Studies On An Edible Rotavirus Vaccine Produced In Transgenic Alfalfa (Medicago Sativa L.)

Elevated expression of the antigen in transgenic plants is a critical factor in the development of a safe and effective rotavirus vaccine. Using codon optimization, a gene that encodes the inner capsid protein VP6 of the human group A rotavirus was synthesized (sVP6). To examine expression in prokaryotic cells, the VP6 and sVP6 genes were inserted into pGEX-4T-l respectively, and the recombined plasmids were transformed into E. coli BL21. SDS-PAGE analysis demonstrated that glutathione S-transferase (GST) fusion proteins of VP6 and sVP6 (GST-VP6 and GST-sVP6) expressed in E. coli BL21 were of equal size (65kDa). The expressed VP6 and sVP6 proteins were purified by GST affinity chromatography, thrombin and fast protein liquid chromatography (FPLC). The purified sVP6 protein had a relative molecular weight of 42kDa under denaturing conditions, but formed trimers (~130kDa) under nondenaturing conditions. Western blot analysis further confirmed that the sVP6 protein and its trimers bound rabbit polyclonal anti-rotavirus VP6 antibodies. These results indicated that the structure and the immunoreactivity of the sVP6 protein were similar to that of native VP6.The sVP6 gene and VP6 gene were transformed into tobacco (Nicotiana tabacum L.) plants using Agrobacterium tumefaciens. The expression level of the sVP6 gene in transgenic plants was 3.8-34-fold higher than that of control containing the non-modified VP6 gene, accounting for up to 0.34% of the total soluble protein (TSP).On these base, the sVP6 gene was inserted into the alfalfa (Medicago sativa L.) genome using Agrobacterium-mediated transformation. As much as 0.28% of the total soluble protein of the pBsVP6-transgenic alfalfa was sVP6.Female BALB/c mice were gavaged weekly with 10mg of transgenic alfalfa extract containing 24μg of sVP6 protein and 10μg of CpG oligodeoxynucleotides as mucosal adjuvant. Immunized mice developed high titers of anti-VP6 serum IgG and mucosal IgA. The proportion of pups with diarrhea symptoms, the duration of diarrhea and the intensity of diarrhea were significantly reduced in the offspring of mice immunized with the transgenic alfalfa after challenge with simian rotavirus SA-11. Complete resolution of diarrhea symptoms occurred five days after virus challenge. These results suggested that the anti-VP6 antibodies generated in orally immunized female mice were passively transferred to the pups and provided heterotypic protection, indicating that an alfalfa-derived sVP6-based oral vaccine could efficiently protect young animals and children from severe acute diarrhea caused by human group A rotaviruses.