Mosaic sarbecovirus nanoparticles elicit cross-reactive responses in pre-vaccinated animals.

Immunization with mosaic-8b (nanoparticles presenting 8 SARS-like betacoronavirus [sarbecovirus] receptor-binding domains [RBDs]) elicits more broadly cross-reactive antibodies than homotypic SARS-CoV-2 RBD-only nanoparticles and protects against sarbecoviruses. To investigate original antigenic sin (OAS) effects on mosaic-8b efficacy, we evaluated the effects of prior COVID-19 vaccinations in non-human primates and mice on anti-sarbecovirus responses elicited by mosaic-8b, admix-8b (8 homotypics), or homotypic SARS-CoV-2 immunizations, finding the greatest cross-reactivity for mosaic-8b. As demonstrated by molecular fate mapping, in which antibodies from specific cohorts of B cells are differentially detected, B cells primed by WA1 spike mRNA-LNP dominated antibody responses after RBD-nanoparticle boosting. While mosaic-8b- and homotypic-nanoparticles boosted cross-reactive antibodies, de novo antibodies were predominantly induced by mosaic-8b, and these were specific for variant RBDs with increased identity to RBDs on mosaic-8b. These results inform OAS mechanisms and support using mosaic-8b to protect COVID-19-vaccinated/infected humans against as-yet-unknown SARS-CoV-2 variants and animal sarbecoviruses with human spillover potential.

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 (RBD-NP) vaccine 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-NPs 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.

Deep Mutational Scanning of SARS-CoV-2 Receptor Binding Domain Reveals Constraints on Folding and ACE2 Binding.

The receptor binding domain (RBD) of the SARS-CoV-2 spike glycoprotein mediates viral attachment to ACE2 receptor and is a major determinant of host range and a dominant target of neutralizing antibodies. Here, we experimentally measure how all amino acid mutations to the RBD affect expression of folded protein and its affinity for ACE2. Most mutations are deleterious for RBD expression and ACE2 binding, and we identify constrained regions on the RBD's surface that may be desirable targets for vaccines and antibody-based therapeutics. But a substantial number of mutations are well tolerated or even enhance ACE2 binding, including at ACE2 interface residues that vary across SARS-related coronaviruses. However, we find no evidence that these ACE2-affinity-enhancing mutations have been selected in current SARS-CoV-2 pandemic isolates. We present an interactive visualization and open analysis pipeline to facilitate use of our dataset for vaccine design and functional annotation of mutations observed during viral surveillance.

Molecular fate-mapping of serum antibody responses to repeat immunization.

The protective efficacy of serum antibodies results from the interplay of antigen-specific B cell clones of different affinities and specificities. These cellular dynamics underlie serum-level phenomena such as original antigenic sin (OAS)-a proposed propensity of the immune system to rely repeatedly on the first cohort of B cells engaged by an antigenic stimulus when encountering related antigens, in detriment to the induction of de novo responses1-5. OAS-type suppression of new, variant-specific antibodies may pose a barrier to vaccination against rapidly evolving viruses such as influenza and SARS-CoV-26,7. Precise measurement of OAS-type suppression is challenging because cellular and temporal origins cannot readily be ascribed to antibodies in circulation; its effect on subsequent antibody responses therefore remains unclear5,8. Here we introduce a molecular fate-mapping approach with which serum antibodies derived from specific cohorts of B cells can be differentially detected. We show that serum responses to sequential homologous boosting derive overwhelmingly from primary cohort B cells, while later induction of new antibody responses from naive B cells is strongly suppressed. Such 'primary addiction' decreases sharply as a function of antigenic distance, allowing reimmunization with divergent viral glycoproteins to produce de novo antibody responses targeting epitopes that are absent from the priming variant. Our findings have implications for the understanding of OAS and for the design and testing of vaccines against evolving pathogens.

ACE2 binding is an ancestral and evolvable trait of sarbecoviruses.

