Structural basis of broad SARS-CoV-2 cross-neutralization by affinity-matured public antibodies

Descendants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant now account for almost all SARS-CoV-2 infections. The Omicron variant and its sublineages have spike glycoproteins that are highly diverged from the pandemic founder and first-generation vaccine strain, resulting in significant evasion from monoclonal antibody therapeutics and vaccines. Understanding how commonly elicited antibodies can broaden to cross-neutralize escape variants is crucial. We isolate IGHV3-53, using ‘‘public’’ monoclonal antibodies (mAbs) from an individual 7 months post infection with the ancestral virus and identify antibodies that exhibit potent and broad cross-neutralization, extending to the BA.1, BA.2, and BA.4/BA.5 sublineages of Omicron. Deep mutational scanning reveals these mAbs’ high resistance to viral escape. Structural analysis via cryoelectron microscopy of a representative broadly neutralizing antibody, CAB-A17, in complex with the Omicron BA.1 spike highlights the structural underpinnings of this broad neutralization. By reintroducing somatic hypermutations into a germline-reverted CAB-A17, we delineate the role of affinity maturation in the development of cross-neutralization by a public class of antibodies

Single-cell longitudinal analysis of SARS-CoV-2 infection in human airway epithelium identifies target cells, alterations in gene expression, and cell state changes.

There are currently limited Food and Drug Administration (FDA)-approved drugs and vaccines for the treatment or prevention of Coronavirus Disease 2019 (COVID-19). Enhanced understanding of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection and pathogenesis is critical for the development of therapeutics. To provide insight into viral replication, cell tropism, and host-viral interactions of SARS-CoV-2, we performed single-cell (sc) RNA sequencing (RNA-seq) of experimentally infected human bronchial epithelial cells (HBECs) in air-liquid interface (ALI) cultures over a time course. This revealed novel polyadenylated viral transcripts and highlighted ciliated cells as a major target at the onset of infection, which we confirmed by electron and immunofluorescence microscopy. Over the course of infection, the cell tropism of SARS-CoV-2 expands to other epithelial cell types including basal and club cells. Infection induces cell-intrinsic expression of type I and type III interferons (IFNs) and interleukin (IL)-6 but not IL-1. This results in expression of interferon-stimulated genes (ISGs) in both infected and bystander cells. This provides a detailed characterization of genes, cell types, and cell state changes associated with SARS-CoV-2 infection in the human airway

Elicitation of broadly protective sarbecovirus immunity by receptor-binding domain nanoparticle vaccines

Understanding vaccine-elicited protection against SARS-CoV-2 variants and other sarbecoviruses is key for guiding public health policies. We show that a clinical stage multivalent SARS-CoV-2 spike receptor-binding domain nanoparticle vaccine (RBD-NP) protects mice from SARS-CoV-2 challenge after a single immunization, indicating a potential dose-sparing strategy. We benchmarked serum neutralizing activity elicited by RBD-NP in non-human primates against a lead prefusion-stabilized SARS-CoV-2 spike (HexaPro) using a panel of circulating mutants. Polyclonal antibodies elicited by both vaccines are similarly resilient to many RBD residue substitutions tested although mutations at and surrounding position 484 have negative consequences for neutralization. Mosaic and cocktail nanoparticle immunogens displaying multiple sarbecovirus RBDs elicit broad neutralizing activity in mice and protect mice against SARS-CoV challenge even in the absence of SARS-CoV RBD in the vaccine. This study provides proof of principle that multivalent sarbecovirus RBD-NPs induce heterotypic protection and motivates advancing such broadly protective sarbecovirus vaccines to the clinic

Improvements in Pulmonary Tissue Engineering: Toward Functional Tracheal and Lung Replacements

