Selection and characterization of neutralizing single domain antibodies against Dengue virus and SARS-CoV2

Abstract

Abstract Antibodies, the secreted adaptor molecules of activated B cells, are one of the critical components of adaptive immunity to help neutralize pathogens at entry sites to achieve the control of infection. Due to their ability to recognize antigens with pin-point precision and amenability to genetic modifications, antibodies serve as the reagents of choice in immunotherapy and diagnosis. The discovery of the smallest variant of antibodies, referred to as single domain antibodies (sdAbs) or nanobodies, has provided the much-needed impetus to the field of antibody engineering to generate custom made products and optimally harness their potential. Such antibodies have unique features such as small size, improved solubility and stability as well as high affinities. The study was planned to select, modify and characterize sdAbs that could neutralize viruses such as Dengue (DenV) and the causative agent of COVID- 19. The first segment of investigation aimed at generating sdAbs against DenV. A universal prophylaxis or a therapy is lacking against DenV and the full-length conventional antibodies, counterintuitively, promote pathogenesis by a phenomenon known as antibody dependent enhancement (ADE) of infection. sdAbs are unlikely to cause ADE because of the missing Fc region, a major contributor to the phenomenon. Therefore, a high-affinity sdAb specific to the envelope (E) protein of DenV-2 was selected using phage display technology. The sdAb specifically interacted with the viral protein and neutralized the infectivity of a non-replicating pseudovirus expressing E protein as well as a virulent DenV. The effect was demonstrated both in vitro in cell culture and in vivo in a highly susceptible IFNRKO mouse. In the second segment of the study, sdAbs against the receptor binding domain (RBD) and the polybasic furin cleavage site (FCS) of the spike (S) protein of SARS-CoV2 were selected and evaluated for anti-viral potential. The FCS was newly acquired by SARS-CoV2 and served as via key virulence factor in enhancing infectivity. The generated antibodies resisted denaturation by elevated temperature and remained stable over a wide range of pH. Neutralizing potential as well as the anti-fusogenic activity of both the sdAbs was demonstrated. Fusion mediated by the viral S protein helps spread the virus to bystander cells to enhance infectivity. Given the shared epitope in the FCS of S protein and the entry mediators of several other viruses, the anti- FCS sdAb was evaluated for its neutralizing potential against multiple viruses of animals and humans. The anti-FCS sdAb cross neutralized viruses such as HSV1 and PPRV which infect humans and animals, respectively. By their mere nature, conventional antibodies function extracellularly but the potential of the sdAbs as intrabodies (IB) was demonstrated in deciphering events in viral biogenesis. Accordingly, IB expressing cells failed to produce infectious virus particles. The study, therefore, highlighted the potential utility of sdAbs as a therapy against DenV and SARS-CoV2 infection.