Two different sarbecoviruses have caused major human outbreaks in the past two decades1,2. Both of these sarbecoviruses, SARS-CoV-1 and SARS-CoV-2, engage ACE2 through the spike receptor-binding domain2-6. However, binding to ACE2 orthologues of humans, bats and other species has been observed only sporadically among the broader diversity of bat sarbecoviruses7-11. Here we use high-throughput assays12 to trace the evolutionary history of ACE2 binding across a diverse range of sarbecoviruses and ACE2 orthologues. We find that ACE2 binding is an ancestral trait of sarbecovirus receptor-binding domains that has subsequently been lost in some clades. Furthermore, we reveal that bat sarbecoviruses from outside Asia can bind to ACE2. Moreover, ACE2 binding is highly evolvable-for many sarbecovirus receptor-binding domains, there are single amino-acid mutations that enable binding to new ACE2 orthologues. However, the effects of individual mutations can differ considerably between viruses, as shown by the N501Y mutation, which enhances the human ACE2-binding affinity of several SARS-CoV-2 variants of concern12 but substantially decreases it for SARS-CoV-1. Our results point to the deep ancestral origin and evolutionary plasticity of ACE2 binding, broadening the range of sarbecoviruses that should be considered to have spillover potential.

SARS-CoV-2 RBD antibodies that maximize breadth and resistance to escape.

An ideal therapeutic anti-SARS-CoV-2 antibody would resist viral escape1-3, have activity against diverse sarbecoviruses4-7, and be highly protective through viral neutralization8-11 and effector functions12,13. Understanding how these properties relate to each other and vary across epitopes would aid the development of therapeutic antibodies and guide vaccine design. Here we comprehensively characterize escape, breadth and potency across a panel of SARS-CoV-2 antibodies targeting the receptor-binding domain (RBD). Despite a trade-off between in vitro neutralization potency and breadth of sarbecovirus binding, we identify neutralizing antibodies with exceptional sarbecovirus breadth and a corresponding resistance to SARS-CoV-2 escape. One of these antibodies, S2H97, binds with high affinity across all sarbecovirus clades to a cryptic epitope and prophylactically protects hamsters from viral challenge. Antibodies that target the angiotensin-converting enzyme 2 (ACE2) receptor-binding motif (RBM) typically have poor breadth and are readily escaped by mutations despite high neutralization potency. Nevertheless, we also characterize a potent RBM antibody (S2E128) with breadth across sarbecoviruses related to SARS-CoV-2 and a high barrier to viral escape. These data highlight principles underlying variation in escape, breadth and potency among antibodies that target the RBD, and identify epitopes and features to prioritize for therapeutic development against the current and potential future pandemics.

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.

The SARS-CoV-2 Delta variant induces an antibody response largely focused on class 1 and 2 antibody epitopes.

Exposure histories to SARS-CoV-2 variants and vaccinations will shape the specificity of antibody responses. To understand the specificity of Delta-elicited antibody immunity, we characterize the polyclonal antibody response elicited by primary or mRNA vaccine-breakthrough Delta infections. Both types of infection elicit a neutralizing antibody response focused heavily on the receptor-binding domain (RBD). We use deep mutational scanning to show that mutations to the RBD's class 1 and class 2 epitopes, including sites 417, 478, and 484-486 often reduce binding of these Delta-elicited antibodies. The anti-Delta antibody response is more similar to that elicited by early 2020 viruses than the Beta variant, with mutations to the class 1 and 2, but not class 3 epitopes, having the largest effects on polyclonal antibody binding. In addition, mutations to the class 1 epitope (e.g., K417N) tend to have larger effects on antibody binding and neutralization in the Delta spike than in the D614G spike, both for vaccine- and Delta-infection-elicited antibodies. These results help elucidate how the antigenic impacts of SARS-CoV-2 mutations depend on exposure history.

A SARS-CoV-2 variant elicits an antibody response with a shifted immunodominance hierarchy.

Many SARS-CoV-2 variants have mutations at key sites targeted by antibodies. However, it is unknown if antibodies elicited by infection with these variants target the same or different regions of the viral spike as antibodies elicited by earlier viral isolates. Here we compare the specificities of polyclonal antibodies produced by humans infected with early 2020 isolates versus the B.1.351 variant of concern (also known as Beta or 20H/501Y.V2), which contains mutations in multiple key spike epitopes. The serum neutralizing activity of antibodies elicited by infection with both early 2020 viruses and B.1.351 is heavily focused on the spike receptor-binding domain (RBD). However, within the RBD, B.1.351-elicited antibodies are more focused on the "class 3" epitope spanning sites 443 to 452, and neutralization by these antibodies is notably less affected by mutations at residue 484. Our results show that SARS-CoV-2 variants can elicit polyclonal antibodies with different immunodominance hierarchies.

