Lack of association between pandemic chilblains and SARS-CoV-2 infection

An increased incidence of chilblains has been observed during the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic and attributed to viral infection. Direct evidence of this relationship has been limited, however, as most cases do not have molecular evidence of prior SARS-CoV-2 infection with PCR or antibodies. We enrolled a cohort of 23 patients who were diagnosed and managed as having SARS-CoV-2–associated skin eruptions (including 21 pandemic chilblains [PC]) during the first wave of the pandemic in Connecticut. Antibody responses were determined through endpoint titration enzyme-linked immunosorbent assay and serum epitope repertoire analysis. T cell responses to SARS-CoV-2 were assessed by T cell receptor sequencing and in vitro SARS-CoV-2 antigen-specific peptide stimulation assays. Immunohistochemical and PCR studies of PC biopsies and tissue microarrays for evidence of SARS-CoV-2 were performed. Among patients diagnosed and managed as “covid toes” during the pandemic, we find a percentage of prior SARS-CoV-2 infection (9.5%) that approximates background seroprevalence (8.5%) at the time. Immunohistochemistry studies suggest that SARS-CoV-2 staining in PC biopsies may not be from SARS-CoV-2. Our results do not support SARS-CoV-2 as the causative agent of pandemic chilblains; however, our study does not exclude the possibility of SARS-CoV-2 seronegative abortive infections.

Concurrent with the rise of COVID-19 cases worldwide during the pandemic in early 2020, reports from different groups on different continents described increased diagnoses of chilblains attributed to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (1–8). Chilblains are an acral inflammatory rash typically affecting the toes and fingers of adults in colder, wetter conditions without a known association with respiratory viruses. Arguments for an association between this entity titled “covid toe” (which we refer to as pandemic chilblains [PC]) and SARS-CoV-2 infection include clustering of chilblains in areas with high incidence of COVID-19, SARS-CoV-2 exposure/symptoms in a significant percentage of PC cases, positive staining of spike (S) antigen in some biopsies, increased incidence in warmer temperatures in spring/summer of 2020, an expanded body distribution and possibly more severe, recalcitrant type of chilblain eruption, and the absence of a history of chilblains and/or other laboratory associations with classic chilblains (9–11).Despite this purported association between PC and SARS-CoV-2 infection, the majority of these patients lack evidence of prior infection (1, 4–8). Although testing was not widely available in early studies, this relationship has nonetheless held in later studies with more comprehensive testing (2, 3, 12). Arguments for this persistent inability to detect prior infection include 1) a missed window, with PCR testing too late and antibody testing too early; 2) loss of antibody positivity over time; and 3) that PC patients may feature a robust SARS-CoV-2 innate immune response that impedes the development of a detectable antibody signal (13). Thus, the association between PC and SARS-CoV-2 critically relies on the expectation that a significant number of these cases without evidence of prior infection did indeed experience infection that has not been successfully detected. We hypothesized that in-depth immunological profiling of both antibody and T cell responses of convalescent patients may resolve this question. Herein, we report our findings from a small cohort of PC patients that do not support an association between PC and prior SARS-CoV-2 infection.     

Extracellular vimentin is an attachment factor that facilitates SARS-CoV-2 entry into human endothelial cells

