Broad and potent activity against SARS-like viruses by an engineered human monoclonal antibody.

The recurrent zoonotic spillover of coronaviruses (CoVs) into the human population underscores the need for broadly active countermeasures. We employed a directed evolution approach to engineer three severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies for enhanced neutralization breadth and potency. One of the affinity-matured variants, ADG-2, displays strong binding activity to a large panel of sarbecovirus receptor binding domains and neutralizes representative epidemic sarbecoviruses with high potency. Structural and biochemical studies demonstrate that ADG-2 employs a distinct angle of approach to recognize a highly conserved epitope that overlaps the receptor binding site. In immunocompetent mouse models of SARS and COVID-19, prophylactic administration of ADG-2 provided complete protection against respiratory burden, viral replication in the lungs, and lung pathology. Altogether, ADG-2 represents a promising broad-spectrum therapeutic candidate against clade 1 sarbecoviruses.

An Engineered Antibody with Broad Protective Efficacy in Murine Models of SARS and COVID-19.

The recurrent zoonotic spillover of coronaviruses (CoVs) into the human population underscores the need for broadly active countermeasures. Here, we employed a directed evolution approach to engineer three SARS-CoV-2 antibodies for enhanced neutralization breadth and potency. One of the affinity-matured variants, ADG-2, displays strong binding activity to a large panel of sarbecovirus receptor binding domains (RBDs) and neutralizes representative epidemic sarbecoviruses with remarkable potency. Structural and biochemical studies demonstrate that ADG-2 employs a unique angle of approach to recognize a highly conserved epitope overlapping the receptor binding site. In murine models of SARS-CoV and SARS-CoV-2 infection, passive transfer of ADG-2 provided complete protection against respiratory burden, viral replication in the lungs, and lung pathology. Altogether, ADG-2 represents a promising broad-spectrum therapeutic candidate for the treatment and prevention of SARS-CoV-2 and future emerging SARS-like CoVs.

Nonsense-containing mRNAs that accumulate in the absence of a functional nonsense-mediated mRNA decay pathway are destabilized rapidly upon its restitution.

Nonsense-mediated mRNA decay (NMD) is a conserved proofreading mechanism that protects eukaryotic cells from the potentially deleterious effects of truncated proteins. Studies of Saccharomyces cerevisiae imply that NMD is a predominantly cytoplasmic decay pathway, while studies of mammalian systems suggest that decay of most substrate mRNAs may occur while they are still associated with the nucleus, possibly during a round of translation that occurs during their export to the cytoplasm. Complete entry of the latter mRNAs into the cytoplasm appears to render them immune to further NMD; i.e., they escape further susceptibility to this decay pathway. To determine if yeast cytoplasmic nonsense-containing mRNAs that evade decay are subsequently immune to NMD, we examined the consequences of placing each of the three UPF/NMD genes under the control of a galactose-inducible promoter. The decay kinetics of ADE2 and PGK1 nonsense-containing mRNAs were then analyzed when expression of UPF1, NMD2, or UPF3 was either repressed or subsequently induced. Results from these experiments demonstrated that activation of NMD caused rapid and immediate degradation of both substrate transcripts, with half-lives of both stable mRNA populations shortened to approximately 7 min. These findings make it unlikely that yeast nonsense-containing mRNAs can escape degradation by NMD and indicate that such mRNAs are available to this decay pathway at each round of translation.

Database-Centric Method for Automated High-Throughput Deconvolution and Analysis of Kinetic Antibody Screening Data.

The state-of-the-art industrial drug discovery approach is the empirical interrogation of a library of drug candidates against a target molecule. The advantage of high-throughput kinetic measurements over equilibrium assessments is the ability to measure each of the kinetic components of binding affinity. Although high-throughput capabilities have improved with advances in instrument hardware, three bottlenecks in data processing remain: (1) intrinsic molecular properties that lead to poor biophysical quality in vitro are not accounted for in commercially available analysis models, (2) processing data through a user interface is time-consuming and not amenable to parallelized data collection, and (3) a commercial solution that includes historical kinetic data in the analysis of kinetic competition data does not exist. Herein, we describe a generally applicable method for the automated analysis, storage, and retrieval of kinetic binding data. This analysis can deconvolve poor quality data on-the-fly and store and organize historical data in a queryable format for use in future analyses. Such database-centric strategies afford greater insight into the molecular mechanisms of kinetic competition, allowing for the rapid identification of allosteric effectors and the presentation of kinetic competition data in absolute terms of percent bound to antigen on the biosensor.

An improved yeast transformation method for the generation of very large human antibody libraries.

Antibody library selection by yeast display technology is an efficient and highly sensitive method to identify binders to target antigens. This powerful selection tool, however, is often hampered by the typically modest size of yeast libraries (approximately 10(7)) due to the limited yeast transformation efficiency, and the full potential of the yeast display technology for antibody discovery and engineering can only be realized if it can be coupled with a mean to generate very large yeast libraries. We describe here a yeast transformation method by electroporation that allows for the efficient generation of large antibody libraries up to 10(10) in size. Multiple components and conditions including CaCl(2), MgCl(2), sucrose, sorbitol, lithium acetate, dithiothreitol, electroporation voltage, DNA input and cell volume have been tested to identify the best combination. By applying this developed protocol, we have constructed a 1.4 x 10(10) human spleen antibody library essentially in 1 day with a transformation efficiency of 1-1.5 x 10(8) transformants/microg vector DNA. Taken together, we have developed a highly efficient yeast transformation method that enables the generation of very large and productive human antibody libraries for antibody discovery, and we are now routinely making 10(9) libraries in a day for antibody engineering purposes.

Toeprint analysis of the positioning of translation apparatus components at initiation and termination codons of fungal mRNAs.

The ability to map the position of ribosomes and their associated factors on mRNAs is critical for an understanding of translation mechanisms. Earlier approaches to monitoring these important cellular events characterized nucleotide sequences rendered nuclease-resistant by ribosome binding. While these approaches furthered our understanding of translation initiation and ribosome pausing, the pertinent techniques were technically challenging and not widely applied. Here we describe an alternative assay for determining the mRNA sites at which ribosomes or other factors are bound. This approach uses primer extension inhibition, or "toeprinting," to map the 3' boundaries of mRNA-associated complexes. This methodology, previously used to characterize initiation mechanisms in prokaryotic and eukaryotic systems, is used here to gain an understanding of two interesting translational regulatory phenomena in the fungi Neurospora crassa and Saccharomyces cerevisiae: (a) regulation of translation in response to arginine concentration by an evolutionarily conserved upstream open reading frame, and (b) atypical termination events that occur as a consequence of the presence of premature stop codons.