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.

Selection Analysis Identifies Clusters of Unusual Mutational Changes in Omicron Lineage BA.1 That Likely Impact Spike Function.

Among the 30 nonsynonymous nucleotide substitutions in the Omicron S-gene are 13 that have only rarely been seen in other SARS-CoV-2 sequences. These mutations cluster within three functionally important regions of the S-gene at sites that will likely impact (1) interactions between subunits of the Spike trimer and the predisposition of subunits to shift from down to up configurations, (2) interactions of Spike with ACE2 receptors, and (3) the priming of Spike for membrane fusion. We show here that, based on both the rarity of these 13 mutations in intrapatient sequencing reads and patterns of selection at the codon sites where the mutations occur in SARS-CoV-2 and related sarbecoviruses, prior to the emergence of Omicron the mutations would have been predicted to decrease the fitness of any virus within which they occurred. We further propose that the mutations in each of the three clusters therefore cooperatively interact to both mitigate their individual fitness costs, and, in combination with other mutations, adaptively alter the function of Spike. Given the evident epidemic growth advantages of Omicron overall previously known SARS-CoV-2 lineages, it is crucial to determine both how such complex and highly adaptive mutation constellations were assembled within the Omicron S-gene, and why, despite unprecedented global genomic surveillance efforts, the early stages of this assembly process went completely undetected.

Pressure-Regulated Ventilator Splitting for Disaster Relief: Design, Testing, and Clinical Experience.

The coronavirus disease 2019 (COVID-19) pandemic has revealed that even the best-resourced hospitals may lack sufficient ventilators to support patients under surge conditions. During a pandemic or mass trauma, an affordable, low-maintenance, off-the-shelf device that would allow health care teams to rapidly expand their ventilator capacity could prove lifesaving, but only if it can be safely integrated into a complex and rapidly changing clinical environment. Here, we define an approach to safe ventilator sharing that prioritizes predictable and independent care of patients sharing a ventilator. Subsequently, we detail the design and testing of a ventilator-splitting circuit that follows this approach and describe our clinical experience with this circuit during the COVID-19 pandemic. This circuit was able to provide individualized and titratable ventilatory support with individualized positive end-expiratory pressure (PEEP) to 2 critically ill patients at the same time, while insulating each patient from changes in the other's condition. We share insights from our experience using this technology in the intensive care unit and outline recommendations for future clinical applications.

A Diverse Set of Single-domain Antibodies (VHHs) against the Anthrax Toxin Lethal and Edema Factors Provides a Basis for Construction of a Bispecific Agent That Protects against Anthrax Infection.

Infection with Bacillus anthracis, the causative agent of anthrax, can lead to persistence of lethal secreted toxins in the bloodstream, even after antibiotic treatment. VHH single-domain antibodies have been demonstrated to neutralize diverse bacterial toxins both in vitro and in vivo, with protein properties such as small size and high stability that make them attractive therapeutic candidates. Recently, we reported on VHHs with in vivo activity against the protective antigen component of the anthrax toxins. Here, we characterized a new set of 15 VHHs against the anthrax toxins that act by binding to the edema factor (EF) and/or lethal factor (LF) components. Six of these VHHs are cross-reactive against both EF and LF and recognize the N-terminal domain (LFN, EFN) of their target(s) with subnanomolar affinity. The cross-reactive VHHs block binding of EF/LF to the protective antigen C-terminal binding interface, preventing toxin entry into the cell. Another VHH appears to recognize the LF C-terminal domain and exhibits a kinetic effect on substrate cleavage by LF. A subset of the VHHs neutralized against EF and/or LF in murine macrophage assays, and the neutralizing VHHs that were tested improved survival of mice in a spore model of anthrax infection. Finally, a bispecific VNA (VHH-based neutralizing agent) consisting of two linked toxin-neutralizing VHHs, JMN-D10 and JMO-G1, was fully protective against lethal anthrax spore infection in mice as a single dose. This set of VHHs should facilitate development of new therapeutic VNAs and/or diagnostic agents for anthrax.