SARS-CoV-2 entry into host cells is a crucial step for virus tropism, transmission, and pathogenesis. Angiotensin-converting enzyme 2 (ACE2) has been identified as the primary entry receptor for SARS-CoV-2; however, the possible involvement of other cellular components in the viral entry has not yet been fully elucidated. Here we describe the identification of vimentin (VIM), an intermediate filament protein widely expressed in cells of mesenchymal origin, as an important attachment factor for SARS-CoV-2 on human endothelial cells. Using liquid chromatography–tandem mass spectrometry, we identified VIM as a protein that binds to the SARS-CoV-2 spike (S) protein. We showed that the S-protein receptor binding domain (RBD) is sufficient for S-protein interaction with VIM. Further analysis revealed that extracellular VIM binds to SARS-CoV-2 S-protein and facilitates SARS-CoV-2 infection, as determined by entry assays performed with pseudotyped viruses expressing S and with infectious SARS-CoV-2. Coexpression of VIM with ACE2 increased SARS-CoV-2 entry in HEK-293 cells, and shRNA-mediated knockdown of VIM significantly reduced SARS-CoV-2 infection of human endothelial cells. Moreover, incubation of A549 cells expressing ACE2 with purified VIM increased pseudotyped SARS-CoV-2-S entry. CR3022 antibody, which recognizes a distinct epitope on SARS-CoV-2-S-RBD without interfering with the binding of the spike with ACE2, inhibited the binding of VIM with CoV-2 S-RBD, and neutralized viral entry in human endothelial cells, suggesting a key role for VIM in SARS-CoV-2 infection of endothelial cells. This work provides insight into the pathogenesis of COVID-19 linked to the vascular system, with implications for the development of therapeutics and vaccines.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19) (1). SARS-CoV-2 binds to host receptors and attachment factors through its spike (S) glycoprotein and mediates membrane fusion and viral entry (2, 3). Although infection of cells along the respiratory tract was almost immediately defined as an important hallmark of the disease, the SARS-CoV-2 virus has also been detected not only in the lungs, but also in the cardiovascular system, brain, liver, kidneys, and intestine (4–7). Angiotensin-converting enzyme 2 (ACE2) is recognized as an important receptor for SARS-CoV-2 (1, 2). However, recent single-cell sequencing studies have demonstrated that ACE2 expression is relatively high in upper respiratory cells but low in the lower respiratory tract (8–12), and this result coupled with the identification of multiple other factors—including AXL receptor tyrosine kinase (11), Neuropilin-1 (13), CD209L/L-SIGN, CD209/DC-SIGN (14), and heparin sulfate (15)—as additional potential receptors or coreceptors for SARS-CoV-2, suggest that multiple receptors/coreceptors may facilitate SARS-CoV-2 entry in a cell type-dependent manner. Therefore, variations in the mechanisms of SARS-CoV-2 entry could account for its robust tropism, transmission, and pathogenesis.To search for unidentified receptors involved in SARS-CoV-2 entry into endothelial cells, we used SARS-CoV-2 S-receptor binding domain (S-RBD) as bait and whole-cell lysate (WCL) of human umbilical vein endothelial cells (HUVEC-TERT) as a source for prey proteins followed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis of proteins that showed an affinity for the SARS-CoV-2 S-RBD. Our analysis identified vimentin (VIM) as a SARS-CoV-2 binding protein. Further biochemical and cell culture studies using pseudotyped viruses expressing SARS-CoV-2 S-protein or infectious SARS-CoV-2 demonstrated that VIM interacts with both SARS-CoV-2 S and ACE2 and acts as a coreceptor for SARS-CoV-2.VIM is a type III intermediate filament protein and is widely expressed in cells of mesenchymal origin, such as endothelial cells, fibroblasts, and monocytes (16). In addition to its key role in intermediate filament formation, VIM is also present at the extracellular surface of endothelial cells and macrophages (17–19). Previous studies have found that extracellular VIM functions as an attachment factor or coreceptor for various viruses, including, SARS-CoV-1 (20), cowpea mosaic virus (21), Japanese encephalitis virus (22), dengue virus (23), and human papillomavirus (24). VIM could play an important role in promoting SARS-CoV-2 entry into the human vascular system, as well as other organs and tissues via cis- and trans-infection mechanisms.     

