Plants are potentially the most economical platforms for the large-scale production of recombinant proteins. Thus, plant-based expression of subunit human immunodeficiency virus type 1 (HIV-1) vaccines provides an opportunity for their global use against the acquired immunodeficiency syndrome pandemic. CTB-MPR(649-684)[CTB, cholera toxin B subunit; MPR, membrane proximal (ectodomain) region of gp41] is an HIV-1 vaccine candidate that has been shown previously to induce antibodies that block a pathway of HIV-1 mucosal transmission. In this article, the molecular characterization of CTB-MPR(649-684) expressed in transgenic Nicotiana benthamiana plants is reported. Virtually all of the CTB-MPR(649-684) proteins expressed in the selected line were shown to have assembled into pentameric, GM1 ganglioside-binding complexes. Detailed biochemical analyses on the purified protein revealed that it was N-glycosylated, predominantly with high-mannose-type glycans (more than 75%), as predicted from a consensus asparagine-X-serine/threonine (Asn-X-Ser/Thr) N-glycosylation sequon on the CTB domain and an endoplasmic reticulum retention signal attached at the C-terminus of the fusion protein. Despite this modification, the plant-expressed protein retained the nanomolar affinity to GM1 ganglioside and the critical antigenicity of the MPR(649-684) moiety. Furthermore, the protein induced mucosal and serum anti-MPR(649-684) antibodies in mice after mucosal prime-systemic boost immunization. Our data indicate that plant-based expression can be a viable alternative for the production of this subunit HIV-1 vaccine candidate.
