Nanobodies as effective therapeutic agents against H5N1 influenza virus - Abstract The highly pathogenic avian influenza virus H5N1, which causes outbreaks in poultry and mammals, has shown increased rates of infection with high mortality in humans, indicating an urgent need to develop effective therapeutic agents against virus infection. The surface hemagglutinin (HA) protein of H5N1, including its HA1 and HA2 subunits, plays a key role in virus infection and pathogenesis, thereby serving as a critical therapeutic target. Nanobodies (single-domain antibodies) are small (in size), possess potent antigen-binding affinity and tissue penetration, and are stable in extreme conditions. Nanobodies can be easily engineered to increase their potency and efficiency, and could be rapidly and cost-effectively produced in large quantities. The proposed studies aim to develop potent and broadly neutralizing anti-H5N1 nanobodies with high stability, rapid production, and cost-effectiveness, being delivered efficiently as aerosols to the lungs, a major organ of H5N1 infection. This proposal is based on the hypothesis that nanobodies targeting the HA1 region, which induces highly potent neutralizing antibodies, or HA2 subunit (a highly conserved region), could be appropriately engineered to have broadly neutralizing activity and extended half-lives for efficient delivery to the lungs. Critical neutralizing domains in the HA of H5N1 have been found to induce potent and broadly neutralizing antibodies against multiple H5N1 strains. In addition, phage display nanobody libraries have been generated, and appropriate assays for nanobody characterization, neutralization, and efficacy evaluation have been developed, providing solid bases for the proposed studies. The overall goal is to rationally design and develop potent, highly stable, cost-effective, and broadly neutralizing nanobodies against H5N1 to prevent and treat current and future outbreaks. To achieve this goal, this proposal will first screen, develop, and engineer nanobodies specific to the H5N1 HA protein. These nanobodies will be characterized and modified to improve their antigen-binding affinity and broad neutralizing activity, as well as enhancing their ability to be delivered in aerosols with protective efficacy. The results of this proposal may be highly significant, allowing the rapid development of much-needed therapeutic agents that can prevent and treat current and future outbreaks of H5N1 infection. These proposed studies may also have implications for the development of effective antiviral agents against other influenza viruses and other pathogens with outbreak or pandemic potential.
