Vaccine 32 (2014) 2050–2055

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First assessment of classical swine fever marker vaccine candidate CP7 E2alf for oral immunization of wild boar under field conditions Francesco Feliziani a , Sandra Blome b , Stefano Petrini a , Monica Giammarioli a , Carmen Iscaro a , Giulio Severi a , Luca Convito c , Jana Pietschmann b , Martin Beer b , Gian Mario De Mia a,∗ a

Istituto Zooprofilattico Sperimentale Umbria e Marche, via Salvemini 1, 06126 Perugia, Italy Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Suedufer 10, 17493 Greifswald-Insel Riems, Germany c Servizio gestione faunistica e protezione ambientale, Provincia di Perugia, via Pievaiola 140, 06132 Perugia, Italy b

a r t i c l e

i n f o

Article history: Received 13 December 2013 Received in revised form 31 January 2014 Accepted 6 February 2014 Available online 22 February 2014 Keywords: Classical swine fever virus (CSFV) Marker vaccine candidate CP7 E2alf Oral immunization Wild boar Field trial

a b s t r a c t Oral vaccination against classical swine fever (CSF) is a potent tool to control disease outbreaks in wild boar. So far, vaccination campaigns have been carried out using live attenuated vaccines that do not allow serological differentiation of infected from vaccinated animals (DIVA). Although this drawback is acceptable for wild boar, the use of marker vaccines would facilitate studies on disease and vaccination dynamics. Recently, the CSF marker vaccine candidate CP7 E2alf was assessed for oral immunization under laboratory conditions. Promising results prompted efforts to study the vaccine candidate under field conditions and in bait formulation. In this context, two oral vaccination campaigns were carried out with CP7 E2alf bait vaccines in two areas called ‘faunistic-hunting farms’ in the region of Umbria, Italy. One campaign was conducted using single vaccination, the second with the routinely employed double vaccination strategy. Both campaigns were carried out before concerted hunting actions were performed. Bait uptake, vaccine virus detection and antibody responses were assessed along with inspections upon gutting. As a comparator, seven wild boar were hand-fed with baits under laboratory conditions. In the field, bait uptake ranged from 63.7% to 98.7%, whereas antibody prevalence reached only 33.3–35.1%. The marker serology showed a strong influence of sample quality on the test outcome with a total of 85% of samples being classified correctly. Vaccine virus was not detectable. Under hand feeding conditions, six out of seven wild boar took up at least one bait, and five of them showed detectable antibody levels seven weeks after vaccination. These results were supplemented by stability tests. Appropriate stability of vaccine virus was shown both under field and laboratory conditions. In total, most results were in line with our expectations. However, optimization of the DIVA assay has to be attempted in the future. Published by Elsevier Ltd.

1. Introduction Classical swine fever (CSF) is a severe, multi-systemic viral disease that can affect all species of the Suidae family. The notifiable disease is caused by a small enveloped RNA virus of the genus Pestivirus within the Flaviviridae family [1]. Over the last decades, several European Union (EU) Member States were confronted with outbreaks of CSF among wild boar that had a clear tendency to

∗ Corresponding author at: Istituto Zooprofilattico Sperimentale, dell’Umbria e delle Marche, via Salvemini 1, 06126 Perugia, Italy. Tel.: +39 075 343239. E-mail addresses: [email protected] (F. Feliziani), (S. Blome), [email protected] (S. Petrini), [email protected] (L. Convito), (M. Beer), [email protected] (G.M. De Mia). 0264-410X/Published by Elsevier Ltd.

establish endemicity. Endemically affected wild boar populations are considered an important virus reservoir [2–10], and retrospective analysis of CSF outbreaks in Germany between 1993 and 1997 showed that approximately 60% of the primary outbreaks in domestic pigs could be linked to direct or indirect contact to affected wild boar [3]. To control the disease in wildlife, and thus to safeguard domestic pigs in affected regions, oral emergency vaccination using a C-strain based bait vaccine has proven to be a powerful tool [9–12]. Despite their uncontested virtues, C-strain vaccines have one major disadvantage: serological discrimination of infected from vaccinated animals (DIVA) is impossible. In the context of wild boar vaccination this drawback is acceptable but hampers full evaluation of outbreaks and the implemented control measures. Especially, dynamics of infection under vaccination coverage and true vaccination efficiency remain elusive. Thus, a

