Display of Porcine Epidemic Diarrhea Virus Spike Protein on Baculovirus to Improve Immunogenicity and Protective Efficacy

Abstract

A new variant of the porcine epidemic diarrhea virus (PEDV) is an emerging swine disease, killing considerable numbers of neonatal piglets in North America and Asia in recent years. To generate immunogens mimicking the complex spike (S) protein folding with proper posttranslational modification to mount a robust immune response against the highly virulent PEDV, two baculoviruses displaying the full-length S protein (S-Bac) and the S1 protein (S1-Bac) of the virulent Taiwan genotype 2b (G2b) PEDV Pintung 52 (PEDV-PT) strain were constructed. Intramuscular immunizations of mice and piglets with the S-Bac and S1-Bac demonstrated significantly higher levels of systemic anti-PEDV S-specific IgG, as compared with control group. Our results also showed that piglets in the S-Bac group elicited superior PEDV-specific neutralizing antibodies than those of the S1-Bac and control groups. The highly virulent PEDV-PT strain challenge experiment showed that piglets immunized with S-Bac and S1-Bac showed milder clinical symptoms with significantly less fecal viral shedding as compared with non-immunized control piglets. More importantly, piglets immunized with the S-Bac exhibited no to mild clinical signs, with a delayed, minimal viral shedding. Our results demonstrated that the S-Bac could serve as a safe, easy to manipulate, and effective vaccine candidate against the PEDV infection.

Keywords: PEDV; baculovirus display system; intramuscular injection; spike vaccine.

Figure 1

The organization of the expression cassette and the construction maps of the plasmids, pTriEx-S and pTriEx-S1, for generation of membrane-anchored recombinant baculovirus, S-Bac and S1-Bac. The full-length spike (S) and S1 genes are driven by the TriEx promoter, followed by the GP64 signal protein and 6xHis-tag. The pTriEx-S contains the codon optimized full-length S gene derived from the Taiwan G2b PEDV-PT strain with its original PEDV S transmembrane domain (TM-S) and the PEDV S cytoplasmic domain (CTD-S) for membrane anchoring. The pTriEx-S1 has the codon optimized S1 gene associated with GP64 transmembrane domain (TM-B) and the GP64 cytoplasmic domain (CTD-B) for membrane anchoring. Both constructs were also inserted with an mCherry fluorescent protein gene driven by the SV40-pag promoter as a reporter.

Figure 2

The detection of porcine epidemic diarrhea virus (PEDV) full-length S and S1 proteins in the cell lysate of S-Bac and S1-Bac infected Sf21 cells at 3 days post-infection with an M.O.I. of 5. Western blotting analysis of PEDV S and S1 proteins expressing by baculoviruses was performed and probed with anti-His tag antibodies. The corresponding molecular weights of S and S1 proteins were approximately 170–200 kDa and 90–100 kDa, respectively. Cell only: the non-infected Sf21 cell; Wild-type: Sf21 cells infected with wild type AcMNPV; S-Bac: Sf21 cells infected with S-Bac; S1-Bac: Sf21 cells infected with S1-Bac; GAPDH: control cellular protein for equal volume loading.

Figure 3

The detection of recombinant SBac and S1-Bac by electron microscopy (immuno-EM). The electron micrographs demonstrated positive colloid gold signals of porcine epidemic diarrhea virus (PEDV) full-length S and S1 proteins on the surface of recombinant S-Bac (a); S1-Bac (b); and wild-type Bac (c), respectively. S-Bac: PEDV S display baculovirus viral particle. S1-Bac: PEDV S1 display baculovirus viral particle. Wild type-Bac: wild-type baculovirus viral particle. The bars represent a reference of 50 nm.

Figure 4

The changes of systemic porcine epidemic diarrhea virus (PEDV) spike (S)-specific IgG levels in S-Bac and S1-Bac vaccinated mice. The serum samples of the mice were collected three times in two-week intervals, including day 0 (pre-priming), 14 (2 weeks post-priming), and 28 (2 weeks post-boosting). The systemic anti-PEDV S protein IgG levels were detected by the PEDV S protein-based ELISA. The X axis represents the day post vaccination; whereas the Y axis shows the sample-to-positive control ratios (S/P ratio) of the optical density (OD) values from ELISA. The S/P ratio was defined as the ratio of the difference between the OD values of sample and negative control and the difference between OD values of positive and negative controls. The error bars represent the SD values of each group in different time points. The solid line with square icon and the gray line with round icon represent the climbing trend of IgG level in the S-Bac group and S1-Bac group, respectively. The IgG levels in the control group was expressed as the dotted line with triangle icon. *: significant difference with the control group (p < 0.05).