Elucidating the Composition and Structure of Uranium Oxide Powders Produced via NO2 Voloxidation.

Voloxidation is a potential alternative reprocessing scheme for spent nuclear fuel that uses gas-solid reactions to minimize aqueous wastes and to separate volatile fission products from the desired actinide phase. The process uses NO2(g) as an oxidant for uranium dioxide (UO2) fuel, ideally producing soluble uranium powders which can then be processed for full recycle. To continue development of the process flowsheet for voloxidation, ongoing examination of the process chemistry and associated process materials is required: discrepancies in the proposed chemical reactions that occur when spent nuclear fuel is exposed to NO2(g) atmospheres must be addressed. The objective of this work is to analyze the intermediate solid phases produced during voloxidation to support verification of the proposed NO2(g) voloxidation reaction mechanisms. This objective was achieved through using (1) powder X-ray diffraction and Raman spectroscopy to identify bulk uranium phases and (2) scanning electron microscopy to describe the morphology and microstructure of the powders at each reaction stage. The initial oxidation of UO2 under NO2(g) reactions produced ε-UO3. Further exposure to NO2(g) did not nitrate the solid to produce uranyl nitrate, as reported in some literature. However, after the powder was hydrated with steam and then further exposed to NO2(g), some traces of uranyl nitrate hexahydrate were found. The results of this study suggest that surface hydration of powders plays a vital role in uranyl nitrate formation under voloxidation conditions and raises questions about the kinetics of the oxide-to-nitrate voloxidation conversion process. Future chemical and engineering design decisions for the voloxidation process may benefit from an improved understanding of these chemical mechanisms.

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.

Organ Boundary Circuits Regulate Sox9+ Alveolar Tuft Cells During Post-Pneumonectomy Lung Regeneration.

Tissue homeostasis is controlled by cellular circuits governing cell growth, organization, and differentation. In this study we identify previously undescribed cell-to-cell communication that mediates information flow from mechanosensitive pleural mesothelial cells to alveolar-resident stem-like tuft cells in the lung. We find mesothelial cells to express a combination of mechanotransduction genes and lineage-restricted ligands which makes them uniquely capable of responding to tissue tension and producing paracrine cues acting on parenchymal populations. In parallel, we describe a large population of stem-like alveolar tuft cells that express the endodermal stem cell markers Sox9 and Lgr5 and a receptor profile making them uniquely sensitive to cues produced by pleural Mesothelium. We hypothesized that crosstalk from mesothelial cells to alveolar tuft cells might be central to the regulation of post-penumonectomy lung regeneration. Following pneumonectomy, we find that mesothelial cells display radically altered phenotype and ligand expression, in a pattern that closely tracks with parenchymal epithelial proliferation and alveolar tissue growth. During an initial pro-inflammatory stage of tissue regeneration, Mesothelium promotes epithelial proliferation via WNT ligand secretion, orchestrates an increase in microvascular permeability, and encourages immune extravasation via chemokine secretion. This stage is followed first by a tissue remodeling period, characterized by angiogenesis and BMP pathway sensitization, and then a stable return to homeostasis. Coupled with key changes in parenchymal structure and matrix production, the cumulative effect is a now larger organ including newly-grown, fully-functional tissue parenchyma. This study paints Mesothelial cells as a key orchestrating cell type that defines the boundary of the lung and exerts critical influence over the tissue-level signaling state regulating resident stem cell populations. The cellular circuits unearthed here suggest that human lung regeneration might be inducible through well-engineered approaches targeting the induction of tissue regeneration and safe return to homeostasis.

Mosaic sarbecovirus nanoparticles elicit cross-reactive responses in pre-vaccinated animals.

