Oral delivery of maize-produced porcine epidemic diarrhea virus spike protein elicits neutralizing antibodies in pigs

Abstract

Porcine Epidemic Diarrhea Virus (PEDV) causes severe diarrhea and mortality in piglets. Robust immunity may break the transmission cycle. Expression of antigens in maize grains is a promising method for producing low-cost vaccines. As a first step, we expressed maize constructs containing PEDV S1 spike protein targeted to various cellular locations including the cell wall, endoplasmic reticulum, and vacuole, and fused to carrier proteins E. coli heat labile subunit (LTB) and a dendritic cell (DC) binding peptide, and obtained sufficient antigen for oral immunization. Constructs targeting S1 to the ER or fused to carrier proteins produced high levels of antigen of greater than 20 mg/kg. Oral administration to pigs elicited serum neutralizing antibodies, supporting oral immunization as a practical and cost-effective PEDV vaccine.

Keywords: Maize-expression; Neutralizing antibodies; Oral vaccines; PEDV; S1 antigen.

Fig. 1

Diagram of constructs. The layout of six transformed constructs is shown. Promoters pr25 and pr44 target expression to the embryo and promoter pr39 targets expression to the endosperm. The BAASS signal sequence targets expression to the cell wall but addition of the KDEL sequence causes the protein to be retained in the ER. Construct PDB contains a signal sequence targeting expression to the vacuole. The spike protein coding region from the Colorado 2013 strain is either amino acids 23–738 (S1) or amino acids 23–789 (S1ext). All constructs contain the PinII terminator sequence. Constructs PDK and PDM have the LTB or DC peptides, respectively, fused to the carboxy terminus

Fig. 2

Western blot of S1 expression in representative seed. Four representative individual seeds for the indicated plant were pulverized and extracted in 1 X PBS + 1%SDS. The protein was transferred to PVDF membrane and incubated with polyclonal rabbit antibody raised to commercially synthesized COE portion of the spike protein (shown as a positive control at the right). Note that in all cases except PDC T3 generation, T1 generation seed crossed to untransformed maize is shown and half of the seed are expected to be positive. PDC T3 has been self-crossed leading to expression in all seeds. The predicted sizes for PDC, PDK, PDM and the COE are 81.2 kDa, 97.6 kDa, 87.4 kDa, and 16.1 kDa respectively. The expected segregation pattern with a robust band in approximately half the seed in T1 generation plants and not in untransformed maize (not shown) is indicative of specificity, while other non-specific bands were observed in all seed. However, the bands for maize-produced S1 run at a higher apparent molecular weight than predicted, possibly due to post-translational modifications such as glycosylation

Fig. 3

PEDV S1 expression is maintained in homozygous plants after a back-cross program. Ten individual seed from plants harboring construct PDC after self-crossing for homozygosity were tested for S1 expression by western blot as described for Fig. 2. After initial analysis (Fig. 2), the recombinant COE standard is not included to maximize gel space when screening individual seed for homozygous lines

Fig. 4

Pig serum neutralizing antibody response. Serum neutralizing antibody response from the indicated groups (n = 4) were assessed by fluorescent focus neutralization assay at 15 days post-challenge (dpc), as described in the methods section. At 15 dpc treatment groups containing S1 (one and two, a) showed significant differences with the control group (three, b) with p-values for each comparison substantially below 0.05. There was no significant difference in the titers between treatment groups one (injected S1) and two (orally-delivered maize S1)