We describe the binding and neutralization properties of a new set of human monoclonal antibodies derived from memory B cells of 10 coronavirus disease 2019 (COVID-19) convalescent donors in the first pandemic wave of prototype SARS-CoV-2. There were 12 antibodies targeting distinct epitopes on spike, including two sites on the RBD and one on the N-terminal domain (NTD), that displayed cross-neutralization of VOCs, for which distinct antibody targets could neutralize discrete variants.

The rapid evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variants has emphasized the need to identify antibodies with broad neutralizing capabilities to inform future monoclonal therapies and vaccination strategies. Herein, we identified S728-1157, a broadly neutralizing antibody (bnAb) targeting the receptor-binding site (RBS) that was derived from an individual previously infected with WT SARS-CoV-2 prior to the spread of variants of concern (VOCs). S728-1157 demonstrated broad cross-neutralization of all dominant variants, including D614G, Beta, Delta, Kappa, Mu, and Omicron (BA.1/BA.2/BA.2.75/BA.4/BA.5/BL.1/XBB). Furthermore, S728-1157 protected hamsters against in vivo challenges with WT, Delta, and BA.1 viruses. Structural analysis showed that this antibody targets a class 1/RBS-A epitope in the receptor binding domain via multiple hydrophobic and polar interactions with its heavy chain complementarity determining region 3 (CDR-H3), in addition to common motifs in CDR-H1/CDR-H2 of class 1/RBS-A antibodies. Importantly, this epitope was more readily accessible in the open and prefusion state, or in the hexaproline (6P)-stabilized spike constructs, as compared with diproline (2P) constructs. Overall, S728-1157 demonstrates broad therapeutic potential and may inform target-driven vaccine designs against future SARS-CoV-2 variants.

The S2 subunit of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike is highly conserved across coronavirus strains and therefore is a potential pan-coronavirus vaccine target. However, antibodies targeting this region are typically non-neutralizing. We report herein that S2-targeting antibodies from patients who recovered from SARS-CoV-2 infection bound only closely related sarbecovirus subgenus strains and, like most known S2 antibodies, none of these were neutralizing. In contrast, first-exposure, severe acutely infected COVID-19 patients predominantly induced back-boosted antibody-secreting cells imprinted against past common cold coronavirus strain OC43 that were cross-reactive to as many as five subgenera of betacoronavirus strains and gave rise to antibodies that were neutralizing and protective. The antibodies targeted two different sites: one defined by competition with stem helix antibodies, and the second to an underdescribed epitope at the apex of S2. These findings suggest that S2-targeted vaccines could strategically exploit controlled OC43 priming followed by SARS-CoV-2 boosting to enhance the breadth and quality of protective antibody responses.

Cell hashing, a nucleotide barcode-based method that allows users to pool multiple samples and demultiplex in downstream analysis, has gained widespread popularity in single-cell sequencing due to its compatibility, simplicity, and cost-effectiveness. Despite these advantages, the performance of this method remains unsatisfactory under certain circumstances, especially in experiments that have imbalanced sample sizes or use many hashtag antibodies. Here, we introduce a hybrid demultiplexing strategy that increases accuracy and cell recovery in multi-sample single-cell experiments. This approach correlates the results of cell hashing and genetic variant clustering, enabling precise and efficient cell identity determination without additional experimental costs or efforts. In addition, we developed HTOreader, a demultiplexing tool for cell hashing that improves the accuracy of cut-off calling by avoiding the dominance of negative signals in experiments with many hashtags or imbalanced sample sizes. When compared to existing methods using real-world datasets, this hybrid approach and HTOreader consistently generate reliable results with increased accuracy and cell recovery.

Artificial mutagenesis and protein engineering have laid the foundation for antigenic characterization and universal vaccine design for influenza viruses. However, many methods used in this process require manual sequence editing and protein expression, limiting their efficiency and utility in high-throughput applications. More streamlined in silico tools allowing researchers to properly analyze and visualize influenza viral protein sequences with accurate nomenclature are necessary to improve antigen design and productivity. To address this need, we developed Librator, a system for analyzing and designing custom protein sequences of influenza virus hemagglutinin (HA) and neuraminidase (NA) glycoproteins. Within Librator’s graphical interface, users can easily interrogate viral sequences and phylogenies, visualize antigen structures and conservation, mutate target residues and design custom antigens. Librator also provides optimized fragment design for Gibson Assembly of HA and NA expression constructs based on peptide conservation of all historical HA and NA sequences, ensuring fragments are reusable and compatible across related subtypes, thereby promoting reagent savings. Finally, the program facilitates single-cell immune profiling, epitope mapping of monoclonal antibodies and mosaic protein design. Using Librator-based antigen construction, we demonstrate that antigenicity can be readily transferred between HA molecules of H3, but not H1, lineage viruses. Altogether, Librator is a valuable tool for analyzing influenza virus HA and NA proteins and provides an efficient resource for optimizing recombinant influenza antigen synthesis.

Summary: The rapid evolution of SARS-CoV-2 Omicron variants has emphasized the need to identify antibodies with broad neutralizing capabilities to inform future monoclonal therapies and vaccination strategies. Herein, we identify S728-1157, a broadly neutralizing antibody (bnAb) targeting the receptor-binding site (RBS) and derived from an individual previously infected with SARS-CoV-2 prior to the spread of variants of concern (VOCs). S728-1157 demonstrates broad cross-neutralization of all dominant variants including D614G, Beta, Delta, Kappa, Mu, and Omicron (BA.1/BA.2/BA.2.75/BA.4/BA.5/BL.1). Furthermore, it protected hamsters against in vivo challenges with wildtype, Delta, and BA.1 viruses. Structural analysis reveals that this antibody targets a class 1 epitope via multiple hydrophobic and polar interactions with its CDR-H3, in addition to common class 1 motifs in CDR-H1/CDR-H2. Importantly, this epitope is more readily accessible in the open and prefusion state, or in the hexaproline (6P)-stabilized spike constructs, as compared to diproline (2P) constructs. Overall, S728-1157 demonstrates broad therapeutic potential, and may inform target-driven vaccine design against future SARS-CoV-2 variants.