SARS-CoV-2 spreads through cell-to-cell transmission

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible coronavirus responsible for the global COVID-19 pandemic. Herein, we provide evidence that SARS-CoV-2 spreads through cell–cell contact in cultures, mediated by the spike glycoprotein. SARS-CoV-2 spike is more efficient in facilitating cell-to-cell transmission than is SARS-CoV spike, which reflects, in part, their differential cell–cell fusion activity. Interestingly, treatment of cocultured cells with endosomal entry inhibitors impairs cell-to-cell transmission, implicating endosomal membrane fusion as an underlying mechanism. Compared with cell-free infection, cell-to-cell transmission of SARS-CoV-2 is refractory to inhibition by neutralizing antibody or convalescent sera of COVID-19 patients. While angiotensin-converting enzyme 2 enhances cell-to-cell transmission, we find that it is not absolutely required. Notably, despite differences in cell-free infectivity, the authentic variants of concern (VOCs) B.1.1.7 (alpha) and B.1.351 (beta) have similar cell-to-cell transmission capability. Moreover, B.1.351 is more resistant to neutralization by vaccinee sera in cell-free infection, whereas B.1.1.7 is more resistant to inhibition by vaccinee sera in cell-to-cell transmission. Overall, our study reveals critical features of SARS-CoV-2 spike-mediated cell-to-cell transmission, with important implications for a better understanding of SARS-CoV-2 spread and pathogenesis.

SARS-CoV-2 is a novel beta-coronavirus that is closely related to two other highly pathogenic human coronaviruses, SARS-CoV and MERS-CoV (1). The spike (S) proteins of SARS-CoV-2 and SARS-CoV mediate entry into target cells, and both use angiotensin-converting enzyme 2 (ACE2) as the primary receptor (2–6). The spike protein of SARS-CoV-2 is also responsible for induction of neutralizing antibodies, thus playing a critical role in host immunity to viral infection (7–10).Similar to HIV and other class I viral fusion proteins, SARS-CoV-2 spike is synthesized as a precursor that is subsequently cleaved and highly glycosylated; these properties are critical for regulating viral fusion activation, native spike structure, and evasion of host immunity (11–15). However, distinct from SARS-CoV, yet similar to MERS-CoV, the spike protein of SARS-CoV-2 is cleaved by furin into S1 and S2 subunits during the maturation process in producer cells (6, 16, 17). S1 is responsible for binding to the ACE2 receptor, whereas S2 mediates viral membrane fusion (18, 19). SARS-CoV-2 spike can also be cleaved by additional host proteases, including transmembrane serine protease 2 (TMPRSS2) on the plasma membrane and several cathepsins in the endosome, which facilitate viral membrane fusion and entry into host cells (20–22).Enveloped viruses spread in cultured cells and tissues via two routes: by cell-free particles and through cell–cell contact (23–26). The latter mode of viral transmission normally involves tight cell–cell contacts, sometimes forming virological synapses, where local viral particle density increases (27), resulting in efficient transfer of virus to neighboring cells (24). Additionally, cell-to-cell transmission has the ability to evade antibody neutralization, accounting for efficient virus spread and pathogenesis, as has been shown for HIV and hepatitis C virus (HCV) (28–32). Low levels of neutralizing antibodies, as well as a deficiency in type I IFNs, have been reported for SARS-CoV-2 (18, 33–37) and may have contributed to the COVID-19 pandemic and disease progression (38–43).In this work, we evaluated cell-to-cell transmission of SARS-CoV-2 in the context of cell-free infection and in comparison with SARS-CoV. Results from this in vitro study reveal the heretofore unrecognized role of cell-to-cell transmission that potentially impacts SARS-CoV-2 spread, pathogenesis, and shielding from antibodies in vivo.     

SARS-CoV-2 spike engagement of ACE2 primes S2′ site cleavage and fusion initiation

