Classical Swine Fever Marker Research Articles

Generation and Efficacy of Two Chimeric Viruses Derived from GPE- Vaccine Strain as Classical Swine Fever Vaccine Candidates.

A previous study proved that vGPE- mainly maintains the properties of classical swine fever (CSF) virus, which is comparable to the GPE- vaccine seed and is a potentially valuable backbone for developing a CSF marker vaccine. Chimeric viruses were constructed based on an infectious cDNA clone derived from the live attenuated GPE- vaccine strain as novel CSF vaccine candidates that potentially meet the concept of differentiating infected from vaccinated animals (DIVA) by substituting the glycoprotein Erns of the GPE- vaccine strain with the corresponding region of non-CSF pestiviruses, either pronghorn antelope pestivirus (PAPeV) or Phocoena pestivirus (PhoPeV). High viral growth and genetic stability after serial passages of the chimeric viruses, namely vGPE-/PAPeV Erns and vGPE-/PhoPeV Erns, were confirmed in vitro. In vivo investigation revealed that two chimeric viruses had comparable immunogenicity and safety profiles to the vGPE- vaccine strain. Vaccination at a dose of 104.0 TCID50 with either vGPE-/PAPeV Erns or vGPE-/PhoPeV Erns conferred complete protection for pigs against the CSF virus challenge in the early stage of immunization. In conclusion, the characteristics of vGPE-/PAPeV Erns and vGPE-/PhoPeV Erns affirmed their properties, as the vGPE- vaccine strain, positioning them as ideal candidates for future development of a CSF marker vaccine.

Construction and efficacy of a new live chimeric C-strain vaccine with DIVA characteristics against classical swine fever.

To develop the new classical swine fever (CSF) vaccine candidate with differentiating infected vaccinated animals (DIVA) characteristics, a chimeric CSF virus (CSFV) was constructed based on an infectious cDNA clone of the CSF vaccine C-strain. The 5'- and 3'-untranslated regions (UTRs) and partial E2 region (residues 690-860) of the C-strain were substituted with the corresponding regions of bovine viral diarrhoea virus (BVDV) to construct the chimeric cDNA clone pC/bUTRs-tE2. The chimeric virus rC/bUTRs-tE2 was generated by several passages of pC/bUTRs-tE2-transfected PK15 cells. Stable growth and genetic properties of rC/bUTRs-tE2 were obtained after 30 serial passages. Compared to parental rC/bUTRs-tE2 (1st passage), two residue mutations (M834K and M979K) located in E2 in rC/bUTRs-tE2 P30 were observed. Compared to the C-strain, rC/bUTRs-tE2 exhibited unchanged cell tropism and decreased plaque-forming ability. Substituting the C-strain UTRs with the BVDV UTRs resulted in significantly increased viral replication in PK15 cells. Compared to CSFV Erns-positive and BVDV tE2-negative antibody responses induced by the CSF vaccine C-strain, immunization of rabbits and piglets with rC/bUTRs-tE2 resulted in serological profiles of CSFV Erns- and BVDV tE2-positive antibodies, which are used to serologically discriminate pigs that are clinically infected and vaccinated. Vaccination of piglets with rC/bUTRs-tE2 conferred complete protection against lethal CSFV challenge. Our results suggest that rC/bUTRs-tE2 is a promising new CSF marker vaccine candidate.

The Development of Classical Swine Fever Marker Vaccines in Recent Years

Classical swine fever (CSF) is a severe disease that has caused serious economic losses for the global pig industry and is widely prevalent worldwide. In recent decades, CSF has been effectively controlled through compulsory vaccination with a live CSF vaccine (C strain). It has been successfully eradicated in some countries or regions. However, the re-emergence of CSF in Japan and Romania, where it had been eradicated, has brought increased attention to the disease. Because the traditional C-strain vaccine cannot distinguish between vaccinated and infected animals (DIVA), this makes it difficult to fight CSF. The emergence of marker vaccines is considered to be an effective strategy for the decontamination of CSF. This paper summarizes the progress of the new CSF marker vaccine and provides a detailed overview of the vaccine design ideas and immunization effects. It also provides a methodology for the development of a new generation of vaccines for CSF and vaccine development for other significant epidemics.

