Broad Sarbecovirus Neutralizing Antibodies Obtained by Computational Design and Synthetic Library Screening.

Members of the Sarbecovirus subgenus of Coronaviridae have twice caused deadly threats to humans. There is increasing concern about the rapid mutation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which has evolved into multiple generations of epidemic variants in 3 years. Broad neutralizing antibodies are of great importance for pandemic preparedness against SARS-CoV-2 variants and divergent zoonotic sarbecoviruses. Here, we analyzed the structural conservation of the receptor-binding domain (RBD) from representative sarbecoviruses and chose S2H97, a previously reported RBD antibody with ideal breadth and resistance to escape, as a template for computational design to enhance the neutralization activity and spectrum. A total of 35 designs were purified for evaluation. The neutralizing activity of a large proportion of these designs against multiple variants was increased from several to hundreds of times. Molecular dynamics simulation suggested that extra interface contacts and enhanced intermolecular interactions between the RBD and the designed antibodies are established. After light and heavy chain reconstitution, AI-1028, with five complementarity determining regions optimized, showed the best neutralizing activity across all tested sarbecoviruses, including SARS-CoV, multiple SARS-CoV-2 variants, and bat-derived viruses. AI-1028 recognized the same cryptic RBD epitope as the parental prototype antibody. In addition to computational design, chemically synthesized nanobody libraries are also a precious resource for rapid antibody development. By applying distinct RBDs as baits for reciprocal screening, we identified two novel nanobodies with broad activities. These findings provide potential pan-sarbecovirus neutralizing drugs and highlight new pathways to rapidly optimize therapeutic candidates when novel SARS-CoV-2 escape variants or new zoonotic coronaviruses emerge. IMPORTANCE The subgenus Sarbecovirus includes human SARS-CoV, SARS-CoV-2, and hundreds of genetically related bat viruses. The continuous evolution of SARS-CoV-2 has led to the striking evasion of neutralizing antibody (NAb) drugs and convalescent plasma. Antibodies with broad activity across sarbecoviruses would be helpful to combat current SARS-CoV-2 mutations and longer term animal virus spillovers. The study of pan-sarbecovirus NAbs described here is significant for the following reasons. First, we established a structure-based computational pipeline to design and optimize NAbs to obtain more potent and broader neutralizing activity across multiple sarbecoviruses. Second, we screened and identified nanobodies from a highly diversified synthetic library with a broad neutralizing spectrum using an elaborate screening strategy. These methodologies provide guidance for the rapid development of antibody therapeutics against emerging pathogens with highly variable characteristics.

Recombinant Full-Length Hepatitis C Virus E1E2 Dimer Elicits Pangenotypic Neutralizing Antibodies.

An effective prophylactic vaccine would be beneficial for controlling and eradicating hepatitis C virus (HCV) infections. However, the high diversity across HCV genotypes is a major challenge for vaccine development. Selection of the appropriate immunogen is critical to elicit broad HCV neutralizing antibodies (NAbs). To increase the antigenic coverage of heterodimer glycoproteins, we designed and produced recombinant E1E2 antigens for genotypes 1a/1b/2a/3a/6a from an IgG Fc-tagged precursor protein in FreeStyle 293-F cells. The recombinant E1 and E2 antigens were localized and associated with the endoplasmic reticulum and co-purified from membrane extracts. By examining the interactions with HCV entry co-receptors and the blockade of HCV infection, we found that these purified Fc-E1E2 proteins displayed correct folding and function. Mouse immunization results showed that each recombinant E1E2 antigen could elicit a pangenotypic antibody response to itself and other genotypes. We also found that the pentavalent formula triggered a relatively higher and more uniform NAb titer and T cell response than monovalent antigens. Taken together, our findings may provide a useful strategy for the vaccine development of HCV and other viruses with highly heterogeneous surface glycoproteins.

An ultrapotent RBD-targeted biparatopic nanobody neutralizes broad SARS-CoV-2 variants.

The wide transmission and host adaptation of SARS-CoV-2 have led to the rapid accumulation of mutations, posing significant challenges to the effectiveness of vaccines and therapeutic antibodies. Although several neutralizing antibodies were authorized for emergency clinical use, convalescent patients derived natural antibodies are vulnerable to SARS-CoV-2 Spike mutation. Here, we describe the screen of a panel of SARS-CoV-2 receptor-binding domain (RBD) targeted nanobodies (Nbs) from a synthetic library and the design of a biparatopic Nb, named Nb1-Nb2, with tight affinity and super-wide neutralization breadth against multiple SARS-CoV-2 variants of concern. Deep-mutational scanning experiments identify the potential binding epitopes of the Nbs on the RBD and demonstrate that biparatopic Nb1-Nb2 has a strong escape-resistant feature against more than 60 tested RBD amino acid substitutions. Using pseudovirion-based and trans-complementation SARS-CoV-2 tools, we determine that the Nb1-Nb2 broadly neutralizes multiple SARS-CoV-2 variants at sub-nanomolar levels, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Lambda (C.37), Kappa (B.1.617.1), and Mu (B.1.621). Furthermore, a heavy-chain antibody is constructed by fusing the human IgG1 Fc to Nb1-Nb2 (designated as Nb1-Nb2-Fc) to improve its neutralization potency, yield, stability, and potential half-life extension. For the new Omicron variant (B.1.1.529) that harbors unprecedented multiple RBD mutations, Nb1-Nb2-Fc keeps a firm affinity (KD < 1.0 × 10-12 M) and strong neutralizing activity (IC50 = 1.46 nM for authentic Omicron virus). Together, we developed a tetravalent biparatopic human heavy-chain antibody with ultrapotent and broad-spectrum SARS-CoV-2 neutralization activity which highlights the potential clinical applications.