Rice-based oral antibody fragment prophylaxis and therapy against rotavirus infection.

Rotavirus-induced diarrhea is a life-threatening disease in immunocompromised individuals and in children in developing countries. We have developed a system for prophylaxis and therapy against rotavirus disease using transgenic rice expressing the neutralizing variable domain of a rotavirus-specific llama heavy-chain antibody fragment (MucoRice-ARP1). MucoRice-ARP1 was produced at high levels in rice seeds using an overexpression system and RNAi technology to suppress the production of major rice endogenous storage proteins. Orally administered MucoRice-ARP1 markedly decreased the viral load in immunocompetent and immunodeficient mice. The antibody retained in vitro neutralizing activity after long-term storage (>1 yr) and boiling and conferred protection in mice even after heat treatment at 94°C for 30 minutes. High-yield, water-soluble, and purification-free MucoRice-ARP1 thus forms the basis for orally administered prophylaxis and therapy against rotavirus infections.

Induction of toxin-specific neutralizing immunity by molecularly uniform rice-based oral cholera toxin B subunit vaccine without plant-associated sugar modification.

Plants have been used as expression systems for a number of vaccines. However, the expression of vaccines in plants sometimes results in unexpected modification of the vaccines by N-terminal blocking and sugar-chain attachment. Although MucoRice-CTB was thought to be the first cold-chain-free and unpurified oral vaccine, the molecular heterogeneity of MucoRice-CTB, together with plant-based sugar modifications of the CTB protein, has made it difficult to assess immunological activity of vaccine and yield from rice seed. Using a T-DNA vector driven by a prolamin promoter and a signal peptide added to an overexpression vaccine cassette, we established MucoRice-CTB/Q as a new generation oral cholera vaccine for humans use. We confirmed that MucoRice-CTB/Q produces a single CTB monomer with an Asn to Gln substitution at the 4th glycosylation position. The complete amino acid sequence of MucoRice-CTB/Q was determined by MS/MS analysis and the exact amount of expressed CTB was determined by SDS-PAGE densitometric analysis to be an average of 2.35 mg of CTB/g of seed. To compare the immunogenicity of MucoRice-CTB/Q, which has no plant-based glycosylation modifications, with that of the original MucoRice-CTB/N, which is modified with a plant N-glycan, we orally immunized mice and macaques with the two preparations. Similar levels of CTB-specific systemic IgG and mucosal IgA antibodies with toxin-neutralizing activity were induced in mice and macaques orally immunized with MucoRice-CTB/Q or MucoRice-CTB/N. These results show that the molecular uniformed MucoRice-CTB/Q vaccine without plant N-glycan has potential as a safe and efficacious oral vaccine candidate for human use.

RNAi suppression of rice endogenous storage proteins enhances the production of rice-based Botulinum neutrotoxin type A vaccine.

Mucosal vaccines based on rice (MucoRice) offer a highly practical and cost-effective strategy for vaccinating large populations against mucosal infections. However, the limitation of low expression and yield of vaccine antigens with high molecular weight remains to be overcome. Here, we introduced RNAi technology to advance the MucoRice system by co-introducing antisense sequences specific for genes encoding endogenous rice storage proteins to minimize storage protein production and allow more space for the accumulation of vaccine antigen in rice seed. When we used RNAi suppression of a combination of major rice endogenous storage proteins, 13 kDa prolamin and glutelin A in a T-DNA vector, we could highly express a vaccine comprising the 45 kDa C-terminal half of the heavy chain of botulinum type A neurotoxin (BoHc), at an average of 100 µg per seed (MucoRice-BoHc). The MucoRice-Hc was water soluble, and was expressed in the cytoplasm but not in protein body I or II of rice seeds. Thus, our adaptation of the RNAi system improved the yield of a vaccine antigen with a high molecular weight. When the mucosal immunogenicity of the purified MucoRice-BoHc was examined, the vaccine induced protective immunity against a challenge with botulinum type A neurotoxin in mice. These findings demonstrate the efficiency and utility of the advanced MucoRice system as an innovative vaccine production system for generating highly immunogenic mucosal vaccines of high-molecular-weight antigens.