impact on animal health and pig ind...
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