Tissue engineering offers a uniquely powerful solution for the regeneration of damaged or diseased tissues, in the form of personalized living replacements. Building such replacements, however, requires a deep appreciation for the biological, chemical, and engineering aspects that govern their function. Currently, the generation of biomimetic trachea and lung replacements is hampered by incomplete recapitulation of certain of these critical aspects, which have been addressed through the work in this thesis. In Chapter 2, I reviewed all reported clinical applications of engineered tracheal grafts to replace long-segment, circumferential damage from 1898 to 2018. From these reports, I identified the current gold-standard in clinical tracheal replacement to be the Leuven protocol, based on patient survival and follow-up time. By collating graft-related causes of mortality, I generated a list of clinical care priorities and critical design criteria that will inform future efforts to generate engineered tracheal replacement grafts. In Chapter 3, I addressed the biomechanics of engineered tracheal grafts, which is a leading cause of tracheal graft failure. I evaluated 3D bulk mechanical properties of native and decellularized tracheas, isolating behaviors of each structural component of the trachea (cartilage, smooth muscle, and connective tissue). I then correlated mechanical deviations from native trachea, to structural changes to the extracellular matrix with decellularization treatment. Taken together, decellularized tracheal grafts possessed significantly impaired mechanical properties and protein structures compared to native, which should discourage their application clinically. In Chapter 4, I evaluated platform-specific effects on the differentiation of a novel population of pharmacologically expanded, primary basal cells (peBC). This work determined that artificial culture platforms, including air-liquid interface (ALI) and organoid cultures, impart non-physiologic artifacts on cellular response in these systems. Conversely, decellularized trachea and lung scaffolds impart region-specific differentiation cues on cultured peBC, which generate more physiologic cellular outcomes. This work represents the first published evaluation of engineered tissues by single-cell RNA sequencing (scRNAseq). In Chapter 5, I leveraged scRNAseq methodologies from Chapter 4 to evaluate SARS-CoV-2 viral infection dynamics in an ALI model of human proximal epithelium. This work identified a novel mechanism of viral entry via ciliated cells, which further elucidated a mechanism of enhanced system vulnerability on infection. In Chapter 6, I characterized the immune landscape of the postnatal developing rat lung by scRNAseq. I identified 26 distinct cell types and elucidated patterns of immune cell colonization, differentiation, and maturation. I also identified putative roles for certain immune cell types in regulating and contributing to the developing lung matrix morphology. In Chapter 7, I leveraged peBC in whole-lung engineered cultures to enhance epithelial barrier function, in co-culture with endothelium, fibroblasts, and/or pulmonary macrophages. I also further improved engineered lung culture paradigms to enhance tissue homogeneity and maturation. By scRNAseq evaluation of engineered lung epithelium, I found that peBC differentiate away from a proximal epithelial phenotype, and rather than gaining canonical distal epithelial character, gains a novel phenotype of regenerative, barrier-forming epithelium that resembles that observed in various disease states. Taken together, the work in this thesis represents significant strides toward generating functional engineered tracheal and lung replacements

Deep mutational scans for ACE2 binding, RBD expression, and antibody escape in the SARS-CoV-2 Omicron BA.1 and BA.2 receptor-binding domains.

SARS-CoV-2 continues to acquire mutations in the spike receptor-binding domain (RBD) that impact ACE2 receptor binding, folding stability, and antibody recognition. Deep mutational scanning prospectively characterizes the impacts of mutations on these biochemical properties, enabling rapid assessment of new mutations seen during viral surveillance. However, the effects of mutations can change as the virus evolves, requiring updated deep mutational scans. We determined the impacts of all single amino acid mutations in the Omicron BA.1 and BA.2 RBDs on ACE2-binding affinity, RBD folding, and escape from binding by the LY-CoV1404 (bebtelovimab) monoclonal antibody. The effects of some mutations in Omicron RBDs differ from those measured in the ancestral Wuhan-Hu-1 background. These epistatic shifts largely resemble those previously seen in the Alpha variant due to the convergent epistatically modifying N501Y substitution. However, Omicron variants show additional lineage-specific shifts, including examples of the epistatic phenomenon of entrenchment that causes the Q498R and N501Y substitutions present in Omicron to be more favorable in that background than in earlier viral strains. In contrast, the Omicron substitution Q493R exhibits no sign of entrenchment, with the derived state, R493, being as unfavorable for ACE2 binding in Omicron RBDs as in Wuhan-Hu-1. Likely for this reason, the R493Q reversion has occurred in Omicron sub-variants including BA.4/BA.5 and BA.2.75, where the affinity buffer from R493Q reversion may potentiate concurrent antigenic change. Consistent with prior studies, we find that Omicron RBDs have reduced expression, and identify candidate stabilizing mutations that ameliorate this deficit. Last, our maps highlight a broadening of the sites of escape from LY-CoV1404 antibody binding in BA.1 and BA.2 compared to the ancestral Wuhan-Hu-1 background. These BA.1 and BA.2 deep mutational scanning datasets identify shifts in the RBD mutational landscape and inform ongoing efforts in viral surveillance