A novel anti-HIV-1 bispecific bNAb-lectin fusion protein engineered in a plant-based transient expression system

The discovery of broadly neutralizing antibodies (bNAbs) has been a major step towards better prophylactic and therapeutic agents against human immunodeficiency virus type 1 (HIV-1). However, effective therapy will likely require a combination of anti-HIV agents to avoid viral evasion. One possible solution to this problem is the creation of bispecific molecules that can concurrently target two vulnerable sites providing synergistic inhibitory effects. Here, we describe the production in plants and anti-HIV activity of a novel bispecific fusion protein consisting of the antigen-binding fragment (Fab) of the CD4 binding site-specific bNAb VRC01 and the antiviral lectin Avaren, which targets the glycan shield of the HIV-1 envelope (VRC01_Fab -Avaren). This combination was justified by a preliminary experiment demonstrating the synergistic HIV-1 neutralization activity of VRC01 and Fc-fused Avaren dimer (Avaren-Fc). Using the GENEWARE^® tobacco mosaic virus vector, VRC01_Fab -Avaren was expressed in Nicotiana benthamiana and purified using a three-step chromatography procedure. Surface plasmon resonance and ELISA demonstrated that both the Avaren and VRC01_Fab moieties retain their individual binding specificities. VRC01_Fab -Avaren demonstrated enhanced neutralizing activity against representative HIV-1 strains from A, B and C clades, compared to equimolar combinations of VRC01_Fab and Avaren. Notably, VRC01_Fab -Avaren showed significantly stronger neutralizing effects than the bivalent parent molecules VRC01 IgG and Avaren-Fc, with IC₅₀ values ranging from 48 to 310 pm. These results support the continued development of bispecific anti-HIV proteins based on Avaren and bNAbs, to which plant-based transient overexpression systems will provide an efficient protein engineering and production platform.