Florian I. Schmidt

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread, with devastating consequences. For passive immunization efforts, nanobodies have size and cost advantages over conventional antibodies. In this study, we generated four neutralizing nanobodies that target the receptor binding domain of the SARS-CoV-2 spike protein. We used x-ray crystallography and cryo-electron microscopy to define two distinct binding epitopes. On the basis of these structures, we engineered multivalent nanobodies with more than 100 times the neutralizing activity of monovalent nanobodies. Biparatopic nanobody fusions suppressed the emergence of escape mutants. Several nanobody constructs neutralized through receptor binding competition, whereas other monovalent and biparatopic nanobodies triggered aberrant activation of the spike fusion machinery. These premature conformational changes in the spike protein forestalled productive fusion and rendered the virions noninfectious.

Inflammasomes are supramolecular complexes that play key roles in immune surveillance. This is accomplished by the activation of inflammatory caspases, which leads to the proteolytic maturation of interleukin 1β (IL-1β) and pyroptosis. Here, we show that nucleotide-binding domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NLRP3)- and pyrin-mediated inflammasome assembly, caspase activation, and IL-1β conversion occur at the microtubule-organizing center (MTOC). Furthermore, the dynein adapter histone deacetylase 6 (HDAC6) is indispensable for the microtubule transport and assembly of these inflammasomes both in vitro and in mice. Because HDAC6 can transport ubiquitinated pathological aggregates to the MTOC for aggresome formation and autophagosomal degradation, its role in NLRP3 and pyrin inflammasome activation also provides an inherent mechanism for the down-regulation of these inflammasomes by autophagy. This work suggests an unexpected parallel between the formation of physiological and pathological aggregates.

The unique class of heavy chain-only antibodies, present in Camelidae, can be shrunk to just the variable region of the heavy chain to yield VHHs, also called nanobodies. About one-tenth the size of their full-size counterparts, nanobodies can serve in applications similar to those for conventional antibodies, but they come with a number of signature advantages that find increasing application in biology. They not only function as crystallization chaperones but also can be expressed inside cells as such, or fused to other proteins to perturb the function of their targets, for example, by enforcing their localization or degradation. Their small size also affords advantages when applied in vivo, for example, in imaging applications. Here we review such applications, with particular emphasis on those areas where conventional antibodies would face a more challenging environment.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evades most innate immune responses but may still be vulnerable to some. Here, we systematically analyze the impact of SARS-CoV-2 proteins on interferon (IFN) responses and autophagy. We show that SARS-CoV-2 proteins synergize to counteract anti-viral immune responses. For example, Nsp14 targets the type I IFN receptor for lysosomal degradation, ORF3a prevents fusion of autophagosomes and lysosomes, and ORF7a interferes with autophagosome acidification. Most activities are evolutionarily conserved. However, SARS-CoV-2 Nsp15 antagonizes IFN signaling less efficiently than the orthologs of closely related RaTG13-CoV and SARS-CoV-1. Overall, SARS-CoV-2 proteins counteract autophagy and type I IFN more efficiently than type II or III IFN signaling, and infection experiments confirm potent inhibition by IFN-γ and -λ1. Our results define the repertoire and selected mechanisms of SARS-CoV-2 innate immune antagonists but also reveal vulnerability to type II and III IFN that may help to develop safe and effective anti-viral approaches.