Gonzalez Llamazares, A. | Search bioRxiv

bioRxiv2021

Single-dose immunisation with a multimerised SARS-CoV-2 receptor binding domain (RBD) induces an enhanced and protective response in mice

The COVID-19 pandemic, caused by the SARS-CoV-2 coronavirus, has triggered a worldwide health emergency. So far, several different types of vaccines have shown strong efficacy. However, both the emergence of new SARS-CoV-2 variants and the need to vaccinate a large fraction of the worlds population necessitate the development of alternative vaccines, especially those that are simple and easy to store, transport and administer. Here, we showed that ferritin-like Dps protein from hyperthermophilic Sulfolobus islandicus can be covalently coupled with different SARS-CoV-2 antigens via the SpyCatcher system, to form extremely stable and defined multivalent dodecameric vaccine nanoparticles that remain intact even after lyophilisation. Immunisation experiments in mice demonstrated that the SARS-CoV-2 receptor binding domain (RBD) coupled to Dps (RBD-S-Dps) shows particular promise as it elicited a higher antibody titre and an enhanced neutralising antibody response compared to the monomeric RBD. Furthermore, we showed that a single immunisation with the multivalent RBD-S-Dps completely protected hACE2-expressing mice from serious illness and led to efficient viral clearance from the lungs upon SARS-CoV-2 infection. Our data highlight that multimerised SARS-CoV-2 subunit vaccines are a highly efficacious modality, particularly when combined with an ultra-stable scaffold.

immunology↗

bioRxiv2019

Bactofilins form non-polar filaments that bind to membranes directly

Bactofilins are small beta-helical proteins that form cytoskeletal filaments in a range of bacteria. Bactofilins have diverse functions: filaments in Caulobacter crescentus are involved in cell stalk formation whereas Myxococcus xanthus filaments aid chromosome segregation and motility. The precise molecular architecture of bactofilin filaments has remained unclear. Here we revealed by sequence analyses and electron microscopy that in addition to wide distribution across bacteria and archaea, bactofilins are also present in a few eukaryotic cells such as oomycetes. The sole bactofilin from Thermus thermophilus was demonstrated to form constitutive filaments and cryo-EM analysis revealed that protofilaments formed through end-to-end association of the beta-helical domains. Using a nanobody against Thermus bactofilin we determined the near-atomic filament structure, showing that the filaments are non-polar, with subunits arranged head-to-head and tail-to-tail. A polymerisation-impaired mutant F105R, that disrupts one of the two protofilament interfaces, enabled crystallisation. The crystal structure also revealed non-polar protofilaments, and the dominance of the beta-stacking interface that formed despite the inhibiting mutation. To confirm the generality of the lack of polarity, we performed co-evolutionary analysis of a large set of sequences. Finally, using Thermus bactofilin, we determined that the N-terminal disordered tail of the protein is responsible for direct binding to lipid membranes both on liposomes and by electron cryotomography in E. coli cells. The tail is conserved, suggesting that membrane binding is likely a general feature of these very common but only recently discovered filaments of the prokaryotic cytoskeleton.

biochemistry↗