HIV-1 Neutralization Profile and Plant-Based Recombinant Expression of Actinohivin, an Env Glycan-Specific Lectin Devoid of T-Cell Mitogenic Activity

The development of a topical microbicide blocking the sexual transmission of HIV-1 is urgently needed to control the global HIV/AIDS pandemic. The actinomycete-derived lectin actinohivin (AH) is highly specific to a cluster of high-mannose-type glycans uniquely found on the viral envelope (Env). Here, we evaluated AH's candidacy toward a microbicide in terms of in vitro anti-HIV-1 activity, potential side effects, and recombinant producibility. Two validated assay systems based on human peripheral blood mononuclear cell (hPBMC) infection with primary isolates and TZM-bl cell infection with Env-pseudotyped viruses were employed to characterize AH's anti-HIV-1 activity. In hPMBCs, AH exhibited nanomolar neutralizing activity against primary viruses with diverse cellular tropisms, but did not cause mitogenicity or cytotoxicity that are often associated with other anti-HIV lectins. In the TZM-bl-based assay, AH showed broad anti-HIV-1 activity against clinically-relevant, mucosally transmitting strains of clades B and C. By contrast, clade A viruses showed strong resistance to AH. Correlation analysis suggested that HIV-1′s AH susceptibility is significantly linked to the N-glycans at the Env C2 and V4 regions. For recombinant (r)AH expression, we evaluated a tobacco mosaic virus-based system in Nicotiana benthamiana plants as a means to facilitate molecular engineering and cost-effective mass production. Biochemical analysis and an Env-mediated syncytium formation assay demonstrated high-level expression of functional rAH within six days. Taken together, our study revealed AH's cross-clade anti-HIV-1 activity, apparent lack of side effects common to lectins, and robust producibility using plant biotechnology. These findings justify further efforts to develop rAH toward a candidate HIV-1 microbicide

Expression of rAH in <i>N. benthamiana</i>.

<p>(A) Representative TMV vector-infiltrated <i>N. benthamiana</i> leaves at 6 dpi. The leaves show only a minor level of tissue necrosis. (B) Electrophoretic analysis of rAH expression in leaf extract. Coomassie-stained SDS-PAGE gel (Lanes 1–3) and western blot analysis (Lanes 4–7) are shown. Total leaf proteins were extracted in a buffer containing 2% SDS and separated under reducing conditions. Lanes 1 and 4: non-infiltrated control leaf extract; lanes 2, 3, 5, and 6: vector-infiltrated leaf extracts made from two independent infiltration events; and lane 7: the dialyzed aqueous fraction of infiltrated leaf proteins prepared by 6 M guanidine buffer extraction. Asterisks indicate the bands corresponding to rAH (Mw: 12.5 kDa). Efficient extraction of gp120-binding rAH required a buffer containing a chaotropic agent such as SDS or guanidine HCl. The fraction contains apparent multimers of rAH (Lanes 5 and 6). These aggregates are largely absent after dialysis, while the monomer protein stays soluble (Lane 7). See text for detail.</p