Development of a classical swine fever subunit marker vaccine and companion diagnostic test

Abstract

The development of a classical swine fever (CSF) subunit marker vaccine, based on viral envelope glycoprotein E2, and a companion diagnostic test, based on a second viral envelope glycoprotein E^RNS, will be described. Important properties of the vaccine, such as onset and duration of immunity, and prevention of horizontal and vertical transmission of virus were evaluated. A single dose of the vaccine protected pigs against clinical signs of CSF, following intranasal challenge with 100LD₅₀ of virulent classical swine fever virus (CSFV) at 2 weeks after vaccination. However, challenge virus transmission to unvaccinated sentinels was not always completely inhibited at this time point. From 3 weeks up to 6 months after vaccination, pigs were protected against clinical signs of CSF, and no longer transmitted challenge virus to unvaccinated sentinels. In contrast, unvaccinated control pigs died within 2 weeks after challenge. We also evaluated transmission of challenge virus in a setup enabling determination of the reproduction ratio (R value) of the virus. In such an experiment, transmission of challenge virus is determined in a fully vaccinated population at different time points after vaccination. Pigs challenged at 1 week after immunization died of CSF, whereas the vaccinated sentinels became infected, seroconverted for E^RNS antibodies, but survived. At 2 weeks after vaccination, the challenged pigs seroconverted for E^RNS antibodies, but none of the vaccinated sentinels did. Thus, at 1 week after vaccination, R>1, and at 2 weeks, R=0, implying no control or control of an outbreak, respectively. Vertical transmission of CSFV to the immune-incompetent fetus may lead to the birth of highly viraemic, persistently infected piglets which are one of the major sources of virus spread. Protection against transplacental transmission of CSFV in vaccinated sows was, therefore, tested in once and twice vaccinated sows. Only one out of nine once-vaccinated sows transmitted challenge virus to the fetus, whereas none of the nine twice-vaccinated sows did. Finally, our data show that the E^RNS test detects CSFV-specific antibodies in vaccinated or unvaccinated pigs as early as 14 days after infection with a virulent CSF strain. This indicates that the E2 vaccine and companion test fully comply with the marker vaccine concept. This concept implies the possibility of detecting infected animals within a vaccinated population.

Introduction

Classical swine fever (CSF, synonym — hog cholera) is a highly contagious and often fatal disease of swine. The disease is of worldwide importance, and is endemic in many countries in Eastern Europe, South-East Asia, and Southern and Central America. Although many Western European countries are free of CSF, countries with pig-dense areas like Germany, The Netherlands, Belgium and Spain, are susceptible to a high risk of CSF outbreaks. These countries have recently suffered or are suffering from large outbreaks of the disease. The European Union (EU) maintains a non-vaccination policy against CSF, even though a safe and efficacious modified live vaccine against CSF is available. Thus, outbreaks of CSF in the EU are controlled by stamping out infected and suspected herds, with concomitant quarantine measures. Such an eradication policy can lead to large economic losses. In control programs for other infectious diseases, such as pseudorabies (Van Oirschot et al., 1990) and infectious bovine rhinotracheitis (Van Oirschot et al., 1996), marker vaccines have been successfully employed to assist with eradication of the causative virus. A marker vaccine against CSF which could be used for ring vaccination in an emergency situation, and a companion diagnostic test to trace residual infections, might contribute to the control of a CSF outbreak by limiting its duration and extent, and thereby reduce the economic losses.

Classical swine fever virus (CSFV), the causative agent of CSF is a member of the genus Pestivirus of the family Flaviviridae (Murphy et al., 1995). Animals infected with a pestivirus invariably develop antibodies against the viral envelope proteins E^RNS and E2, and the nonstructural protein NS3 (formerly called p80) (Kwang et al., 1992, Paton et al., 1991, Wensvoort et al., 1988). It has been shown in different ways that E2 alone may protect pigs against CSF (van Zijl et al., 1991, Hulst et al., 1993, Konig et al., 1995, van Rijn et al., 1996). Furthermore, neutralizing epitopes conserved among CSFV (Wensvoort, 1989) have been identified on this protein. A subunit vaccine developed on the basis of E2 may thus not only confer a broad protection against CSF field virus strains, but is also a potential marker vaccine since it allows the discrimination between vaccinated and infected pigs on the basis of detection of antibodies against E^RNS or NS3. Since epitopes conserved among pestiviruses are present on NS3 (Edwards et al., 1991), this antigen is not suitable to develop a CSFV-specific diagnostic test accompanying the E2 vaccine. However, as we will demonstrate, a test based on the E^RNS antigen is highly CSFV-specific and is suitable for detection of a CSFV infection in a population vaccinated with the E2 subunit marker vaccine. Furthermore, data will be presented to show that the CSF E2 subunit marker vaccine confers early and long-lasting protection, and prevents horizontal and vertical transmission of CSFV.