Oral Vaccination with a DNA Vaccine Encoding Capsid Protein of Duck Tembusu Virus Induces Protection Immunity

Abstract

The emergence of duck tembusu virus (DTMUV), a new member of the Flavivirus genus, has caused great economical loss in the poultry industry in China. Since the outbreak and spread of DTMUV is hard to control in a clinical setting, an efficient and low-cost oral delivery DNA vaccine SL7207 (pVAX1-C) based on the capsid protein of DTMUV was developed and evaluated in this study. The antigen capsid protein was expressed from the DNA vaccine SL7207 (pVAX1-C), both in vitro and in vivo. The humoral and cellular immune responses in vivo were observed after oral immunization with the SL7207 (pVAX1-C) DNA vaccine. High titers of the specific antibody against the capsid protein and the neutralizing antibody against the DTMUV virus were both detected after inoculation. The ducks were efficiently protected from lethal DTMUV exposure by the SL7207 (pVAX1-C) vaccine in this experiment. Taken together, we demonstrated that the capsid protein of DTMUV possesses a strong immunogenicity against the DTMUV infection. Moreover, an oral delivery of the DNA vaccine SL7207 (pVAX1-C) utilizing Salmonella SL7207 was an efficient way to protect the ducks against DTMUV infection and provides an economic and fast vaccine delivery strategy for a large scale clinical use.

Keywords: Flavivirus; capsid protein; duck tembusu virus; immunogenicity; oral DNA vaccine.

Figure 1

(A) Schedule of vaccination and sample collection. Ducks were orally vaccinated with 10¹⁰ CFU of SL7207 (pVAX-C) and SL7207 (pVAX) at 8-day-old and 24-day-old, respectively. Animals were sacrificed at 3, 24, 32 and 40 dpi to collect the spleen (n = 3 of each time point). Sera were collected at 8, 16, 24, 32, 40 and 48 dpi (n = 3 of each time point); (B) Schedule of challenge experiment. 10 ducks of each group were randomly selected at 32 dpi for the immune protection test and mortality was recorded for continuous 10 days after exposure of duck tembusu virus (DTMUV).

Figure 2

Expression of the capsid protein gene in vitro and in vivo. (A) COS7 cells were transfected with the DNA vaccine plasmids pVAX-C or pVAX. At 48 h post transfection, the cells were processed by indirect immunofluorescence assay using the anti-DTMUV-capsid protein rabbit polyclonal antibody as the first antibody and the Alexa Fluor 488-conjugated (green fluorescence) goat anti-rabbit as the secondary antibody. Scale bar 200 μm; (B) Spleens collected from the SL7207 (pVAX) (a) or the SL7207 (pVAX-C) vaccination group (b) at 3 dpi (n = 3). The capsid protein was detected by immunohistochemistry assay using the anti-DTMUV-capsid protein rabbit polyclonal antibody as the primary antibody and the horseradish peroxidase-conjugated goat anti-rabbit secondary antibody. The brown dots directed by the arrow indicated the capsid protein. Scale bar 50 μm.

Figure 3

Immune responses stimulated by the vaccine. (A) Expression of IL-4 and IL-6 was measured by quantitative real-time polymerase chain reaction (RT-PCR) to evaluate the cellular immune responses. Data from RT-PCR was analyzed using 2^−ΔΔCt method. The expression level of IL-4 and IL-6 in the SL7207 (pVAX-C) vaccinated group was presented as changed fold reference to those in the SL7207 (pVAX) group. Data are shown as the mean ± standard deviations (n = 3 of each time point); (B) The specific antibody IgY in the serum against the DTMUV capsid protein was detected by using indirect ELISA. The serum samples were incubated with the capsid proteins and detected by using the horseradish peroxidase conjugated goat anti-bird IgY. OD₄₅₀ value of each well was measured. The titers of the specific antibody were presented as the means ± standard deviations (n = 3 of each time point); (C) Neutralizing antibodies against DTMUV in the serum was detected by neutralizing assay. The titers of neutralizing antibodies against DTMUV were detected and presented as the log₂ changed folds (Y-axis) reference to the negative control group SL7207 (pVAX). Data are shown as the means ± standard deviations (n = 3 of each time point). The dash line indicates the lowest threshold value for positive reaction in the neutralizing assay. All data were graphed by GraphPad Prism v5.0 (La Jolla, CA, USA).

Figure 4

Survival post challenge with lethal doses of virulent DTMUV. The ducks (n = 10/group) were challenged with 10^4.5-fold ELD₅₀ DTMUV at 16 days after the second immunization. The death number of ducks was recorded for consecutive 10 days after virus intravenous injection and graphed by GraphPad Prism v5.0.