F. Feliziani et al. / Vaccine 32 (2014) 2050–2055

marker vaccine is preferable also under these circumstances. So far, no marker vaccine was available for oral immunization as the currently available E2 subunit vaccines are not suitable for oral application. Now, new concepts of vaccine generation have been followed and potent vaccine candidates are available [13]. One vaccine candidate is the chimeric Pestivirus “CP7 E2alf”. This live marker vaccine candidate is based on the cytopathogenic Bovine viral diarrhoea virus (BVDV) strain “CP7” that carries the structural protein E2 of CSFV strain “Alfort/187” [14]. This chimaera proved to be a safe and efficacious marker vaccine candidate for oral and intramuscular vaccination of domestic pigs and European wild boar [15–19], and was chosen after large comparative trials [20,21] as candidate for a licensing request within the EU funded research project “Improve tools and strategies for the prevention and control of classical swine fever” (CSFV goDIVA, KBBE-227003). Together with the bait currently in use with C-strain “Riems”, CP7 E2alf is suitable for emergency vaccination of wild boar. As mentioned above, trials addressing efficacy and safety of oral vaccination were already conducted successfully, but none of these studies was carried out with a bait formulation. While oral immunization through syringe administration ensured comparability of data, conditions in real life were barely mirrored. Prompted by the promising results, efforts were undertaken to conduct studies with bait formulations, and under field conditions. In this context, two oral vaccination campaigns were carried out with CP7 E2alf bait vaccines in two areas called ‘faunistic-hunting farms’ in the region of Umbria, Italy. One campaign was conducted using single vaccination, the second with the routinely employed double vaccination strategy. Both campaigns were carried out before concerted hunting actions were performed. Bait uptake, vaccine virus detection and antibody responses were assessed along with inspections upon gutting. Along with these field trials, a proof-of-principle study was conducted under controlled laboratory conditions with the final bait formulation to assess uptake, safety, and seroconversion upon vaccination with single bait. To supplement these data, stability of vaccine virus was assessed under different ambient temperatures.

2. Materials and methods 2.1. Experimental settings 2.1.1. Field vaccination trials Two areas called ‘faunistic-hunting farms’ were selected for the field trials in the region of Umbria in central Italy. These areas with 5.8 km2 and 11.9 km2 , respectively, are private regional territories where agriculture, cattle breeding, and hunting represent common practices. Wild boar in these areas are not fenced but attracted by feeding in confined places. The scientific field trials were carried out in accordance with Directive 2001/18/EC on the deliberate release of genetically modified organisms into the environment (notification B/IT/10/01). The immunization scheme consisted of two distinct vaccination campaigns. The first was started in September 2011 consisting of a single vaccination carried out one month before concerted hunting. Prior to distribution of vaccine baits, maize was laid out at eleven predetermined feeding places. Depending on the estimated density of the wild boar population, 35–40 vaccine baits per feeding place were laid out manually mixed with maize. In total, 425 baits were distributed. Disappearance of vaccine baits at feeding grounds was checked the day after each distribution. Bait uptake as well as the presence of competitors was monitored by camera traps. One year later (September 2012), an amended protocol was carried out based on a vaccination campaign consisting of a double vaccination with 1-week interval. This scheme was carried out to get closer to current field approaches with C-strain vaccine,


