Identification of broad, potent antibodies to functionally constrained regions of SARS-CoV-2 spike following a breakthrough infection.

The antiviral benefit of antibodies can be compromised by viral escape especially for rapidly evolving viruses. Therefore, durable, effective antibodies must be both broad and potent to counter newly emerging, diverse strains. Discovery of such antibodies is critically important for SARS-CoV-2 as the global emergence of new variants of concern (VOC) has compromised the efficacy of therapeutic antibodies and vaccines. We describe a collection of broad and potent neutralizing monoclonal antibodies (mAbs) isolated from an individual who experienced a breakthrough infection with the Delta VOC. Four mAbs potently neutralize the Wuhan-Hu-1 vaccine strain, the Delta VOC, and also retain potency against the Omicron VOCs through BA.4/BA.5 in both pseudovirus-based and authentic virus assays. Three mAbs also retain potency to recently circulating VOCs XBB.1.5 and BQ.1.1 and one also potently neutralizes SARS-CoV-1. The potency of these mAbs was greater against Omicron VOCs than all but one of the mAbs that had been approved for therapeutic applications. The mAbs target distinct epitopes on the spike glycoprotein, three in the receptor-binding domain (RBD) and one in an invariant region downstream of the RBD in subdomain 1 (SD1). The escape pathways we defined at single amino acid resolution with deep mutational scanning show they target conserved, functionally constrained regions of the glycoprotein, suggesting escape could incur a fitness cost. Overall, these mAbs are unique in their breadth across VOCs, their epitope specificity, and include a highly potent mAb targeting a rare epitope outside of the RBD in SD1.

Cytoplasmic Tail Truncation Stabilizes S1-S2 Association and Enhances S Protein Incorporation into SARS-CoV-2 Pseudovirions.

Truncations of the cytoplasmic tail (CT) of entry proteins of enveloped viruses dramatically increase the infectivity of pseudoviruses (PVs) bearing these proteins. Several mechanisms have been proposed to explain this enhanced entry, including an increase in cell surface expression. However, alternative explanations have also been forwarded, and the underlying mechanisms for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S protein remain undetermined. Here, we show that the partial or complete deletion of the CT (residues 19 to 35) does not modify SARS-CoV-2 S protein expression on the cell surface when the S2 subunit is measured, whereas it is significantly increased when the S1 subunit is measured. We also show that the higher level of S1 in these CT-truncated S proteins reflects the decreased dissociation of the S1 subunit from the S2 subunit. In addition, we demonstrate that CT truncation further promotes S protein incorporation into PV particles, as indicated by biochemical analyses and cryo-electron microscopy. Thus, our data show that two distinct mechanisms contribute to the markedly increased infectivity of PVs carrying CT-truncated SARS-CoV-2 S proteins and help clarify the interpretation of the results of studies employing such PVs. IMPORTANCE Various forms of PVs have been used as tools to evaluate vaccine efficacy and study virus entry steps. When PV infectivity is inherently low, such as that of SARS-CoV-2, a CT-truncated version of the viral entry glycoprotein is widely used to enhance PV infectivity, but the mechanism underlying this enhanced PV infectivity has been unclear. Here, our study identified two mechanisms by which the CT truncation of the SARS-CoV-2 S protein dramatically increases PV infectivity: a reduction of S1 shedding and an increase in S protein incorporation into PV particles. An understanding of these mechanisms can clarify the mechanistic bases for the differences observed among various assays employing such PVs.

Identification of broad, potent antibodies to functionally constrained regions of SARS-CoV-2 spike following a breakthrough infection.

The antiviral benefit of antibodies can be compromised by viral escape especially for rapidly evolving viruses. Therefore, durable, effective antibodies must be both broad and potent to counter newly emerging, diverse strains. Discovery of such antibodies is critically important for SARS-CoV-2 as the global emergence of new variants of concern (VOC) has compromised the efficacy of therapeutic antibodies and vaccines. We describe a collection of broad and potent neutralizing monoclonal antibodies (mAbs) isolated from an individual who experienced a breakthrough infection with the Delta VOC. Four mAbs potently neutralize the Wuhan-Hu-1 vaccine strain, the Delta VOC, and also retain potency against the Omicron VOCs through BA.4/BA.5 in both pseudovirus-based and authentic virus assays. Three mAbs also retain potency to recently circulating VOCs XBB.1.5 and BQ.1.1 and one also potently neutralizes SARS-CoV-1. The potency of these mAbs was greater against Omicron VOCs than all but one of the mAbs that had been approved for therapeutic applications. The mAbs target distinct epitopes on the spike glycoprotein, three in the receptor binding domain (RBD) and one in an invariant region downstream of the RBD in subdomain 1 (SD1). The escape pathways we defined at single amino acid resolution with deep mutational scanning show they target conserved, functionally constrained regions of the glycoprotein, suggesting escape could incur a fitness cost. Overall, these mAbs are novel in their breadth across VOCs, their epitope specificity, and include a highly potent mAb targeting a rare epitope outside of the RBD in SD1.

Cryo-ET of Env on intact HIV virions reveals structural variation and positioning on the Gag lattice.

HIV-1 Env mediates viral entry into host cells and is the sole target for neutralizing antibodies. However, Env structure and organization in its native virion context has eluded detailed characterization. Here, we used cryo-electron tomography to analyze Env in mature and immature HIV-1 particles. Immature particles showed distinct Env positioning relative to the underlying Gag lattice, providing insights into long-standing questions about Env incorporation. A 9.1-Å sub-tomogram-averaged reconstruction of virion-bound Env in conjunction with structural mass spectrometry revealed unexpected features, including a variable central core of the gp41 subunit, heterogeneous glycosylation between protomers, and a flexible stalk that allows Env tilting and variable exposure of neutralizing epitopes. Together, our results provide an integrative understanding of HIV assembly and structural variation in Env antigen presentation.

