Nanobodies targeting norovirus capsid reveal functional epitopes and potential mechanisms of neutralization

Abstract

Norovirus is the leading cause of gastroenteritis worldwide. Despite recent developments in norovirus propagation in cell culture, these viruses are still challenging to grow routinely. Moreover, little is known on how norovirus infects the host cells, except that histo-blood group antigens (HBGAs) are important binding factors for infection and cell entry. Antibodies that bind at the HBGA pocket and block attachment to HBGAs are believed to neutralize the virus. However, additional neutralization epitopes elsewhere on the capsid likely exist and impeding the intrinsic structural dynamics of the capsid could be equally important. In the current study, we investigated a panel of Nanobodies in order to probe functional epitopes that could trigger capsid rearrangement and/ or interfere with HBGA binding interactions. The precise binding sites of six Nanobodies (Nano-4, Nano-14, Nano-26, Nano-27, Nano-32, and Nano-42) were identified using X-ray crystallography. We showed that these Nanobodies bound on the top, side, and bottom of the norovirus protruding domain. The impact of Nanobody binding on norovirus capsid morphology was analyzed using electron microscopy and dynamic light scattering. We discovered that distinct Nanobody epitopes were associated with varied changes in particle structural integrity and assembly. Interestingly, certain Nanobody-induced capsid morphological changes lead to the capsid protein degradation and viral RNA exposure. Moreover, Nanobodies employed multiple inhibition mechanisms to prevent norovirus attachment to HBGAs, which included steric obstruction (Nano-14), allosteric interference (Nano-32), and violation of normal capsid morphology (Nano-26 and Nano-85). Finally, we showed that two Nanobodies (Nano-26 and Nano-85) not only compromised capsid integrity and inhibited VLPs attachment to HBGAs, but also recognized a broad panel of norovirus genotypes with high affinities. Consequently, Nano-26 and Nano-85 have a great potential to function as novel therapeutic agents against human noroviruses.

Norovirus GII.17 Virus-Like Particles Bind to Different Histo-Blood Group Antigens and Cross-React with Genogroup II-Specific Mouse Sera

“…blocking epitope F with ´NERK´ motif (24), which may be less prone to sequence variations than HBGA binding sites and still block NoV binding to HBGA through allosteric interference. Moreover, two residues of ´NERK´ have been linked to GII.10 NoV blocking mechanism, suggesting possible involvement of this motif on NoV neutralization on larger scale (20). However, comparison of five putative GII.4 blockade antibody epitope sequences (12,25) (Table 4) did not provide more insights to superiority of GII.4-99 to induce GII.17 blocking antibodies, as GII.17 shared 12 aa residues with genetically more closely related GII.12 and only 6 aa with GII.4-99.…”

Antigen recognition by single-domain antibodies: structural latitudes and constraints

“…Serotype cross-neutralizing antibodies targeting the CD155-binding ‘canyon’ of the poliovirus capsid are rarely produced by the murine or human humoral immune systems, 79,80 but are apparently common in llama heavy-chain only responses. 81 Likewise, V H Hs targeting the HBGA-binding pocket of norovirus VP1 neutralized a broad range of genotypes, 82 while larger conventional antibodies also made contact with antigenically variable residues surrounding the HBGA pocket and were thus strain-specific. 83 …”

Atomic Structure of the Murine Norovirus Protruding Domain and Soluble CD300lf Receptor Complex

“…Human noroviruses interact with histo-blood group antigens (HBGAs) (6)(7)(8)(16)(17)(18)(19)(20). Indeed, two recently developed human norovirus cell culture systems have shown that HBGAs and HBGA secretor status are important for virus infection (21,22).…”