where three double vaccinations are carried out with an interval of 7–14 days [22] and with a four-week interval [9] respectively. This campaign was conducted in the study area covering 5.8 km2 . As in the previous campaign, maize was laid out before each distribution of vaccine baits at eight predetermined feeding places. At these spots, 47–50 vaccine baits were laid out manually mixed with maize. During the hunting action (driven hunt, beatings), all animals shot were submitted to a full inspection upon gutting, and clinical specimens (tonsil, serum/fluid from the chest cavity) were collected. Individual data sets including e.g. sex and estimated age of the wild boar were recorded. 2.1.2. Experimental baiting efficiency After one pre-baiting attempt with empty baits, oral immunization of seven adult wild boar (2 boars, 5 sows) was carried out with one hand-fed “CP7 E2alf” bait per animal in the breeding unit of the Friedrich-Loeffler-Institut (FLI), Isle of Riems, Germany. All animals were used to human handling and thus tolerated to be offered the bait by hand. Upon vaccination, animals were monitored for adverse effects. Seven weeks after vaccination, animals were briefly immobilized with Zoletil® 100 (Virbac) at a dose of 6–9 mg/kg, and native blood samples were collected to assess of seroconversion. 2.1.3. Stability tests In order to evaluate the stability of vaccine virus and thus also the possible persistence in the environment, baits were subjected to different ambient temperatures, namely room temperature (20–23 ◦ C), 37 ◦ C (incubator), 4–6 ◦ C (fridge), and −20 ◦ C (freezer). At days 0, 1–7, 10, 14, 17, 21, 28, 35, 42, 49, and 56, three blisters were opened and the pooled vaccine solution titrated. To supplement these experiments under laboratory conditions with field data, baits recollected 24 h after distribution in the above mentioned field trials were back titrated. 2.2. Vaccine virus The CP7 E2alf vaccine virus stock was produced in bioreactors with swine kidney cells (SK-6) under ‘Good Manufacturing Practice (GMP)’ conditions by Pfizer Olot S.L.U. (Spain). The GMP-produced CP7 E2alf had a virus titre of 105.2 tissue culture infectious doses 50% (TCID50 ) per ml. The vaccine was filled into blisters at a volume of 1.6 ml. Thereafter, blisters were incorporated into the traditional cereal-based bait matrix that is in use also for C-strain vaccines. Filling and preparation of final pilot bait vaccines was done by IDT Biologika GmbH, Dessau-Rosslau, Germany. The size of baits was 3.5 cm × 3.5 cm × 0.7 cm. Baits were shipped on dry ice and stored at −80 ◦ C until their release. 2.3. Laboratory investigations 2.3.1. Virus detection Tonsil samples were tested for vaccine virus genome in reverse transcription polymerase chain reaction (RT-PCR). Extraction of viral RNA from tonsil homogenates was performed using the RNeasy Mini Kit (QIAGEN GmbH, Hilden, Germany) according to manufacturer’s recommendations. Subsequently, viral RNA was detected using the RT-PCR protocol published by Paton et al. in 2000 [24]. The assay amplifies a 190 nucleotides fragment of the E2 envelope glycoprotein. Virus titrations were performed by end point dilution on PK15 cells using standard procedures as laid down in the Technical Annex of the EU Diagnostic Manual (Commission Decision 2002/106/EC). The titres expressed as TCID50 /ml were obtained by indirect immuno-peroxidase staining of heat-fixated cells which


F. Feliziani et al. / Vaccine 32 (2014) 2050–2055

was performed 72 h post inoculation using a mouse-anti-CSFVE2 monoclonal antibody mix and a polyclonal goat anti-mouse horseradish peroxidase conjugated secondary antibody (Thermo Fisher Scientific, Waltham, USA). 2.3.2. Antibody detection Serum or chest cavity fluid samples were tested for CSFV antibodies. All samples 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 on PK15 cells. 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 HerdChek® CSFV Ab ELISA (IDEXX Laboratories, Ludwigsburg, Germany). A set of 20 antibody positive (neutralization tests and/or ELISA) serum samples from the hunting season in 2012 was additionally subjected to the Erns specific antibody using the PrioCHECK® Erns ELISA (Prionics Lelystad BV, Lelystad, The Netherlands) according to the manufacturer’s instructions. The latter accompanies CP7 E2alf as discriminatory assay. 3. Results and discussion 3.1. Field observations 3.1.1. Bait uptake and behavioural evaluation under field conditions The behavioural studies using camera trapping showed that non-target species, such as badgers, roe deer, porcupine and birds were rarely observed consuming baits. According to the season and taking into account the weather conditions it can be stated that the risk of oak mast competing with baits was very low. All wild boars involved in the study showed interest in the baits; however, bait consumption was generally lower in piglets. Disappearance of vaccine baits at feeding grounds was checked 24 h after each distribution. Overall uptake rates ranged from 63.7% to 98.7% on average. In some feeding places, even 100% bait uptake was observed. No noticeable pattern was observed that could explain the observed differences. Taken together, uptake rates were fairly good to excellent with almost no interference with other species. However, these results may have an inherent bias as baiting was done for only 24 h at known wild boar feeding places with animals that were used to maize as feed. Experiences from C-strain vaccination campaigns with the same bait show that uptake can be much lower under truly natural conditions. 3.1.2. Hunting During the first vaccination campaign, nine beatings took place into the defined areas, two into the smaller farm, and seven into the bigger one. Hunting areas, wider in the bigger farm, showed a variable distance from the baiting/feeding places. A total of 168 wild boar was shot and the corresponding tonsil and serum samples (n = 150) were submitted to laboratory investigations. For n = 18 animals, chest cavity fluids replaced serum samples. After the double vaccination campaign, 6 beatings took place and a total of 81 wild boars were shot. It has to be stated that it cannot be guaranteed that only animals were shot that had access to oral immunization. The use of biomarkers could have ensured traceability. However, incorporation of biomarkers such as oxytetracycline (OTC) or iophenoxic acids (IPA) was not possible due to consumer protection issues. For the latter in particular, despite IPA residues in the muscle are supposed to be very low [23], it cannot be excluded the possible long term adverse effect after chronic exposure of humans.

