Oral Administration of a Seed-based Bivalent Rotavirus Vaccine Containing VP6 and NSP4 Induces Specific Immune Responses in Mice

Abstract

Rotavirus is the leading cause of severe diarrheal disease among newborns. Plant-based rotavirus vaccines have been developed in recent years and have been proven to be effective in animal models. In the present study, we report a bivalent vaccine candidate expressing rotavirus subunits VP6 and NSP4 fused with the adjuvant subunit B of E. coli heat-labile enterotoxin (LTB) in maize seeds. The RT-PCR and Western blot results showed that VP6 and LTB-NSP4 antigens were expressed and accumulated in maize seeds. The expression levels were as high as 0.35 and 0.20% of the total soluble protein for VP6 and LTB-NSP4, respectively. Oral administration of transgenic maize seeds successfully stimulated systemic and mucosal responses, with high titers of serum IgG and mucosal IgA antibodies, even after long-term storage. This study is the first to use maize seeds as efficient generators for the development of a bivalent vaccine against rotavirus.

Keywords: NSP4; VP6; oral vaccine; plant-based vaccine; rotavirus.

FIGURE 1

Schematic map of DNA fragments used for maize transformation. (A) Structure of target genes frangment. P 27: promoter of maize 27 KD γ-zzein; T 27: terminator of maize 27 KD γ-zein; sVP6: synthesized rotavirus VP6 gene; sLTB-SNP4: synthesized fusion gene encoding the B subunit of E. coli heat-labile enterotoxin and the 114–135 peptide of rotavirus NSP4; (B) Structure of marker gene fragment. P 35: cauliflower mosaic virus 35S promoter; T 35: 35S: cauliflower mosaic virus 35S terminator; Bar: coding sequence of phosphinothricin acetyltransferase.

FIGURE 2

PCR and RT-PCR analysis of transgenic maize. (A) PCR identification of transgenic maize. Genomic DNA was used as template. WT: wild type maize plant; Line 1, 2, 3, 4, 7, 8, 9: transgenic maize lines; PC: positive control. Arrows on the left indicate the positive bands. (B) RT-PCR analysis of the expression of exogenous genes. Total RNA was extracted from developing seeds. WT: wild type maize plant; Line 1, 2, 3, 8, 9: transgenic maize lines; PC: positive control. β-actin was used as internal control. Arrows on the right indicate the positive bands.

FIGURE 3

Analysis of protein accumulation in transgenic maize seeds. (A) Western blot analysis of VP6 and LTB-NSP4 protein in the seeds of transgenic maize. Line 1, 3, 7: transgenic maize lines; WT: wild type maize plant; PC: recombinant GST-VP6 and HIS-LTB-NSP4 protein expressed in E. coli were used as positive controls. The expected molecular weights of target proteins are indicated on the right and antibodies used in the detection are showed below. (B) Quantitation of VP6 and LTB-NSP4 proteins expressed in transgenic maize seeds by ELISA. WT: wild type maize plant; Line 1, 2, 3, 7, 8, 9: seed samples from individual transgenic maize plants. TSP: total soluble protein.

FIGURE 4

Tissue specific expression of target genes. (A) RT-PCR examination of the specificity of target genes. Total RNAs were extracted from root, stem, leaf and developing seed of wild type (WT) or transgenic maize plants. Maize β-actin was used as internal control. Arrows on the right indicate the positive bands. (B) Western blot analysis of target protein accumulation specificity. Total soluble proteins were extracted from root, stem, leaf and seed of wild type (WT) or transgenic maize plants. Antibodies used are indicated on the right and expected molecular weight of target proteins are indicated on the left. β-actin was used as loading control.

FIGURE 5

Anti-VP6 and anti-LTB-NSP4 antibody titers determined by ELISA in mice after oral immunization. (A) VP6-specific and LTB-NSP4-secific serum IgG examined at 36 day post the initial immunization. (B) Specific IgA titers against VP6 and LTB-NSP4 in the small intestine at 60 day post the initial immunization. (C) Saliva IgA titers against VP6 and LTB-NSP4 determined at 1 and 3 week post the initial immunization. All groups immunized with VP6 and LTB-NSP4 antigens induced significantly higher antibody titers compared with the two groups gavaged with wild type maize seeds (P < 0.01), and the significances are not indicated in the figures. The significances showed in the figures above represent certain group compared to group 3. ^∗P < 0.05 and ^∗∗P < 0.01. Group 1: mice gavaged with purified protein from wild type maize seed; Group 2: mice gavaged with wild type maize seed powder; Group 3: mice gavaged with purified protein from E. coli; Group 4: mice gavaged with purified protein from transgenic maize seed; Group 5: mice gavaged with seed powder from newly harvested transgenic maize; Group 6: mice gavaged with seed powder from transgenic maize stored for 2 years. The results are presented as the arithmetic means ± SD.