Methods in antibody discovery

Antibodies are an essential part of adaptive immunity and can be raised against almost any molecule. The ability to bind a wide variety of molecules with exquisite specificity, causes antibodies to be highly desirable tools for research and in medicine. Beginning with the discovery of antibodies in 1890 by Behring and Kitasato, the scientific community was aware of the enormous therapeutic potential of molecules that can be induced to bind any disease-causing factor. However, the realization of this potential was stifled by the lack of techniques to isolate and produce antibodies of defined specificity. Since then, versatile antibody discovery methods have been developed, however, initial antibody isolation and characterization remain a bottleneck in the antibody discovery process. The work presented here improves upon existing methods of antibody discovery. Development of a novel yeast surface display strain reduces selection times by half and expedites downstream development by use of a native antibody format that translates effortlessly into full-length antibodies. The combination of antibody yeast surface display and mass-spectrometry based protein sequencing of serum antibodies successfully isolates SARS-CoV-2 binders from infected donors. Additionally, examination of the role of antibody light chains leads to insights into repertoire diversity and sparks an innovative approach to antibody heavy and light chain pairing. Cumulatively, the synergistic use of these antibody discovery methods yields 49 SARS-CoV-2 neutralizing antibodies against a variety of spike protein epitopes. Four antibodies and their binding mode were structurally characterized, revealing overlap with known antigenic supersites of the spike protein, and a unique quaternary binding mode at the receptor binding domain.Biochemistr

Prevalent, protective, and convergent IgG recognition of SARS-CoV-2 non-RBD spike epitopes

The molecular composition and binding epitopes of the immunoglobulin G (IgG) antibodies that circulate in blood plasma following SARS-CoV-2 infection are unknown. Proteomic deconvolution of the IgG repertoire to the spike glycoprotein in convalescent subjects revealed that the response is directed predominantly (>80%) against epitopes residing outside the receptor-binding domain (RBD). In one subject, just four IgG lineages accounted for 93.5% of the response, including an N-terminal domain (NTD)-directed antibody that was protective against lethal viral challenge. Genetic, structural, and functional characterization of a multi-donor class of “public” antibodies revealed an NTD epitope that is recurrently mutated among emerging SARS-CoV-2 variants of concern. These data show that “public” NTD-directed and other non-RBD plasma antibodies are prevalent and have implications for SARS-CoV-2 protection and antibody escape

Plasmacytoid Dendritic Cells and Type I Interferon Promote Extrafollicular B Cell Responses to Extracellular Self-DNA

International audienceClass-switched antibodies to double-stranded DNA (dsDNA) are prevalent and pathogenic in systemic lupus erythematosus (SLE), yet mechanisms of their development remain poorly understood. Humans and mice lacking secreted DNase DNASE1L3 develop rapid anti-dsDNA antibody responses and SLE-like disease. We report that anti-DNA responses in Dnase1l3-/- mice require CD40L-mediated T cell help, but proceed independently of germinal center formation via short-lived antibody-forming cells (AFCs) localized to extrafollicular regions. Type I interferon (IFN-I) signaling and IFN-I-producing plasmacytoid dendritic cells (pDCs) facilitate the differentiation of DNA-reactive AFCs in vivo and in vitro and are required for downstream manifestations of autoimmunity. Moreover, the endosomal DNA sensor TLR9 promotes anti-dsDNA responses and SLE-like disease in Dnase1l3-/- mice redundantly with another nucleic acid-sensing receptor, TLR7. These results establish extrafollicular B cell differentiation into short-lived AFCs as a key mechanism of anti-DNA autoreactivity and reveal a major contribution of pDCs, endosomal Toll-like receptors (TLRs), and IFN-I to this pathway