Author Correction: Highly efficient multiplex human T cell engineering without double-strand breaks using Cas9 base editors.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Highly efficient multiplex human T cell engineering without double-strand breaks using Cas9 base editors.

The fusion of genome engineering and adoptive cellular therapy holds immense promise for the treatment of genetic disease and cancer. Multiplex genome engineering using targeted nucleases can be used to increase the efficacy and broaden the application of such therapies but carries safety risks associated with unintended genomic alterations and genotoxicity. Here, we apply base editor technology for multiplex gene modification in primary human T cells in support of an allogeneic CAR-T platform and demonstrate that base editor can mediate highly efficient multiplex gene disruption with minimal double-strand break induction. Importantly, multiplex base edited T cells exhibit improved expansion and lack double strand break-induced translocations observed in T cells edited with Cas9 nuclease. Our findings highlight base editor as a powerful platform for genetic modification of therapeutically relevant primary cell types.

Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template.

The CRISPR-Cas9 system is a powerful genome-editing tool in which a guide RNA targets Cas9 to a site in the genome, where the Cas9 nuclease then induces a double-stranded break (DSB). The potential of CRISPR-Cas9 to deliver precise genome editing is hindered by the low efficiency of homology-directed repair (HDR), which is required to incorporate a donor DNA template encoding desired genome edits near the DSB. We present a strategy to enhance HDR efficiency by covalently tethering a single-stranded oligodeoxynucleotide (ssODN) to the Cas9-guide RNA ribonucleoprotein (RNP) complex via a fused HUH endonuclease, thus spatially and temporally co-localizing the DSB machinery and donor DNA. We demonstrate up to a 30-fold enhancement of HDR using several editing assays, including repair of a frameshift and in-frame insertions of protein tags. The improved HDR efficiency is observed in multiple cell types and target loci and is more pronounced at low RNP concentrations.

The Molecular Mechanism of Notch Activation.

Research in the last several years has shown that Notch proteolysis, and thus Notch activation, is conformationally controlled by the extracellular juxtamembrane NRR of Notch, which sterically occludes the S2 protease site until ligand binds. The question of how conformational exposure of the protease site is achieved during physiologic activation, and thus how normal activation is bypassed in disease pathogenesis, has been the subject of intense study in the last several years, and is the subject of this chapter. Here, we summarize the structural features of the NRR domains of Notch receptors that establish the autoinhibited state and then review a number of recent studies aimed at testing the mechanotransduction model for Notch signaling using force spectroscopy and molecular tension sensors.

Sequence-Directed Covalent Protein-DNA Linkages in a Single Step Using HUH-Tags.

We present a robust strategy to covalently link proteins and DNA using HUH-endonuclease domains as fusion partners (HUH-tags). We show that HUH-tags react robustly with specific sequences of unmodified single-stranded DNA, and we have identified five tags that react orthogonally with distinct DNA sequences. We demonstrate the versatility of HUH-tags as fusion partners in Cas9-mediated gene editing and the construction of doubly DNA-tethered proteins for single-molecule studies. Finally we demonstrate application to cellular imaging in live and fixed cells.

Comparisons of Shewanella strains based on genome annotations, modeling, and experiments.

BACKGROUND: Shewanella is a genus of facultatively anaerobic, Gram-negative bacteria that have highly adaptable metabolism which allows them to thrive in diverse environments. This quality makes them an attractive bacterial target for research in bioremediation and microbial fuel cell applications. Constraint-based modeling is a useful tool for helping researchers gain insights into the metabolic capabilities of these bacteria. However, Shewanella oneidensis MR-1 is the only strain with a genome-scale metabolic model constructed out of 21 sequenced Shewanella strains. RESULTS: In this work, we updated the model for Shewanella oneidensis MR-1 and constructed metabolic models for three other strains, namely Shewanella sp. MR-4, Shewanella sp. W3-18-1, and Shewanella denitrificans OS217 which span the genus based on the number of genes lost in comparison to MR-1. We also constructed a Shewanella core model that contains the genes shared by all 21 sequenced strains and a few non-conserved genes associated with essential reactions. Model comparisons between the five constructed models were done at two levels - for wildtype strains under different growth conditions and for knockout mutants under the same growth condition. In the first level, growth/no-growth phenotypes were predicted by the models on various carbon sources and electron acceptors. Cluster analysis of these results revealed that the MR-1 model is most similar to the W3-18-1 model, followed by the MR-4 and OS217 models when considering predicted growth phenotypes. However, a cluster analysis done based on metabolic gene content revealed that the MR-4 and W3-18-1 models are the most similar, with the MR-1 and OS217 models being more distinct from these latter two strains. As a second level of comparison, we identified differences in reaction and gene content which give rise to different functional predictions of single and double gene knockout mutants using Comparison of Networks by Gene Alignment (CONGA). Here, we showed how CONGA can be used to find biomass, metabolic, and genetic differences between models. CONCLUSIONS: We developed four strain-specific models and a general core model that can be used to do various in silico studies of Shewanella metabolism. The developed models provide a platform for a systematic investigation of Shewanella metabolism to aid researchers using Shewanella in various biotechnology applications.