Improving the potential of influenza A virus-targeting VHHs by antiviral peptide conjugation and multimerization

Abstract

Influenza A virus (IAV) is a global healthcare burden, causing annual epidemics with high mortality rates. Highly pathogenic avian influenza (HPAI) poses a risk for a new zoonotic outbreak, underscoring the need for an “universal therapy”. IAV is characterized by its surface glycoproteins neuraminidase (NA) and hemagglutinin (HA), offering potential targets for an “universal therapy”. VHHs are small antigen binding fragments, exhibiting similar binding strength to full size monoclonal antibodies. Their small size allows VHHs for easy formatting, which in previous studies enhanced the binding breadth and neutralisation for IAV-targeting VHHs, making VHHs an interesting therapeutic for an universal IAV therapy. In this study previously selected VHHs targeting NA and HA were optimised to improve their possible therapeutic usage. Optimisation was pursued by the novel approach of conjugating various antiviral peptides to an HA stem-targeting VHH. Further optimisation included the multimerization of HA stem and NA-targeting VHHs, by fusion to an IgG1 Fc domain or adding a GCN4 multimerization region. It was examined that the antiviral peptides showed neutralisation against H1N1 pdm09 in a newly designed IAV neutralisation assay, but no conjugation could be seen with the HA stem-targeting VHH. Multimerization of the HA stem VHH lead to a modest improvement in binding affinity and neutralising property. Multimerization of the NA-targeting VHHs with a GCN4 tetramerization domain enhanced their neutralisation property, surpassing the effectiveness of currently used small drug molecule Oseltamivir. This emphasizes the therapeutic potential of optimised IAV-targeting VHHs in the clinic.