Immunogenicity of recombinant bacterial antigens expressed as fusion proteins in transgenic rice seeds

Rice-based vaccines do not require high-cost purification. They are stable at room temperature, can eliminate the risk of attenuated vaccine strains, and are resistant to gastrointestinal degradation. We tested the applicability of an oral delivery system for tuberculosis (TB) and cholera antigens in transgenic rice for induction of immune responses in the mucosal compartment as well as in the systemic circulation. For vaccine development, we selected mycobacterial Ag85B antigen and immunoprotective P4 epitope of TcpA fused to the nontoxic cholera toxin B (CTB) subunit for immunization against TB and cholera, respectively, in independent constructs. The expression levels of CTB, CTB-TcpA, and CTB-Ag85B in transgenic lines containing stably integrated, chimeric genes showed up to 0.64%, 0.34%, and 0.02% of total rice seed protein, respectively. Oral immunization of mice with each of the three seed lines resulted in significantly increased levels of both anti-CTB IgG and IgA responses in the serum and IgA responses in the bronchoalveolar lavage (BAL) fluid. This indicated the capacity for oral immunization to elicit immune responses in the respiratory mucosal compartment. Plant-expressed TcpA could be detected in immunoblot analysis by using TcpA-specific commercial antibody, while there was no recognition of rice-expressed Ag85B by the commercial antibody raised against the latter antigen, where both antibodies were produced against the antigens expressed in the bacterial system. This study focused on identifying antigens resistant to both posttranslational modifications in plants and immunogenic under the proposed delivery system in animals for boosting the mucosal and systemic humoral immune response against enteric as well as respiratory pathogens.