Discovering Novel Therapeutic VHHs 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. 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 (VHHs). These proteins can neutralize or sequester viral particles, preventing or reducing infection. However, due to the evolution of viruses, there is a need to isolate new antibodies and direct their binding to particular epitopes on the virus. In vitro selections offer a promising pathway for the selection of therapeutic antibodies, but as we demonstrate, the choice of a target for these selections is key to obtaining the desired viral binding characteristics. Here we report four novel "human" VHHs 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 VHHs 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 VHHs. Though non-neutralizing, when immobilized, these VHHs bind to and sequester VEEV viral particles preventing infection, demonstrating the potential of these VHHs to perform viral "sponging". The selection strategies we report may have applications to further antibody developments against other viruses.