Concerted deletions eliminate a neutralizing supersite in SARS-CoV-2 BA.2.87.1 spike.

BA.2.87.1 represents a major shift in the BA.2 lineage of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is unusual in having two lengthy deletions of polypeptide in the spike (S) protein, one of which removes a beta-strand. Here we investigate its neutralization by a variety of sera from infected and vaccinated individuals and determine its spike (S) ectodomain structure. The BA.2.87.1 receptor binding domain (RBD) is structurally conserved and the RBDs are tightly packed in an "all-down" conformation with a small rotation relative to the trimer axis as compared to the closest previously observed conformation. The N-terminal domain (NTD) maintains a remarkably similar structure overall; however, the rearrangements resulting from the deletions essentially destroy the so-called supersite epitope and eliminate one glycan site, while a mutation creates an additional glycan site, effectively shielding another NTD epitope. BA.2.87.1 is relatively easily neutralized but acquisition of additional mutations in the RBD could increase antibody escape allowing it to become a dominant sub-lineage.

Structural insights into Frizzled3 through nanobody modulators.

The Wnt receptor Frizzled3 (FZD3) is important for brain axonal development and cancer progression. We report structures of FZD3 in complex with extracellular and intracellular binding nanobodies (Nb). The crystal structure of Nb8 in complex with the FZD3 cysteine-rich domain (CRD) reveals that the nanobody binds at the base of the lipid-binding groove and can compete with Wnt5a. Nb8 fused with the Dickkopf-1 C-terminal domain behaves as a FZD3-specific Wnt surrogate, activating ß-catenin signalling. The cryo-EM structure of FZD3 in complex with Nb9 reveals partially resolved density for the CRD, which exhibits positional flexibility, and a transmembrane conformation that resembles active GPCRs. Nb9 binds to the cytoplasmic region of FZD3 at the putative Dishevelled (DVL) or G protein-binding site, competes with DVL binding, and inhibits GαS coupling. In combination, our FZD3 structures with nanobody modulators map extracellular and intracellular interaction surfaces of functional, and potentially therapeutic, relevance.

A structure-function analysis shows SARS-CoV-2 BA.2.86 balances antibody escape and ACE2 affinity.

BA.2.86, a recently described sublineage of SARS-CoV-2 Omicron, contains many mutations in the spike gene. It appears to have originated from BA.2 and is distinct from the XBB variants responsible for many infections in 2023. The global spread and plethora of mutations in BA.2.86 has caused concern that it may possess greater immune-evasive potential, leading to a new wave of infection. Here, we examine the ability of BA.2.86 to evade the antibody response to infection using a panel of vaccinated or naturally infected sera and find that it shows marginally less immune evasion than XBB.1.5. We locate BA.2.86 in the antigenic landscape of recent variants and look at its ability to escape panels of potent monoclonal antibodies generated against contemporary SARS-CoV-2 infections. We demonstrate, and provide a structural explanation for, increased affinity of BA.2.86 to ACE2, which may increase transmissibility.

Emerging variants develop total escape from potent monoclonal antibodies induced by BA.4/5 infection.

The rapid evolution of SARS-CoV-2 is driven in part by a need to evade the antibody response in the face of high levels of immunity. Here, we isolate spike (S) binding monoclonal antibodies (mAbs) from vaccinees who suffered vaccine break-through infections with Omicron sub lineages BA.4 or BA.5. Twenty eight potent antibodies are isolated and characterised functionally, and in some cases structurally. Since the emergence of BA.4/5, SARS-CoV-2 has continued to accrue mutations in the S protein, to understand this we characterize neutralization of a large panel of variants and demonstrate a steady attrition of neutralization by the panel of BA.4/5 mAbs culminating in total loss of function with recent XBB.1.5.70 variants containing the so-called 'FLip' mutations at positions 455 and 456. Interestingly, activity of some mAbs is regained on the recently reported variant BA.2.86.

The SARS-CoV-2 neutralizing antibody response to SD1 and its evasion by BA.2.86.

Under pressure from neutralising antibodies induced by vaccination or infection the SARS-CoV-2 spike gene has become a hotspot for evolutionary change, leading to the failure of all mAbs developed for clinical use. Most potent antibodies bind to the receptor binding domain which has become heavily mutated. Here we study responses to a conserved epitope in sub-domain-1 (SD1) of spike which have become more prominent because of mutational escape from antibodies directed to the receptor binding domain. Some SD1 reactive mAbs show potent and broad neutralization of SARS-CoV-2 variants. We structurally map the dominant SD1 epitope and provide a mechanism of action by blocking interaction with ACE2. Mutations in SD1 have not been sustained to date, but one, E554K, leads to escape from mAbs. This mutation has now emerged in several sublineages including BA.2.86, reflecting selection pressure on the virus exerted by the increasing prominence of the anti-SD1 response.

Structural and mechanistic characterization of bifunctional heparan sulfate N-deacetylase-N-sulfotransferase 1.

Heparan sulfate (HS) polysaccharides are major constituents of the extracellular matrix, which are involved in myriad structural and signaling processes. Mature HS polysaccharides contain complex, non-templated patterns of sulfation and epimerization, which mediate interactions with diverse protein partners. Complex HS modifications form around initial clusters of glucosamine-N-sulfate (GlcNS) on nascent polysaccharide chains, but the mechanistic basis underpinning incorporation of GlcNS itself into HS remains unclear. Here, we determine cryo-electron microscopy structures of human N-deacetylase-N-sulfotransferase (NDST)1, the bifunctional enzyme primarily responsible for initial GlcNS modification of HS. Our structures reveal the architecture of both NDST1 deacetylase and sulfotransferase catalytic domains, alongside a non-catalytic N-terminal domain. The two catalytic domains of NDST1 adopt a distinct back-to-back topology that limits direct cooperativity. Binding analyses, aided by activity-modulating nanobodies, suggest that anchoring of the substrate at the sulfotransferase domain initiates the NDST1 catalytic cycle, providing a plausible mechanism for cooperativity despite spatial domain separation. Our data shed light on key determinants of NDST1 activity, and describe tools to probe NDST1 function in vitro and in vivo.

Reduced neutralization of SARS-CoV-2 B.1.617 by vaccine and convalescent serum (preprint)

A pesquisa aponta que o soro de pessoas previamente infectadas por outras cepas é menos potente contra esta variante viral. O problema é observado de forma marcante entre os indivíduos anteriormente infectados pela variante Gama, identificada originalmente em Manaus e atualmente dominante no Brasil, assim como pela variante Beta, detectada pela primeira vez na África do Sul. Nestes casos, a capacidade de neutralizar a cepa Delta é onze vezes menor. O soro de pessoas vacinadas também tem potência reduzida contra a variante originária da Índia, mas os dados apontam que as vacinas continuam efetivas. A capacidade de neutralizar a cepa é 2,5 vezes menor para o imunizante da Pfizer e 4,3 vezes menor para o da Astrazeneca. Os autores do trabalho ressaltam que os índices são semelhantes aos verificados com as variantes Gama e Alfa ­ que emergiram no Brasil e no Reino Unido, respectivamente. Não há evidência de fuga generalizada da neutralização, diferentemente do registrado com a variante Beta ­ com origem na África do Sul.