Discovering novel therapeutic V_HHs for emerging viruses: perspectives from VEEV selection strategies

Evolution or emergence of a new viral variant is a significant public health concern. Alphaviruses, such as Venezuelan equine encephalitis virus (VEEV), are mosquito-borne viruses which are becoming more prevalent due to expansion of vector habitats. Despite this, there are currently no antiviral therapies or FDA-approved vaccines available to treat or prevent VEEV infection. The increased prevalence of such viruses provides opportunities for novel variants to evolve. Key therapeutic molecules that could be developed against viral pathogens are recombinant antibodies or antibody fragments, such as the variable heavy domain of heavy chain antibodies (V_HHs).

In vitro selections offer a promising pathway for identification of therapeutic antibodies, here we explored isolation of V_HHs using phage and yeast display methodology with three antigen formats 1) recombinant E2, 2) linear peptides of E2, selected based on molecular dynamics analysis, and 3) UV inactivated virus.

Here we report four novel "human" V_HHs which bind to the VEEV E2 protein selected using different strategies that include both computational and biochemical design of suitable antigens and whole virus selections. These V_HHs have distinct complementarity-determining regions (CDRs). Multiple VHHs bind to the VEEV viral particles in ELISAs, and we report the peptide epitope recognized by these V_HHs.

Though non-neutralizing, these V_HHs bind to and sequester VEEV viral particles preventing infection, demonstrating the potential of these V_HHs to perform viral "sponging" which represents a novel therapeutic approach. The selection strategies we report may have applications to further antibody developments against other viruses.