Systemic and Oral Immunogenicity of Porcine Epidemic Diarrhea Virus Antigen Fused to Poly-Fc of Immunoglobulin G and Expressed in ΔXT/FT Nicotiana benthamiana Plants

Abstract

Porcine epidemic diarrhea virus (PEDV), a member of the Coronaviridae family has become increasingly probelmatic in the pig farming industry. Currently, there are no effective, globally applicable vaccines against PEDV. Here, we tested a recombinant PEDV vaccine candidate based on the expression of the core neutralising epitope (COE) of PEDV conjugated to polymeric immunoglobulin G scaffold (PIGS) in glycoengineered Nicotiana be nthamiana plants. The biological activity of COE-PIGS was demonstrated by binding to C1q component of the complement system, as well as the surface of antigen-presenting cells (APCs) in vitro. The recombinant COE-PIGS induced humoral and cellular immune responses specific for PEDV after both systemic and mucosal vaccination. Altogether, the data indicated that PEDV antigen fusion to poly-Fc could be a promising vaccine platform against respiratory PEDV infection.

Keywords: PEDV; immunity; mucosal; plant; vaccine.

FIGURE 1

Vector construction for plant expression of COE-mPIGS and a schematic diagram of poly-Fc platform (A) the structure of S protein of PEDV and plant expression vector: three vectors were used to produce COE-PIGS simultaneously. pMYKT10, 3′ pro-vector containing COE-mPIGS gene; pICH20155, 5′ pro-vector containing apoplast signal peptide sequence; and pICH14011 vector for expression of the integrase. LB and RB, left border and right border of T-DNA; P-Act2, Arabidopsis actin 2 promoter; P-hsp, Arabidopsis heat shock protein hsp 81.1 promoter; NOS, nos terminator; 3′NTR, 3′ nontranslated regions of TMV; MP, movement protein; Apo, apoplast targeting pre-sequence; int, intron; AttP and AttB, recombination sites; NLS, nuclear localization signal; T, terminator. (B) Homodimeric structure of COE-PIGS presenting biologically active sites. COE, antigen of PEDV; Fcγ, Fc gamma chain based on mouse IgG2a; µ tp, µ tail piece of human IgM; PIGS, Polymeric IgG Scaffold. The process of polymerization from a single chain is shown in a lower diagram. Polymers bind to both high and low affinity Fc receptors (FcγI, FcγII, and FcγIII) embedded in antigen presenting cell (APC) resulting in enhancement of the immune responses, while monomeric IgG binds to only high affinity Fc receptor (FcγI). Polymers can be denatured into single chain under reducing conditions.

FIGURE 2

Expression of COE-mPIGS protein in tobacco (A) Immunoblot analysis of COE-PIGS co-expressed without/with J chain: the protein extracts from infiltrated and non-infiltrated leaf tissues were analyzed by probing with anti-mouse IgG gamma chain specific or anti-COE polyclonal antibodies under non-reducing and reducing conditions. PC, commercial mouse IgG or COE recombinant protein produced in E. coli used as positive controls; NC, non-infiltrated protein extract used as the negative control; lanes 2, 4, and 6, protein extracts from the harvested leaves at 2, 4, and 6 days post-infiltration (dpi). Molecular weight markers are shown on the left. (B) Indirect ELISA probing by anti-mouse IgG gamma chain specific antibody. The level of expression (approximately 0.7% of total soluble protein) was estimated by comparing to commercial IgG protein by ELISA.

FIGURE 3

Purification of protein and a functional characterization assay (A) Recombinant COE-mPIGS was purified from plant extracts using protein A/G affinity chromatography methods. The proteins were separated by SDS-PAGE under non-reducing conditions and visualized by Coomassie brilliant blue staining. Lane BSA, bovine serum albumin; lane E1-E3: fractions of eluted protein. Pre-stained protein molecular weight standards and polymers (P), homodimer (D), and single chain (S) shown on the left and right sides of the figure. (B) C1q complement binding by ELISA shown as 2-fold serial dilutions of COE-mPIGS (5 μg/ml). The protein extracts from wild-type plant were used as the negative control. (C) Fc gamma receptor binding in J774 cells by FACS analysis. The COE-mPIGS binding histogram was overlaid to that obtained for mouse IgG2a antibody or secondary antibody alone, used as negative controls.

FIGURE 4

Mouse immune responses induced by systemic injection (A) Schedule for immunization and sample collection. The BALB/c mice were randomly distributed into four groups. Mice were subcutaneously injected with PBS only, or with 10 µg of the purified COE-mPIGS protein alone or with 5 µg of bacterial cholera toxin (bCT) as adjuvant. The blood samples were collected at 3 and 6 weeks post-immunization. (B) Detection of COE-specific serum IgG. COE-mPIGS and COE-mPIGS + bCT significantly enhanced IgG antibody responses after boosting, while COE and PBS induced only a low level or no response, respectively. The endpoint antibody titers were detected at approximately 1:51,200 and 1:204,800 for COE-mPIGS alone and COE-mPIGS with bCT, respectively. The results were shown as the mean of triplicate measurements ±SD of the 1:100 dilution (left panel), and serial 2-fold dilutions starting from 1:100 dilution (right panel). (C) Cellular immune responses. Spot forming units after bacterial COE antigen stimulation in pooled (n = 4) cultures were detected by ELISpot assay (left panel) and the results shown as the mean values of the number of antibody-secreting cells from triplicate cultures. bCOE protein induced only low-frequency antibody-secreting cells, while COE-mPIGS and COE-mPIGS adjuvanted with bCT induced high-levels of the antibody-secreting cells. T cell proliferation was assessed by [3H] thymidine incorporation assay (right panel). Stimulation index was calculated by dividing the CMP (counts per minute) of Ag-stimulated cultures by CPM of control cultures from three separate experiments. **p < 0.01; ****p < 0.0005, by Tukey’s test.

FIGURE 5

Mouse immune responses induced by oral administration (A) Protocol for immunization and sampling (B) Detection of COE-specific serum IgG (top two panels) and fecal IgA (bottom two panels) in pooled samples for each group (left panels) and the individual mice (right panels), in 1:100 diluted serum and 1:4 diluted fecal extracts. COE-mPIGS and COE-mPIGS + Saponin induced significant IgG and IgA antibody titres after boosting. Only background responses were detected for COE and PBS. The results are shown as the mean of triplicate measurements ±SD. *p < 0.05, **p < 0.01 and ***p < 0.001, by Tukey’s test.