Molecularly Engineered Lectins as Anti-Influenza Agents

Abstract

Influenza viruses pose a major threat to public health worldwide, causing both seasonal epidemics, which infect millions and kill ~500,000 people annually, and pandemics, which are unpredictable and have the potential to kill millions, as was illustrated most recently in 2009 with the “swine flu” pandemic and most dramatically in 1918 with the emergence of the “Spanish flu” that resulted in fifty million deaths. There is a strong need for a new broad-spectrum anti-influenza therapeutic, as current prevention and treatment modalities, though helpful, suffer from distinct limitations and are at best only moderately effective. Lectins (carbohydrate-binding proteins) have been regarded as potential antiviral agents as they can selectively bind to specific glycans on viral surface glycoproteins, including those present on influenza virus. However, clinical development of lectins has been stalled by their mitogenicity, which is the ability to stimulate proliferation, especially of immune cells. We previously demonstrated that the mitogenic and antiviral activities of a lectin (banana lectin, BanLec) can be separated via a single amino acid mutation, histidine to threonine at position 84 (H84T). The resulting lectin, H84T BanLec (H84T), is virtually non-mitogenic but retains broad-spectrum antiviral activity against human immunodeficiency virus (HIV), hepatitis C virus (HCV), and multiple influenza strains, including pandemic and avian. In this work, we found that in a lethal mouse model H84T is indeed non-mitogenic, and both early and delayed therapeutic administration of H84T intraperitoneally are highly protective, as is H84T administered subcutaneously. Mechanistically, attachment, which we anticipated to be inhibited by H84T, was only somewhat decreased by the lectin. Instead, H84T is internalized into the late endosomal/lysosomal compartment and inhibits virus-endosome fusion. These studies reveal that H84T is efficacious against influenza virus in vivo, and that the loss of mitogenicity seen previously in tissue culture is also seen in vivo, underscoring the potential utility of H84T as a new broad-spectrum anti-influenza agent. Decreased mitogenicity in H84T was associated with disruption of pi-pi stacking between two aromatic amino acids. To examine whether we could provide further proof-of-principle of the ability to reduce mitogenicity without removing antiviral activity, we identified another lectin, Malaysian banana lectin (Malay BanLec), with similar structural features as BanLec, including pi-pi stacking, and showed that it is both mitogenic to peripheral blood mononuclear cells and potently antiviral. We therefore engineered an F84T mutation expected to disrupt pi-pi stacking, analogous to H84T. As predicted based on structural considerations, F84T Malay BanLec (F84T) was less mitogenic than Malay BanLec. However, F84T maintained strong antiviral activity and inhibited replication of HIV, influenza, Ebola, and other viruses. The F84T mutation disrupted pi-pi stacking without disrupting the overall lectin structure. Taken together, this work highlights the possibility of reducing mitogenicity by rational engineering to unlock the therapeutic potential of antiviral lectins in general, and demonstrates that the non-mitogenic, broad-spectrum antiviral agent H84T BanLec is a candidate to move forward clinically.