P44.06
Background: Entry inhibitors could offer promising means for non-vaccine biomedical prevention against HIV infection, such as topical microbicides. Here, we aim to create a novel entry inhibitor by translationally fusing a broadly HIV-1 neutralizing monoclonal antibody (mAb) and a carbohydrate-binding antiviral protein targeting the glycan shield of viral envelope. We hypothesize that such a bispecific inhibitor may effectively block and control HIV-1 infection.
Methods: We engineered a prototype bispecific fusion protein based on the antigen-binding fragment (Fab) of the CD4 binding site-specific mAb VRC01 and the oligomannose-specific, non-cytotoxic/inflammatory/mitogenic antiviral lectin Avaren (VRC01-Av). VRC01-Av was produced in Nicotiana benthamiana plants due to the prospective scalability and cost effectiveness of plant-based expression systems. The fusion protein was purified by a series of chromatography steps, and tested for the binding properties and anti-HIV activity by surface plasmon resonance (SPR) and Env-pseudotyped virus neutralization assays, respectively.
Results: The fusion protein was efficiently purified to>95% homogeneity by a two-step chromatography process. SPR demonstrated that both active sites of the VRC01-Av fusion retained affinity to a recombinant HIV-1 Env protein gp120. VRC01-Av had significantly stronger HIV-1-neutralizing activity than a 1:1 equimolar mixture of each component (i.e., VRC01Fab and Avaren) and respective bivalent parental molecules (i.e., VRC01 IgG and Avaren-Fc fusion) against multiple strains. We are currently developing a dry powder formulation of VRC01-Av to enhance stability and facilitate delivery system development.
Conclusions: These results highlight the advantages of VRC01-Av bispecific fusion protein over a mixture of the two original entry inhibitors in terms of efficacy, manufacturing and drug development. Our data warrant further preclinical efficacy and toxicity studies of VRC01-Av.