Characteristics of Classical Swine Fever Virus Variants Derived from Live Attenuated GPE- Vaccine Seed.

The GPE− strain is a live attenuated vaccine for classical swine fever (CSF) developed in Japan. In the context of increasing attention for the differentiating infected from vaccinated animals (DIVA) concept, the achievement of CSF eradication with the GPE− proposes it as a preferable backbone for a recombinant CSF marker vaccine. While its infectious cDNA clone, vGPE−, is well characterized, 10 amino acid substitutions were recognized in the genome, compared to the original GPE− vaccine seed. To clarify the GPE− seed availability, this study aimed to generate and characterize a clone possessing the identical amino acid sequence to the GPE− seed. The attempt resulted in the loss of the infectious GPE− seed clone production due to the impaired replication by an amino acid substitution in the viral polymerase NS5B. Accordingly, replication-competent GPE− seed variant clones were produced. Although they were mostly restricted to propagate in the tonsils of pigs, similarly to vGPE−, their type I interferon-inducing capacity was significantly lower than that of vGPE−. Taken together, vGPE− mainly retains ideal properties for the CSF vaccine, compared with the seed variants, and is probably useful in the development of a CSF marker vaccine.

Protection against transplacental transmission of moderately virulent classical swine fever virus using live marker vaccine “CP7_E2alf”

Classical swine fever (CSF) remains as one of the most important infectious diseases of swine. While prophylactic vaccination is usually prohibited in free countries with industrialized pig production, emergency vaccination is still foreseen. In this context, marker vaccines are preferred as they can reduce the impact on trade.The live-attenuated Suvaxyn® CSF Marker vaccine by Zoetis (based on pestivirus chimera “CP7_E2alf”), was recently licensed by the European Medicines Agency. Its efficacy for the individual animal had been shown in prior studies, but questions remained regarding protection against transplacental transmission. To answer this question, a trial with eight pregnant sows and their offspring was performed as prescribed by the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. Six of the sows were intramuscularly vaccinated on day 44 of gestation, while the other two remained as unvaccinated controls. All sows were challenged with the moderately virulent CSFV strain ”Roesrath” and euthanized shortly before the calculated farrowing date. Sows and piglets were grossly examined and necropsied. Organs (spleen, tonsil, lymph node, and kidney), EDTA-blood and serum were collected from all animals. All samples were tested for antibodies against CSFV glycoproteins E2 and Erns as well as CSFV (virus, antigen and genome). It could be demonstrated that the vaccine complies with all requirements, i.e. no virus was found in the blood of vaccinated sows and their fetuses, and no antibodies were found in the serum of the fetuses from the vaccinated sows. All controls were valid.Thus, it was demonstrated that a single dose vaccination in the sows efficiently protected the offspring against transplacental infection with a moderately virulent CSFV strain.

Kinetics of maternally derived antibodies upon intramuscular vaccination against classical swine fever with Suvaxyn® CSF Marker (CP7_E2alf)

Classical swine fever (CSF) remains one of the most important viral diseases that impact on sustainable pig production world wide. To control the disease in either endemic situations or in case of large, high-impact contingencies, safe and highly efficacious live attenuated vaccines exist since decades. However, until recently, the available live vaccines did not allow a serological marker concept that would be important to circumvent long-term trade restrictions. Recently, a new live attenuated marker vaccine, Suvaxyn® CSF Marker (Zoetis), was licensed by the European Medicines Agency (EMA). To supplement the data that are necessary for the design of appropriate vaccination strategies, a trial was carried out with single “emergency-type” vaccination of two pregnant sows. Focus was laid on the kinetics of maternally derived antibodies (MDA) in the screening assays of their offspring that would be used in case of a CSF outbreaks, i.e. CSFV E2 and Erns antibody ELISA. Neutralization peroxidase linked antibody assays were carried out to allow a rough estimate of protection.Upon vaccination with Suvaxyn® CSF Marker 21days before farrowing, MDAs were measurable in all piglets born to the vaccinated sows. The E2 ELISA reactivities showed an almost linear decrease over 10 weeks after which all piglets were tested negative in the ELISA again. No problems were observed in DIVA assays (Erns antibodies) when heat-inactivated sera were used. The protective effect of MDA needs further investigations as the titers were found to be lower than reported for C-strain vaccines.