Immunization with mosaic-8b [60-mer nanoparticles presenting 8 SARS-like betacoronavirus (sarbecovirus) receptor-binding domains (RBDs)] elicits more broadly cross-reactive antibodies than homotypic SARS-CoV-2 RBD-only nanoparticles and protects against sarbecoviruses. To investigate original antigenic sin (OAS) effects on mosaic-8b efficacy, we evaluated effects of prior COVID-19 vaccinations in non-human primates and mice on anti-sarbecovirus responses elicited by mosaic-8b, admix-8b (8 homotypics), or homotypic SARS-CoV-2 immunizations, finding greatest cross-reactivity for mosaic-8b. As demonstrated by molecular fate-mapping in which antibodies from specific cohorts of B cells are differentially detected, B cells primed by WA1 spike mRNA-LNP dominated antibody responses after RBD-nanoparticle boosting. While mosaic-8b- and homotypic-nanoparticles boosted cross-reactive antibodies, de novo antibodies were predominantly induced by mosaic-8b, and these were specific for variant RBDs with increased identity to RBDs on mosaic-8b. These results inform OAS mechanisms and support using mosaic-8b to protect COVID-19 vaccinated/infected humans against as-yet-unknown SARS-CoV-2 variants and animal sarbecoviruses with human spillover potential.

Influenza virus transcription and progeny production are poorly correlated in single cells.

The ultimate success of a viral infection at the cellular level is determined by the number of progeny virions produced. However, most single-cell studies of infection quantify the expression of viral transcripts and proteins, rather than the amount of progeny virions released from infected cells. Here, we overcome this limitation by simultaneously measuring transcription and progeny production from single influenza virus-infected cells by embedding nucleotide barcodes in the viral genome. We find that viral transcription and progeny production are poorly correlated in single cells. The cells that transcribe the most viral mRNA do not produce the most viral progeny and often represent aberrant infections that fail to express the influenza NS gene. However, only some of the discrepancy between transcription and progeny production can be explained by viral gene absence or mutations: there is also a wide range of progeny production among cells infected by complete unmutated virions. Overall, our results show that viral transcription is a relatively poor predictor of an infected cell's contribution to the progeny population.

SARS-CoV-2 leverages airway epithelial protective mechanism for viral infection.

Despite much concerted effort to better understand severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral infection, relatively little is known about the dynamics of early viral entry and infection in the airway. Here we analyzed a single-cell RNA sequencing dataset of early SARS-CoV-2 infection in a humanized in vitro model, to elucidate key mechanisms by which the virus triggers a cell-systems-level response in the bronchial epithelium. We find that SARS-CoV-2 virus preferentially enters the tissue via ciliated cell precursors, giving rise to a population of infected mature ciliated cells, which signal to basal cells, inducing further rapid differentiation. This feedforward loop of infection is mitigated by further cell-cell communication, before interferon signaling begins at three days post-infection. These findings suggest hijacking by the virus of potentially beneficial tissue repair mechanisms, possibly exacerbating the outcome. This work both elucidates the interplay between barrier tissues and viral infections and may suggest alternative therapeutic approaches targeting non-immune response mechanisms.

Decellularization compromises mechanical and structural properties of the native trachea.

Tracheal replacement using tissue engineering technologies offers great potential to improve previously intractable clinical interventions, and interest in this area has increased in recent years. Many engineered airway constructs currently rely on decellularized native tracheas to serve as the scaffold for tissue repair. Yet, mechanical failure leading to airway narrowing and collapse remains a major cause of morbidity and mortality following clinical implantation of decellularized tracheal grafts. To understand better the factors contributing to mechanical failure in vivo, we characterized the histo-mechanical properties of tracheas following two different decellularization protocols, including one that has been used clinically. All decellularized tracheas deviated from native mechanical behavior, which may provide insights into observed in vivo graft failures. We further analyzed protein content by western blot and analyzed microstructure by histological staining and found that the specific method of decellularization resulted in significant differences in the depletion of proteoglycans and degradation of collagens I, II, III, and elastin. Taken together, this work demonstrates that the heterogeneous architecture and mechanical behavior of the trachea is severely compromised by decellularization. Such structural deterioration may contribute to graft failure clinically and limit the potential of decellularized native tracheas as viable long-term orthotopic airway replacements.