A Japanese Encephalitis Virus Vaccine Inducing Antibodies Strongly Enhancing In Vitro Infection Is Protective in Pigs

Abstract

The Japanese encephalitis virus (JEV) is responsible for zoonotic severe viral encephalitis transmitted by Culex mosquitoes. Although birds are reservoirs, pigs play a role as amplifying hosts, and are affected in particular through reproductive failure. Here, we show that a lentiviral JEV vector, expressing JEV prM and E proteins (TRIP/JEV.prME), but not JEV infection induces strong antibody-dependent enhancement (ADE) activities for infection of macrophages. Such antibodies strongly promoted infection via Fc receptors. ADE was found at both neutralizing and non-neutralizing serum dilutions. Nevertheless, in vivo JEV challenge of pigs demonstrated comparable protection induced by the TRIP/JEV.prME vaccine or heterologous JEV infection. Thus, either ADE antibodies cause no harm in the presence of neutralizing antibodies or may even have protective effects in vivo in pigs. Additionally, we found that both pre-infected and vaccinated pigs were not fully protected as low levels of viral RNA were found in lymphoid and nervous system tissue in some animals. Strikingly, the virus from the pre-infection persisted in the tonsils throughout the experiment. Finally, despite the vaccination challenge, viral RNA was detected in the oronasal swabs in all vaccinated pigs. These latter data are relevant when JEV vaccination is employed in pigs.

Keywords: Fc receptor; Japanese encephalitis virus; antibody-dependent enhancement of infection; lentiviral vector vaccine; mucosal virus shedding; persistence; vaccine-induced protection.

Figure 1

Antibody-dependent enhancement (ADE) of macrophage infection. Sera from piglets immunized with lentiviral vector TRIP/JEV which expressed JEV G3 prM and E proteins (TRIP/JEV.prME) or infected with Japanese encephalitis virus (JEV) G3 Nakayama (focus 50% reduction neutralization test; FRNT50 1:160 and 1:320, respectively) were 10-fold diluted (from 1:10 to 1:1000) and incubated with JEV G3 Nakayama at multiplicity of infection (MOI) 0.1 of 50% tissue culture infective dose per mL (TCID50) per cell during 30 min at 37 °C, and then added to the cells. The percentage of cells expressing JEV E protein as a measure of ADE of infection in macrophages is shown. Statistical significance was calculated using a two-way ANOVA followed by Dunnets’s multiple comparison. The results are representative of triplicate cultures repeated in three independent experiments. * p < 0.05, ** p < 0.002, *** p < 0.001.

Figure 2

ADE of infection in SK6-CD16 cells. Sera from TRIP/JEV.prME-immunized, JEV Laos- and JEV Nakayama-infected pigs were tested for ADE activity in the porcine kidney cell line SK6 expressing CD16. ADE of infection was tested as described in Figure 1 using (a) JEV G1 Laos; (b) JEV G3 Nakayama and (c) JEV G5/G3, representing a chimeric virus expressing a G5 prM/E. The percentage of infected cells was determined after 24 h. Statistical significance was calculated using a two-way ANOVA followed by Dunnets’s multiple comparison. The results are representative of triplicate cultures repeated in two independent experiments. * p < 0.05, ** p < 0.002, *** p < 0.001.

Figure 3

Neutralizing antibody response in piglets immunized with JEV G1 Laos or TRIP/JEV.prME. Groups of three pigs were either infected with JEV Laos or immunized with TRIP/JEV.prME or mock-inoculated, and serum was collected at the indicated time points (x-axis). (a) viral RNA load determined by RT-qPCR in sera from all nine animals; (b) JEV Laos- and (c) TRIP/JEV.prME-induced neutralizing antibody responses of sera against homologous JEV Laos (blue circles) and JEV Nakayama (red squares). Mean and standard deviations are shown. Statistical significance was determined after Log2 transformation of the data using two-way ANOVA and Sidak’s multiple comparison. * p < 0.05, ** p < 0.002, *** p < 0.001.

Figure 3

Neutralizing antibody response in piglets immunized with JEV G1 Laos or TRIP/JEV.prME. Groups of three pigs were either infected with JEV Laos or immunized with TRIP/JEV.prME or mock-inoculated, and serum was collected at the indicated time points (x-axis). (a) viral RNA load determined by RT-qPCR in sera from all nine animals; (b) JEV Laos- and (c) TRIP/JEV.prME-induced neutralizing antibody responses of sera against homologous JEV Laos (blue circles) and JEV Nakayama (red squares). Mean and standard deviations are shown. Statistical significance was determined after Log2 transformation of the data using two-way ANOVA and Sidak’s multiple comparison. * p < 0.05, ** p < 0.002, *** p < 0.001.

Figure 4

TRIP/JEV.prME vaccine and previous JEV infection induce protection but do not prevent virus shedding. Groups of three pigs were either mock-inoculated (Neg. CTRL, cross), immunized by previous infection with JEV Laos (red square) or vaccinated with TRIP/JEV.prME (green circle), and then challenged infected with JEV G3 Nakayama. Data post challenge is shown. (a) viral RNA loads determined by RT-qPCR in sera from all nine animals; (b) body temperature; (c) viral RNA load in oro-nasal swabs collected daily.

Figure 5

TRIP/JEV.prME vaccine or previous JEV infection induce protection but do not prevent organ infection. Groups of three pigs were either unvaccinated/infected (negative control; Neg. CTRL, crosses), immunized by previous infection with JEV Laos (squares) or vaccinated with TRIP/JEV.prME (circles), and then challenge infected with JEV G3 Nakayama. At 10 days p.i., the animals were euthanized and organ samples tested for viral RNA by RTqPCR. (a) viral RNA load in pigs from the Neg. CTRL group; (b) viral RNA load from the JEV Laos pre-infected group; (c) viral RNA load from the TRIP/JEVprME-vaccinated group.