For this reason, only antibody and virus detection was possible as indicator. 3.1.3. Observations upon gutting of hunted animals None of the animals showed signs indicative for CSF or any other lesions that could be attributed to vaccine virus intake. Thus, safety was also confirmed in the field. 3.1.4. Vaccination efficiency (serological and virological responses) Antibody detection. After the first vaccination campaign with single distribution of baits, 59 out of 168 serum samples were found positive in NPLA and/or E2 antibody ELISA (35.1%). Detailed results are given in Table 1. Upon double vaccination, 27 out of 81 animals were detected positive for CSFV antibodies (33.3%). Interestingly, 23 wild boars were found antibody positive out of a total of 49 animals shot in the first 4 beatings (46.9%), whereas only 4 wild boar were found positive out of a hunting bag of 32 shot in the last 2 beatings (12.5%) which took place respectively 47 and 58 days after the previous ones. At least under these settings, double vaccination was not superior over the single approach. Judging on seroprevalence in terms of vaccination efficiency is difficult as the true status of the animals was unknown. The fact that the later beatings resulted in lower seroprevalence may indicate that animals from outside moved into the hunting area. Moreover, antibody titres below the detection limit do not necessarily mean that no protection was conferred. Especially in the early phase upon vaccination, titres of protected vaccinees are usually low [20]. On the other hand, it has been shown that CP7 E2alf vaccine administered orally confers a rapid onset of partial protection by interfering with the effects induced by several cytokines [25]. Comparison with field observations from C-strain vaccination is hampered by the fact that these took place in affected regions where immunity after field virus infection was mixed with vaccine induced antibody titres. Comparing detection methods, the overall concordance between the CSFV E2 antibody ELISA and the NPLA was 78.4%. Discrepant results were observed for 56 out of the 259 serum samples (21.6%) tested. In particular, 55 samples were detected positive using NPLA (with low neutralizing antibody titres ranging from 1/5 to 1/10, data not shown) and negative with the CSFV E2 antibody ELISA. This confirms that NPLA is the most sensitive method to detect CSFV antibodies. Virus detection. Viral genome was not detected. This is in contrast to other studies where vaccine virus RNA was found up to 42 days post vaccination in the tonsil [15]. It can be speculated that assay sensitivity might have influenced the outcome. 3.1.5. DIVA properties From the hunting season in 2012, 20 serum samples were chosen for additional testing in the Erns antibody ELISA. These sera had been found positive in either NPLA and/or E2 antibody ELISA. It has to be noted that at the time of the trial, the Erns ELISA was under revalidation and only a limited number of tests was possible. In total, three out of 20 samples were found positive (see Fig. 1). One of the samples was just above the cut-off of 40% inhibition, two others were clearly positive (see Fig. 1). Based on the very limited sample, specificity of the test amounts to 85% (with 15% false positive reactions). The false positive reactions were linked to a certain extent to suboptimal sample quality (haemolytic serum). However, room for improvement is clearly visible. This could include more stringent ELISA washing conditions or cut-off amendments, e.g. inclusion of a doubtful range. In addition to future optimization, additional Erns tests could be most beneficial. Such tests have been recently described and are under further development [26].