Efficacy of Suvaxyn CSF Marker (CP7_E2alf) in the presence of pre-existing antibodies against Bovine viral diarrhea virus type 1

Classical swine fever (CSF) is still one of the most important viral diseases of pigs worldwide and outbreaks are notifiable to the OIE. The different control options also include (emergency) vaccination, preferably with a vaccine that allows differentiation of infected from vaccinated animals (DIVA principle). Recently, the chimeric pestivirus “CP7_E2alf” (Suvaxyn® CSF Marker, Zoetis) was licensed as live attenuated marker vaccine by the European Medicines Agency (EMA). In the context of risk assessments for an emergency vaccination scenario, the question has been raised whether pre-existing anti-pestivirus antibodies, especially against the vaccine backbone Bovine viral diarrhea virus type 1 (BVDV-1), would interfere with “CP7_E2alf” vaccination and the accompanying DIVA diagnostics.To answer this question, a vaccination-challenge-trial was conducted with Suvaxyn® CSF Marker and the “gold-standard” of live-modified CSF vaccines C-strain (RIEMSER® Schweinepestvakzine) as comparator. Pre-existing antibodies against BVDV-1 were provoked in a subset of animals through intramuscular inoculation of a recent field isolate from Germany (two injections with an interval of 2weeks). Twenty-seven days after the first injection, intramuscular vaccination of pre-exposed and naïve animals with either “CP7_E2alf” or C-strain “Riems” was performed. Seven days later, all vaccinated animals and two additional controls were oro-nasally challenged with highly virulent CSF virus (CSFV) strain Koslov.It was demonstrated that pre-existing BVDV-1 antibodies do not impact on the efficacy of live attenuated vaccines against CSF. Both C-strain “Riems” and marker vaccine “CP7_E2alf” were able to confer full protection against highly virulent challenge seven days after vaccination. However, slight interference was seen with serological DIVA diagnostics accompanying the vaccination with CP7_E2alf. Amended sample preparation and combination of test systems was able to resolve most cases of false positive reactions. However, in such a co-infection scenario, optimization and embedding in a well-defined surveillance strategy is clearly needed.

Classical swine fever virus marker vaccine strain CP7_E2alf: Shedding and dissemination studies in boars

Over the last decade, pestivirus chimaera CP7_E2alf has proven to be a most promising marker vaccine candidate against classical swine fever (CSF). To provide further background data for the risk assessment towards licensing and release, especially on presence of the vaccine chimaera in faeces, urine, and organs of the male reproductive tract, supplementary studies were carried out under controlled laboratory conditions. In detail, the shedding and dissemination pattern of Suvaxyn® CSF Marker (“CP7_E2alf”) was assessed in 12 adult boars after single intramuscular vaccination with a tenfold vaccine dose. Four and seven days post vaccination, six animals were subjected to necropsy and triplicate samples were obtained from reproductive and lymphatic organs as well as urine, faeces, blood, and several additional organs and matrices. The sampling days were chosen based on pre-existing data that indicated the highest probability of virus detection. Upon vaccination, neither local nor systemic adverse effects were observed in the experimental animals. It was confirmed that primary replication is restricted to the lymphatic tissues and especially the tonsil. While viral genome was detectable in several samples from lymphatic tissues at four and seven days post vaccination, infectious virus was only demonstrated at four days post vaccination in one tonsil sample and one parotid lymphnode. Sporadic detection at a very low level occurred in some replicates of liver, lung, bone marrow, and salivary gland samples. In contrast, viral genome was not detected in any sample from reproductive organs and accessory sex glands, in faeces, urine, or bile.The presented data on the dissemination of the vaccine virus CP7_E2alf in adult boars are supplementing existing safety and efficacy studies and indicate that the use of the vaccine is also safe in reproductive boars.

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 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.

