Fab molecules are used as therapeutic agents, and are invaluable tools in structural biology. We report here a method for production of recombinant Fab in Drosophila S2 cells for use in structural biology. Stably transfected S2 cell lines expressing the Fab were created within weeks. The recombinant Fab was secreted, and after affinity and size exclusion chromatography, 16 mg of pure protein were obtained from a liter of cell culture. The Fab was functional and formed a complex with its cognate antigen as demonstrated by co-precipitation and size exclusion chromatography. Biochemical characterization indicated that the Fab from S2 cells is less extensively glycosylated than the Fab obtained by digestion of antibody produced in hybridoma cells, a feature that may be advantageous for the purposes of crystallogenesis. Taken together, obtaining recombinant Fab from the S2 cells has been a faster and considerably more cost-effective method compared with the enzymatic digestion of the monoclonal antibody.

Immunological and inflammatory reactions have been suggested to have a role in the development of schizophrenia, a hypothesis that has recently been supported by genetic data. The aim of our study was to perform an unbiased search for autoantibodies in patients with a first psychotic episode, and to explore the association between any seroreactivity and the development of a Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV) disorder characterized by chronic or relapsing psychotic symptoms. We collected plasma samples from 53 patients when they were treated for their first-episode psychosis, and 41 non-psychotic controls, after which the patients were followed for a mean duration of 7 years. Thirty patients were diagnosed with schizophrenia, delusional disorder, schizoaffective disorder, bipolar disorder or a long-term unspecified nonorganic psychosis during follow-up, whereas 23 patients achieved complete remission. At the end of follow-up, plasma samples were analyzed for IgG reactivity to 2304 fragments of human proteins using a multiplexed affinity proteomic technique. Eight patient samples showed autoreactivity to the N-terminal fragment of the PAGE (P antigen) protein family (PAGE2B/PAGE2/PAGE5), whereas no such autoreactivity was seen among the controls. PAGE autoreactivity was associated with a significantly increased risk of being diagnosed with schizophrenia during follow-up (odds ratio 6.7, relative risk 4.6). An immunohistochemistry analysis using antisera raised against the N-terminal fragment stained an unknown extracellular target in human cortical brain tissue. Our findings suggest that autoreactivity to the N-terminal portion of the PAGE protein family is associated with schizophrenia in a subset of patients with first-episode psychosis.

The threat of emergence of further SARS-CoV-2 variants, and the future spillover potential of other sarbecoviruses has prompted continued efforts to isolate broadly reactive monoclonal antibodies for therapeutic use. In this study, we generated monoclonal antibodies from immunised cattle, primarily because of their ability to produce antibodies with ultra-long heavy chain complementarity determining region 3 (CDRH3) domains. Such antibodies have been shown to have potent and cross-reactive neutralisation phenotypes in other virus infections. Following extended immunisation with different forms of spike protein and using single B-cell sorting and phage display techniques, we isolated 33 mAbs, including 10 with ultra-long CDRH3s (>50 amino acids). Of these, mAbs P7 and 99 exhibited remarkable neutralisation breadth and potency. Notably, mAb P7, which possessed an ultra-long CDRH3, neutralised all tested variants, including SARS-CoV-1, with IC₅₀ values ranging from 0.01 µg/mL to 1.06 µg/mL. This antibody was also cross-reactive against a panel of RBDs from diverse sarbecovirus species. Structural studies revealed that mAb 99 targets a region of the receptor-binding domain (RBD) of the spike protein that overlaps with the ACE2 binding site. Although the structure of the P7 Fab-RBD complex was not resolvable, data suggest P7 induces trimer dissociation by binding to an occluded RBD epitope, likely mediated by the extended CDRH3 structure. Syrian hamster challenge experiments, using several VOCs, showed that mAbs P7 and 99 significantly reduced lung viral load. These findings highlight the potential of bovine-derived, especially those possessing ultra-long CDRH3s, as effective therapeutics against current and future sarbecovirus threats. One Sentence Summary Monoclonal antibodies derived from SARS-CoV-2 immunised cows exhibit pan-sarbecovirus reactivity, highlighting their potential use as therapeutics