F. Feliziani et al. / Vaccine 32 (2014) 2050–2055


Table 1 Proportion of antibody positive wild boar after oral immunization against CSF. Immunization period



Proportion (%) of CSFV antibody positive animals per age group ≤1 year

>1 ≤ 2 years

>2 years (adults)

On average

First vaccination campaign 07/09/2011

Promano 1 Promano 2 Promano 3 Promano 4 Promano 5 Promano 6 Promano 7 Spoleto 8 Spoleto 9

07/10/2011 21/10/2011 22/10/2011 25/11/2011 14/11/2011 26/11/2011 30/12/2011 30/10/2011 29/12/2011

0/4 0/3 0/2 3/9 10/26 1/1 8/15 2/4 0/0

0/1 0/2 2/2 0/0 0/1 0/0 0/0 0/0 0/0

0/5 3/14 5/15 6/10 6/26 0/3 3/9 7/11 3/5

0/10 3/19 7/19 9/19 16/53 1/4 11/24 9/15 3/5

24/64 (37.50)

2/6 (33.33)

33/98 (33.67)

59/168 (35.11)

0/0 1/2 4/10 0/0 0/6 0/1

1/6 2/2 0/0 2/3 0/4 2/8

3/5 7/10 3/9 0/2 2/10 0/3

4/11 10/14 7/19 2/5 2/20 2/12

5/19 (26.31)

7/23 (30.43)

15/39 (38.46)

27/81 (33.33)

Total Double vaccination campaign 06/09/2012 13/09/2012

Promano 1 Promano 2 Promano 3 Promano 4 Promano 5 Promano 6

19/10/2012 20/10/2012 16/11/2012 17/11/2012 07/12/2012 18/12/2012


In direct comparison, 11 out of 19 tested samples were positive in the E2 antibody ELISA (see Fig. 1). No correlation was seen between high E2 antibodies and false positive reactions in the Erns ELISA. 3.2. Observations under experimental conditions While all females ate the one bait that was offered, one male was not interested at all. While trying to force-feed this animal, the second male took up a total of 5 baits. No adverse effects occurred upon vaccination. After seven weeks, all but one animal and the male that had not eaten the bait had seroconverted. Antibody titres mirrored the uptake ranging from 6 to 480 ND50 , the latter in the animal that received 5 vaccine doses. An overview is given in Table 2. As expected, oral vaccination using a bait is much more error prone and dependent on the animal’s attitude than oral syringe administration, even under controlled experimental conditions.

However, it could be demonstrated that oral uptake of one bait led to seroconversion in most animals (five out of six that took at least one bait). The antibody titre increased with the uptake of more than one bait. In general it can be concluded that titres after seven weeks were low but detectable. 3.3. Stability of vaccine virus under different environmental conditions As shown in Fig. 2, the bait vaccines proved to be highly stable at −20 ◦ C and 4 ◦ C. Up to D.56, only biological variance (±0.5 log titre) occurred. At room temperature, titres decreased slowly until no virus was detectable at D.35. Assuming that 103 –104 TCID50 present the minimum for reliable induction of protective immunity upon oral vaccination [17, and personal observations with C-strain vaccines], baits would fulfil this prerequisite for approximately two

Fig. 1. Comparison of E2 and Erns antibody ELISA results based on a set of 20 samples that were found positive in NPLA and/or E2 ELISA during hunting season 2012. Both ELISAs have a positive cut-off of 40% inhibition (presented by a line).


F. Feliziani et al. / Vaccine 32 (2014) 2050–2055

Fig. 2. Vaccine virus stability under different ambient temperatures (laboratory conditions). Titres for different time points were obtained by double end-point dilution of three pooled blisters. Titres are expressed as logTCID50 . Table 2 Baiting efficiency using CP7 E2alf bait preparations. Seroconversion was assessed 49 days post vaccination (dpv) using a commercial antibody ELISA (IDEXX CSFV Ab) and a neutralization peroxidase linked assay (NPLA). Results of the ELISA are stated qualitatively with the related inhibition percentages in brackets. Neutralizing antibody titres are presented as neutralization doses 50% (ND50 ). Animal




WB 1, 49 dpv WB 2, 49 dpv WB 3, 49 dpv WB 4, 49 dpv WB 5, 49 dpv WB 6, 49 dpv WB 7, 49 dpv

1 Bait 1 Bait 1 Bait No uptake 1 Bait 1 Bait 5 Baits

Positive (64%) Positive (78%) Positive (80%) Negative (−8%) Negative (−12%) Doubtful (35%) Positive (92%)

20 ND50 80 ND50 80 ND50

First assessment of classical swine fever marker vaccine candidate CP7_E2alf for oral immunization of wild boar under field conditions.

Oral vaccination against classical swine fever (CSF) is a potent tool to control disease outbreaks in wild boar. So far, vaccination campaigns have be...
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