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.

Intramuscular Immunization with Chemokine-Adjuvanted Inactive Porcine Epidemic Diarrhea Virus Induces Substantial Protection in Pigs

“…To evaluate the levels of PEDV-specific plasma IgG and fecal IgA, we performed an in-house recombinant PEDV S protein-based indirect enzyme-linked immunosorbent assay (ELISA), as previously described [9,35]. The MicroWell TM 96-well microplates (Nunc, Rochester, NY, USA) were coated with 2 µg/mL purified recombinant PEDV S protein and incubated overnight at 4 • C. The microplates were washed six times with 300 µL washing buffer (Kirkegaard and Perry Laboratories (KPL), Milford, MA, USA) by a microplate washer (BioTek Instruments, Inc., Winooski, Vermont, USA) and blocked with blocking buffer (KPL) at RT for 1 h. For detection of plasma IgG, plasma samples at 40-fold dilution in blocking buffer (KPL) were incubated at RT for 1 h; for fecal IgA, fecal suspensions at 2-fold dilution in blocking buffer (KPL) were placed overnight at 4 • C. After six washes, 100 µL of horseradish peroxidase (HRP)-conjugated goat anti-pig IgG (KPL) at a dilution of 1:1000 or HRP-conjugated goat anti-pig IgA (Abcam, Cambridge, UK) at a dilution of 1:5000 were added to detect the levels of porcine IgG and IgA, respectively.…”

“…The procedures of RNA extraction and cDNA reverse-transcription using the Cador Pathogen 96 QIAcube HT Kit (Qiagen) and QuantiNova Reverse Transcription Kit (Qiagen), respectively, were carried out according to manufacturer's instructions. The real-time PCR was performed according to our established protocol [9] using the PEDV-N forward primer (3 -CGCAAAGACTGAACCCACTAAC-5 ), PEDV-N reverse primer (3 -TTGCCTCTGTTGTTACTTGGAGAT-5 ), and PEDV specific probe (3 -FAM-TGYYACCAYYACCACGACTCCTGC-BHQ1-5 ). The cycle conditions were as follows: initial denaturation was at 95 • C for 2 min, followed by 45 cycles at 95 • C for 15 s and 55 • C for 15 s.…”

“…The plasma samples of piglets were first incubated at 56 • C for 30 min to inactivate the complement. The neutralizing assay was performed in accordance with the procedure which was previously published [9,35]. The plasma samples were diluted from 10-fold to 320-fold by freshly prepared post-inoculation (PI) medium containing DMEM (Gibco, Gaithersburg, MD, USA) supplemented with 0.3% tryptose phosphate broth (Sigma, St. Louis, MO, USA), 0.02% yeast extract (Acumedia, Lansing, CA, USA), and 10 µg/mL of trypsin (Gibco).…”

“…PEDV strains cause porcine epidemic diarrhea (PED), characterized by vomiting, severe diarrhea, and dehydration in seronegative pigs at all ages, with high mortality in suckling pigs [3]. Many attempts have been made to develop a safe and protective vaccine for controlling PED [4][5][6][7][8][9][10]. However, similar to studies on most enterotropic and mucosal transmissible viral diseases [11][12][13][14], using intramuscular (IM) administration of inactive or subunit vaccines combined with commercial adjuvants, such as multiple emulsions of water/oil/water or a B subunit of Escherichia coli heat-labile enterotoxin (LTB) induced production of systemic IgG and neutralizing antibodies but failed to elicit a mucosal IgA response and efficient protection [6,7,[15][16][17].…”

Oral administration of porcine epidemic diarrhea virus spike protein expressing in silkworm pupae failed to elicit immune responses in pigs

“…KY929405) were codon optimized (GenBank Accession No. MN586852) for the insect protein expression system and synthesized (ProTech, Taipei, Taiwan) as previously described (Chang et al 2018a). In attempt to deliver the interest gene to the BmN cells, the gene of full-length S was cloned into pBPxhE transfer vector (pBPxhE-S-Bm), following the suggested protocol of the In-Fusion ® HD Cloning Kit (Clontech Laboratories Inc., Fremont, CA, USA) (Chang et al 2012).…”