Figure 5

The neutralizing titers of systemic antibody of mice in control, S-Bac and S1-Bac groups at day 0 (pre-priming) and day 28 (2 weeks post-boosting). The shift of neutralizing titers of S-Bac and S1-Bac vaccinated mice are represented as a solid line with square icons and a gray line with round icons, respectively. The neutralizing titers in the blood of control mice are labeled with triangle icons on a dotted line. The error bars represent the SD values of each group at different time points. The background of this neutralizing assay was 1:20, and the area under detection background is marked as a gray zone.

Figure 6

The systemic anti-porcine epidemic diarrhea (PEDV) spike (S)-specific IgG levels in piglets after S-Bac or S1-Bac vaccination. The systemic IgG levels of piglets were detected every two weeks at day 0 (pre-priming), day 14 (2 weeks post-priming) and day 28 (2 weeks post-boosting) by using the PEDV S-based ELISA. The data is shown as the mean values of the sample-to-positive control ratios (S/P ratio), which was defined as the difference between the optical density (OD) values of the sample and the negative control and divided by the difference between OD values of the positive and negative control. The error bars represented the SD values of each group in different time points. The solid line with square icon and the gray line with round icon represent the climbing trend of IgG level in the S-Bac group and S1-Bac group, respectively. The IgG levels in the control group are expressed with the dotted line with triangle icon. *: significant difference with the control group (p < 0.05).

Figure 7

The anti-porcine epidemic diarrhea (PEDV) spike (S)-specific fecal IgA levels in piglets after S-Bac or S1-Bac immunizations. The mucosal IgA levels of pigs were evaluated every two weeks at day 0 (pre-priming), day 14 (2 weeks post-priming) and day 28 (2 weeks post-boosting) from rectal swabs by using PEDV S-based ELISA. The data is presented as mean OD values from five pigs. The error bars represent the SD values of each group at different time points. The solid line with square icons and the gray line with round icons represent the climbing trend of IgA level in the S-Bac group and S1-Bac group, respectively. The IgA levels in the control group is expressed with the dotted line with triangle icon.

Figure 8

The neutralizing titers against porcine epidemic diarrhea virus (PEDV) in each group at day 0 (pre-priming) and day 28 (2 weeks post-boosting). A solid line with square icons, a gray line with round icons, and a dotted line with triangle icons represent the titers of anti-PEDV neutralizing antibodies of pigs in the S-Bac, S1-Bac, and control groups, respectively. Values are presented as means ± SD. The gray zone represents the background of the neutralizing assay. *: significant difference with the control group (p < 0.05).

Figure 9

The average body weight of piglets in each group. The body weight of the piglets was measured in two-week intervals since the piglets were 4 weeks old. The X axis is the time line, indicating the age of the piglets, days post vaccination, and days post challenge. The piglets were vaccinated twice at 5 weeks old and 7 weeks old (labeled with white arrows); the piglets were challenged with 5 × 10⁵ TCID50 PEDVPT-P6&7 at 9 weeks old (labeled with a black solid arrow). The Y axis is the averaged body weights of five piglets in each group. The error bars represent the SD values of each group in different time points. The solid line with square icons and the gray line with round icons represent the average body weights of pigs in the S-Bac group and S1-Bac group, respectively. The average body weight of pigs in the control group is expressed as the dotted line with triangle icon.

Figure 10

The accumulations of stool scores in the control group (a); S1-Bac group (b); and S-Bac (c) groups after the highly virulent porcine epidemic diarrhea virus (PEDV-PT) challenge. The clinical signs were scored by the following rules: 0, normal stool; 1, loose consistency of the stool; 2, semi-fluid consistency of the stool; 3, watery diarrhea. Each piglet was orally challenged with 5 × 10⁵ TCID50 PEDV-PT-P6&7 at day post vaccination 28 (day post challenge 0). A total of 15 days’ observation period was taken after challenge. The blank square represents the score 1 (loose consistent stool); the light-gray square represents the score 2 (semi-fluid stool); the dark-gray square represents the score 3 (watery diarrhea).

Figure 11

The detection of fecal viral shedding after the highly virulent porcine epidemic diarrhea virus (PEDV-PT) challenge. The limit of detection for this probe-based quantitative real-time RT PCR was 1.8 log¹⁰ and is labeled as the gray zone. The error bars represent the SD values of each group at different time points. The solid line with the square icons and the gray line with round icons represent the average fecal viral shedding copy number of pigs in the S-Bac group and S1-Bac groups, respectively. The average fecal viral shedding copy number of pigs in the control group is expressed as the dotted line with the triangle icons.