Shark nanobodies with potent SARS-CoV-2 neutralizing activity and broad sarbecovirus reactivity.

Despite rapid and ongoing vaccine and therapeutic development, SARS-CoV-2 continues to evolve and evade, presenting a need for next-generation diverse therapeutic modalities. Here we show that nurse sharks immunized with SARS-CoV-2 recombinant receptor binding domain (RBD), RBD-ferritin (RFN), or spike protein ferritin nanoparticle (SpFN) immunogens elicit a set of new antigen receptor antibody (IgNAR) molecules that target two non-overlapping conserved epitopes on the spike RBD. Representative shark antibody variable NAR-Fc chimeras (ShAbs) targeting either of the two epitopes mediate cell-effector functions, with high affinity to all SARS-CoV-2 viral variants of concern, including the divergent Omicron strains. The ShAbs potently cross-neutralize SARS-CoV-2 WA-1, Alpha, Beta, Delta, Omicron BA.1 and BA.5, and SARS-CoV-1 pseudoviruses, and confer protection against SARS-CoV-2 challenge in the K18-hACE2 transgenic mouse model. Structural definition of the RBD-ShAb01-ShAb02 complex enabled design and production of multi-specific nanobodies with enhanced neutralization capacity, and picomolar affinity to divergent sarbecovirus clade 1a, 1b and 2 RBD molecules. These shark nanobodies represent potent immunotherapeutics both for current use, and future sarbecovirus pandemic preparation.

Serological evidence of a pararubulavirus and a betacoronavirus in the geographically isolated Christmas Island flying-fox (Pteropus natalis).

Due to their geographical isolation and small populations, insular bats may not be able to maintain acute immunizing viruses that rely on a large population for viral maintenance. Instead, endemic transmission may rely on viruses establishing persistent infections within hosts or inducing only short-lived neutralizing immunity. Therefore, studies on insular populations are valuable for developing broader understanding of viral maintenance in bats. The Christmas Island flying-fox (CIFF; Pteropus natalis) is endemic on Christmas Island, a remote Australian territory, and is an ideal model species to understand viral maintenance in small, geographically isolated bat populations. Serum or plasma (n = 190), oral swabs (n = 199), faeces (n = 31), urine (n = 32) and urine swabs (n = 25) were collected from 228 CIFFs. Samples were tested using multiplex serological and molecular assays, and attempts at virus isolation to determine the presence of paramyxoviruses, betacoronaviruses and Australian bat lyssavirus. Analysis of serological data provides evidence that the species is maintaining a pararubulavirus and a betacoronavirus. There was little serological evidence supporting the presence of active circulation of the other viruses assessed in the present study. No viral nucleic acid was detected and no viruses were isolated. Age-seropositivity results support the hypothesis that geographically isolated bat populations can maintain some paramyxoviruses and coronaviruses. Further studies are required to elucidate infection dynamics and characterize viruses in the CIFF. Lastly, apparent absence of some pathogens could have implications for the conservation of the CIFF if a novel disease were introduced into the population through human carriage or an invasive species. Adopting increased biosecurity protocols for ships porting on Christmas Island and for researchers and bat carers working with flying-foxes are recommended to decrease the risk of pathogen introduction and contribute to the health and conservation of the species.

A betacoronavirus multiplex microsphere immunoassay detects early SARS-CoV-2 seroconversion and antibody cross reactions.

Sensitive and specific SARS-CoV-2 antibody assays remain critical for community and hospital-based SARS-CoV-2 surveillance. Here, we developed and applied a multiplex microsphere-based immunoassay (MMIA) for COVD-19 antibody studies that incorporates spike protein trimers of SARS-CoV-2, SARS-CoV-1, MERS-CoV, and the seasonal human betacoronaviruses, HCoV-HKU1 and HCoV-OC43, that enables measurement of off-target pre-existing cross-reactive antibodies. The MMIA performances characteristics are: 98% sensitive and 100% specific for human subject samples collected as early as 10 days from symptom onset. The MMIA permitted the simultaneous identification of SARS-CoV-2 seroconversion and the induction of SARS-CoV-2 IgG antibody cross reactions to SARS-CoV-1 and MERS-CoV. Further, synchronous increases of HCoV-OC43 IgG antibody levels was detected with SARS-CoV-2 seroconversion in a subset of subjects for whom early infection sera were available prior to their SARS-CoV-2 seroconversion, suggestive of an HCoV-OC43 memory response triggered by SARS-CoV-2 infection.