Human neutralizing antibodies elicited by SARS-CoV-2 infection.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents a global health emergency that is in urgent need of intervention1-3. The entry of SARS-CoV-2 into its target cells depends on binding between the receptor-binding domain (RBD) of the viral spike protein and its cellular receptor, angiotensin-converting enzyme 2 (ACE2)2,4-6. Here we report the isolation and characterization of 206 RBD-specific monoclonal antibodies derived from single B cells from 8 individuals infected with SARS-CoV-2. We identified antibodies that potently neutralize SARS-CoV-2; this activity correlates with competition with ACE2 for binding to RBD. Unexpectedly, the anti-SARS-CoV-2 antibodies and the infected plasma did not cross-react with the RBDs of SARS-CoV or Middle East respiratory syndrome-related coronavirus (MERS-CoV), although there was substantial plasma cross-reactivity to their trimeric spike proteins. Analysis of the crystal structure of RBD-bound antibody revealed that steric hindrance inhibits viral engagement with ACE2, thereby blocking viral entry. These findings suggest that anti-RBD antibodies are largely viral-species-specific inhibitors. The antibodies identified here may be candidates for development of clinical interventions against SARS-CoV-2.

Dual regulation of cytochrome P450 gene expression by two distinct small RNAs, a novel tasiRNA and miRNA, in Marchantia polymorpha.

The miR390-derived TAS3 trans-acting short-interfering RNAs (tasiRNAs) module represents a conserved RNA silencing pathway in the plant kingdom; however, its characterization in the bryophyte Marchantia polymorpha is limited. This study elucidated that MpDCL4 processes MpTAS3 double-stranded RNA (dsRNA) to generate tasiRNAs, primarily from the 5'- and 3'-ends of dsRNA. Notably, we discovered a novel tasiRNA, tasi78A, can negatively regulate a cytochrome P450 gene, MpCYP78A101. Additionally, tasi78A was abundant in MpAGO1, and transient expression assays underscored the role of tasi78A in repressing MpCYP78A101. A microRNA, miR11700, also regulates MpCYP78A101 expression. This coordinate regulation suggests a role in modulating auxin signaling at apical notches of gemma, influencing the growth and sexual organ development of M. polymorpha and emphasizing the significance of RNA silencing in MpCYP78A101 regulation. However, phylogenetic analysis identified another paralog of the CYP78 family, Mp1g14150, which may have a redundant role with MpCYP78A101, explaining the absence of noticeable morphological changes in loss-of-function plants. Taken together, our findings provide new insights into the combined regulatory roles of miR390/MpTAS3/miR11700 in controlling MpCYP78A101 and expand our knowledge about the biogenesis and regulation of tasiRNAs in M. polymorpha.

Engineered Histidine-Rich Peptides Enhance Endosomal Escape for Antibody-Targeted Intracellular Delivery of Functional Proteins.

Currently, the clinical application of protein/peptide therapeutics is mainly limited to the modulation of diseases in extracellular spaces. Intracellular targets are hardly accessed, owing largely to the endosomal entrapment of internalized proteins/peptides. Here, we report a strategy to design and construct peptides that enable endosome-to-cytosol delivery based on an extension of the "histidine switch" principle. By substituting the Arg/Lys residues in cationic cell-penetrating peptides (CPPs) with histidine, we obtained peptides with pH-dependent membrane-perturbation activity. These peptides do not randomly penetrate cells like CPPs, but imitate the endosomal escape of CPPs following cellular uptake. Working with one such 16-residue peptide (hsLMWP) with high endosomal escape capacity, we engineered modular fusion proteins and achieved antibody-targeted delivery of diverse protein cargoes-including the pro-apoptotic protein BID (BH3-interacting domain death agonist) and Cre recombinase-into the cytosol of multiple cancer cell types. After extensive in vitro testing, an in vivo analysis with xenograft mice ultimately demonstrated that a trastuzumab-hsLMWP-BID fusion conferred strong anti-tumor efficacy without apparent side effects. Notably, our fusion protein features a modular design, allowing flexible applications for any antibody/cargo combination of choice. Therefore, the potential applications extend throughout life science and biomedicine, including gene editing, cancer treatment, and immunotherapy.

Extracellular Metabolites of Heterotrophic Auxenochlorella protothecoides: A New Source of Bio-Stimulants for Higher Plants.

