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. PLOS Author summaryWe determined the binding sites of six novel human norovirus specific Nanobodies (Nano-4, Nano-14, Nano-26, Nano-27, Nano-32, and Nano-42) using X-ray crystallography. The unique Nanobody recognition epitopes were correlated with their potential neutralizing capacities. We showed that one Nanobody (Nano-26) bound numerous genogroup II genotypes and interacted with highly conserved capsid residues. Four Nanobodies (Nano-4, Nano-26, Nano-27, and Nano-42) bound to occluded regions on the intact particles and impaired normal capsid morphology and particle integrity. One Nanobody (Nano-14) bound contiguous to the HBGA pocket and interacted with several residues involved in binding HBGAs. We found that the Nanobodies delivered multiple inhibition mechanisms, which included steric obstruction, allosteric interference, and disruption of the capsid stability. Our data suggested that the HBGA pocket might not be an ideal target for drug development, since the surrounding regio...

Heterologous Expression of Human Norovirus GII.4 VP1 Leads to Assembly of T=4 Virus-Like Particles

“…GII.4 VLPs and their corresponding P domains have been extensively examined for binding to co-factors (HBGAs and bile acid), antibodies, human milk oligosaccharides (HMOs), and Nanobodies (21, 31, 37, 39-41). NSW-2012 P domains were capable of binding numerous HBGA types and the VLPs cross-reacted with Nanobodies and antibodies that were raised against other genotypes or GII.4 variants.…”

“…The NSW-2012 and CHDC-1974 VLPs (Genebank accession numbers JX459908 and ACT76142, respectively) were expressed in a baculovirus system as previously described (29-31). Briefly, the bacmid containing the recombinant VP1 gene was transfected in Sf9 insect cells.…”

Novel Structural Features of Human Norovirus Capsid

“…Therefore, this might suggest that the host could produces neutralizing antibodies against epitopes on the T=4 VLPs that were not as accessible on T=3 virions and that efficacy is difficult. Indeed, we have identified several Nanobodies that bind to occluded regions on the T=4 GII.4 VLPs (30, 33).…”

“…The NSW-2012 and CHDC-1974 VLPs (Genebank accession numbers JX459908 and ACT76142, respectively) were expressed in a baculovirus system as previously described (19–21). Briefly, the bacmid containing the recombinant VP1 gene was transfected in Sf9 insect cells.…”

Human Norovirus Neutralized by a Monoclonal Antibody Targeting the HBGA Pocket

“…Moreover, the IgA was GI.1 specific and lacked blocking activity against other GI genotypes. Likewise, we discovered a Nanobody (VHH) that bound at the GII.10 HBGA pocket and showed convincing HBGA blocking potential, but was also genotype specific (27). Other Nanobodies were shown to inhibit norovirus binding to HBGAs by several distinct mechanisms, including allosteric inhibition and disruption of capsid integrity (27).…”