Abstract
Neutralizing antibodies are the key mediators of protective immune response to flaviviruses after both infection and vaccination. Plaque reduction neutralization test (PRNT) is considered the “gold standard” for measurement of the immunity. To date, little is known regarding neutralizing antibody response to Tembusu virus (TMUV), a novel flavivirus emerging in ducks in 2010. Here, we developed a PRNT for detection of TMUV neutralizing antibodies. Following optimization and validation, the PRNT was applied to test serum samples from different flocks of ducks. Using sera prepared in experimental conditions, the levels of 50% end point titer (neutralizing dose, ND50) generated from positive sera (5,012–79,433) were significantly higher than those from mock-infected sera (10 to 126), indicating that the test can be used in the detection of TMUV-specific neutralizing antibodies. Dose-dependent efficacy test of a cell-derived 180th passage of a plaque-purified virus of the PS TMUV isolate (PS180) in combined with immunization-challenge experiments revealed that ND50 titer of ~1,258 is the minimum capable of providing adequate protection against challenge with virulent TMUV. In the investigation of serum samples collected from three flocks infected by TMUV and four flocks vaccinated with a licensed attenuated vaccine (the 120th passage virus), ND50 titers peaked at 1 week after both disease onset (7,943–125,893) and vaccination (3,612–79,432), and high levels of ND50 titer were detected in sera collected at 15 weeks after disease onset (5,012–63,095) and 17 weeks after vaccination (3,981–25,119). Together these findings demonstrated that spontaneous and experimental infections by TMUV and vaccination with the licensed TMUV attenuated vaccine elicit high, long-lasting neutralizing antibodies. The highest ND50 titer of neutralizing antibodies elicited by PS180 was determined to be 3,162, suggesting that attenuation of TMUV by more passages has a dramatic impact on the neutralizing antibody response of the virus.
Highlights
Humoral immune response plays a significant role in protection of the host from flavivirus infections [1]
To evaluate the humoral immune response induced by Tembusu virus (TMUV) attenuated vaccine, the Plaque reduction neutralization test (PRNT) assay was applied to test serum samples collected from three different flocks at 1 week after immunization
To investigate further the kinetics of neutralizing antibodies induced by TMUV attenuated vaccine, the PRNT was employed to test serum samples from a flock of Cherry Valley Pekin ducks at 1 week before immunization and at different time points after immunization
Summary
Humoral immune response plays a significant role in protection of the host from flavivirus infections [1]. Neutralizing antibodies are thought to be the key mediators of protection against flaviviruses following both infection and vaccination [2, 3]. Neutralizing Antibody of Tembusu Virus enhancement (ADE) of infection [4, 5]. In the case of dengue virus (DENV), the ADE phenomenon most frequently occurs in secondary infections with different DENV serotypes and in children with maternal antibodies declined to sub-neutralizing concentrations [6, 7]. Any antibody that neutralizes at sufficiently high concentrations can enhance flavivirus infectivity at sub-neutralizing concentrations [3]. High levels of neutralizing antibody are crucial to exerting a protective effect, especially in the context of vaccine-mediated humoral immunity
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