The biodiversity of microalgal species is enormous, and their versatile metabolism produces a wide diversity of compounds that can be used in food, healthcare, and other applications. Microalgae are also a potential source of bio-stimulants that enhance nutrition efficiency, abiotic stress tolerance, and/or crop quality traits. In this study, the extracellular metabolites of Auxenochlorella protothecoides (EAp) were prepared using three different culture strategies, and their effects on plant growth were examined. Furthermore, the composition of EAp was analyzed by GC-MS. The elongation of lateral roots and the cold-tolerance of Arabidopsis thaliana and Nicotiana benthamiana were promoted by EAp. Moreover, EAp from high-cell-density fermentation stimulated the growth of the leafy vegetables Brassica rapa and Lactuca sativa at dilutions as high as 500- and 1000-fold. Three major groups of compounds were identified by GC-MS, including organic acids or organic acid esters, phenols, and saccharides. Some of these compounds have known plant-stimulating effects, while the rest requires further investigation in the future. Our study demonstrates that EAp is a potential bio-stimulant, while also providing an environmentally friendly and economical microalgae fermentation process.

Impact of the N501Y substitution of SARS-CoV-2 Spike on neutralizing monoclonal antibodies targeting diverse epitopes.

The emergence and rapid spread of the B.1.1.7 lineage (VOC-202012/01) SARS-CoV-2 variant has aroused global concern. The N501Y substitution is the only mutation in the interface between the RBD of B.1.1.7 and ACE2, raising concerns that its recognition by neutralizing antibodies may be affected. Here, we assessed the neutralizing activity and binding affinity of a panel of 12 monoclonal antibodies against the wild type and N501Y mutant SARS-CoV-2 pseudovirus and RBD protein, respectively. We found that the neutralization activity and binding affinity of most detected antibodies (10 out of 12) were unaffected, although the N501Y substitution decreased the neutralizing and binding activities of CB6 and increased that of BD-23. These findings could be of value in the development of therapeutic antibodies.

Engineering pluripotent stem cells with synthetic biology for regenerative medicine.

Pluripotent stem cells (PSCs), characterized by self-renewal and capacity of differentiating into three germ layers, are the programmable building blocks of life. PSC-derived cells and multicellular systems, particularly organoids, exhibit great potential for regenerative medicine. However, this field is still in its infancy, partly due to limited strategies to robustly and precisely control stem cell behaviors, which are tightly regulated by inner gene regulatory networks in response to stimuli from the extracellular environment. Synthetic receptors and genetic circuits are powerful tools to customize the cellular sense-and-response process, suggesting their underlying roles in precise control of cell fate decision and function reconstruction. Herein, we review the progress and challenges needed to be overcome in the fields of PSC-based cell therapy and multicellular system generation, respectively. Furthermore, we summarize several well-established synthetic biology tools and their applications in PSC engineering. Finally, we highlight the challenges and perspectives of harnessing synthetic biology to PSC engineering for regenerative medicine.

A key F27I substitution within HCDR1 facilitates the rapid maturation of P2C-1F11-like neutralizing antibodies in a SARS-CoV-2-infected donor.

Although thousands of anti-SARS-CoV-2 monoclonal neutralizing antibodies (nAbs) have been identified and well characterized, some crucial events in the development of these nAbs during viral infection remain unclear. Using deep sequencing, we explore the dynamics of antibody repertoire in a SARS-CoV-2-infected donor, from whom the potent and broad nAb P2C-1F11 (the parent version of Brii-196) was previously isolated. Further analysis shows a rapid clonal expansion of some SARS-CoV-2-specific antibodies in early infection. Longitudinal tracing of P2C-1F11 lineage antibodies reveals that these elite nAbs were rare. Using sequence alignment, structure modeling, and bioactivity analysis based on site-mutated assay, we demonstrate that a key substitution F27I in heavy chain contributes significantly to the maturation of P2C-1F11-like antibodies. Overall, our findings elucidate the developmental process and maturation pathway of P2C-1F11, providing some important information for the design of novel immunogens to elicit more potent nAbs against SARS-CoV-2 infection.

Potent and protective IGHV3-53/3-66 public antibodies and their shared escape mutant on the spike of SARS-CoV-2.

Neutralizing antibodies (nAbs) to SARS-CoV-2 hold powerful potentials for clinical interventions against COVID-19 disease. However, their common genetic and biologic features remain elusive. Here we interrogate a total of 165 antibodies from eight COVID-19 patients, and find that potent nAbs from different patients have disproportionally high representation of IGHV3-53/3-66 usage, and therefore termed as public antibodies. Crystal structural comparison of these antibodies reveals they share similar angle of approach to RBD, overlap in buried surface and binding residues on RBD, and have substantial spatial clash with receptor angiotensin-converting enzyme-2 (ACE2) in binding to RBD. Site-directed mutagenesis confirms these common binding features although some minor differences are found. One representative antibody, P5A-3C8, demonstrates extraordinarily protective efficacy in a golden Syrian hamster model against SARS-CoV-2 infection. However, virus escape analysis identifies a single natural mutation in RBD, namely K417N found in B.1.351 variant from South Africa, abolished the neutralizing activity of these public antibodies. The discovery of public antibodies and shared escape mutation highlight the intricate relationship between antibody response and SARS-CoV-2, and provide critical reference for the development of antibody and vaccine strategies to overcome the antigenic variation of SARS-CoV-2.