Oral vaccination against classical swine fever with a chimeric Pestivirus: comparative investigations of liquid and lyophilized virus

The goal of this study was to evaluate the efficacy of the chimeric marker vaccine candidate CP7_E2alf in different formulations for oral immunization against classical swine fever (CSF). In the first experiment, three wild boars were vaccinated orally with the liquid chimeric virus CP7_E2alf, whereas in the second experiment, four wild boars and four domestic pigs were immunized with the lyophilized chimera administered in gelatine capsules and new baits. The chimera was safe for wild boars and domestic pigs. All animals vaccinated orally with the liquid chimera were seropositive 21 days after vaccination, and neither viraemia nor virus shedding were observed after challenge with a highly virulent CSF virus. At necropsy, viral genome of the challenge virus was detected in some organs of all surviving wild boars. Lyophilized chimera administered orally did not protect the animals. The reasons for the inefficacy of the lyophilized vaccine virus are discussed. Irrespective of the negative result with lyophilized virus, the study and previous experiences (Reimann et al., Virology, 307:213–227, 2004) suggest that the chimera CP7_E2alf is a potential CSF marker vaccine candidate.

Classical swine fever (CSF) marker vaccine: Trial II. Challenge study in pregnant sows

The efficacy of two marker vaccines against classical swine fever (CSF) was tested in a large scale laboratory trial in several National Swine Fever Laboratories (NSFL) of the EU member states. The vaccines were: BAYOVAC CSF Marker (Vaccine A) from Bayer, Leverkusen, Germany and PORCILIS PESTI (Vaccine B) from Intervet, Boxmeer, The Netherlands. At the NSFL of Belgium, The Netherlands and Germany experiments were carried out to examine the ability of the vaccines to prevent transplacental transmission of CSF virus. In Belgium and The Netherlands pregnant sows were vaccinated once and challenged with virulent CSF virus 14 days later, which was around day 60 of gestation. At the NSFL in Germany sows were vaccinated twice, on days 25 and 46 of pregnancy and were challenged fourteen days after booster vaccination (day 60 of gestation). Apart from minor inflammatory reactions in some sows, no reactions post vaccination were noticed in either vaccine group. Sows vaccinated with Vaccine A were better protected against clinical CSF than sows vaccinated with Vaccine B. The antibody response after vaccination with Vaccine A was more pronounced than after vaccination with Vaccine B. After single vaccination six out of eight sows vaccinated with Vaccine A and all eight sows vaccinated with Vaccine B had viraemic piglets. After double vaccination one out of four litters from sows vaccinated with Vaccine A and four out of five litters from sows vaccinated with Vaccine B were found to be viraemic. However, both vaccines reduced the transmission probability significantly (Vaccine A: P=0.004, Vaccine B: P=0.024) after booster vaccination. However, Vaccine A appeared in this regard more potent as the estimated probability of fetal infections was lower. Nevertheless the risk of virus spreading after vaccination via transplacental transmission is still present and has to be addressed from an epidemiological point of view.

Classical swine fever (CSF) marker vaccine: Trial III. Evaluation of discriminatory ELISAs

The objective of the marker vaccine trial was to test the two available CSF marker vaccines in scenarios which are likely to occur in the field and to evaluate the reliability of the discriminatory tests. The evaluation of the discriminatory tests was of special importance because there is no requirement for formal data concerning their performance by the European Medicinal Products Evaluation Agency (EMEA) in London. EMEA is responsible for the licensing procedure of the marker vaccines within the EU. Sixteen National Swine Fever Laboratories (NSFL) participated in testing the discriminatory ELISAs. They were tested for sensitivity, specificity, reproducibility and practicability. Reference sera (CSFV and BVDV antibody positive) and field sera were used as well as sera from the weaner and sow experiments produced during the marker vaccine trial. Both discriminatory ELISAs were less sensitive than conventional CSF antibody ELISAs, although there was considerable variation between them. One discriminatory ELISA was less specific than the other, but more sensitive, and vice versa. Neither discriminatory ELISA consistently detected the marker-vaccinated, CSF-challenged weaner pigs correctly as ‘CSF positive’, although CSF-challenged pregnant sows were identified correctly. The limitations of the discriminatory ELISAs used in the trial was the major factor that would prevent the use of these two marker vaccines under emergency field conditions.