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has resulted in tremendous loss worldwide. Although viral spike (S) protein binding of angiotensin-converting enzyme 2 (ACE2) has been established, the functional consequences of the initial receptor binding and the stepwise fusion process are not clear. By utilizing a cell–cell fusion system, in complement with a pseudoviral infection model, we found that the spike engagement of ACE2 primed the generation of S2′ fragments in target cells, a key proteolytic event coupled with spike-mediated membrane fusion. Mutagenesis of an S2′ cleavage site at the arginine (R) 815, but not an S2 cleavage site at arginine 685, was sufficient to prevent subsequent syncytia formation and infection in a variety of cell lines and primary cells isolated from human ACE2 knock-in mice. The requirement for S2′ cleavage at the R815 site was also broadly shared by other SARS-CoV-2 spike variants, such as the Alpha, Beta, and Delta variants of concern. Thus, our study highlights an essential role for host receptor engagement and the key residue of spike for proteolytic activation, and uncovers a targetable mechanism for host cell infection by SARS-CoV-2.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has exceeded 240 million cases across the globe, but the molecular mechanisms of viral infection and host pathogenesis remain elusive. The SARS-CoV-2 spike (S) glycoprotein is a class I fusion protein decorated on the viral lipid envelope and is a key determinant of viral entry (1). The SARS-CoV-2 spike monomer contains two fragments: The amino terminus S1 subunit contains a receptor binding domain (RBD) (2–5), which recognizes the host receptor angiotensin-converting enzyme 2 (ACE2) for initial docking, while the carboxyl terminus S2 subunit catalyzes the fusion of viral and cell membranes (6, 7), enabling the subsequent release of viral RNA genome and downstream replication within the infected cells (8). Although many studies have captured the stationary phases of spike binding to human ACE2 (9–11), key molecular and cellular processes downstream of receptor recognition have not been explored.Spike can be proteolytically processed (12). SARS-CoV-2 spike encodes a polybasic cleavage site at its S1/S2 junction, and is posttranslationally processed by the endopeptidase furin (13, 14); cleaved S1 and S2 subunits remain noncovalently attached and fusion competent (15). Furin-cleaved S1 also exposes a C-terminal motif recognized by the host receptor neuropilin-1 (NRP1) (16, 17), which can facilitate SARS-CoV-2 entry. Although spike protein is autoprocessed, additional proteolytic cleavage event within the S2 subunit is proposed to be responsible for the subsequent membrane fusion (18, 19). This cleavage can be mediated at the plasma membrane by the type II transmembrane serine proteases (TMPRSS2) (20–23), or processed by the lysosomal cathepsins during the endocytosis of viral particles (24). Secreted tissue proteases, such as elastase and trypsin, can also facilitate this cleavage event and promote infection (25). As a result, this proteolytic event within the S2 subunit induces the release of a highly conserved hydrophobic region, known as the fusion peptide (18), which subsequently anchors the target host cell membrane (6, 26). A conformational reconfiguration within the S2 subunit then pulls the viral and host membranes into close proximity, allowing lipid membranes to fuse (7, 27–29). The unilateral change of the S2 subunit is of the utmost importance during viral entry, but molecular events regulating the spike processing and activation have not been demonstrated.Cells infected with SARS-CoV-2 drive the fusion with adjacent ACE2-expressing cells, producing morphologically distinct multinuclear giant cells, also known as syncytia (2, 30, 31). Spike-mediated syncytia have been reported in the postmortem lung samples of severe COVID-19 patients (32, 33). Apart from virus to cell transmission, spike-driven syncytia formation may provide an additional route for cell–cell transmission of SARS-CoV-2. Here, by using a cell–cell fusion system, in complement with a pseudoviral particle infection model, we study the functional and molecular requirements of spike activation. Through analyzing the prefusion and postfusion spike protein products, we show that proteolytic cleavage at the S2′ site is triggered by human cell receptor recognition in a range of immortalized cell lines and humanized primary cells. Generation of the S2′ fragment specifically requires spike recognition of functional host ACE2 and is conserved in the several variants of concern. We highlight that arginine 815, but not arginine residues at the S1/S2 cleavage site, is indispensable for the S2′ cleavage and syncytia formation in wild type (WT), as well as the more infectious Alpha, Beta, and Delta spike variants. Hence, these data highlight that both receptor recognition and proteolytic event at the S2′ site are functionally important for spike-mediated membrane fusion and SARS-CoV-2 infection.     

Inhalable nanocatchers for SARS-CoV-2 inhibition

The global coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome (SARS)–like coronavirus (SARS-CoV-2), presents an urgent health crisis. More recently, an increasing number of mutated strains of SARS-CoV-2 have been identified globally. Such mutations, especially those on the spike glycoprotein to render its higher binding affinity to human angiotensin-converting enzyme II (hACE2) receptors, not only resulted in higher transmission of SARS-CoV-2 but also raised serious concerns regarding the efficacies of vaccines against mutated viruses. Since ACE2 is the virus-binding protein on human cells regardless of viral mutations, we design hACE2-containing nanocatchers (NCs) as the competitor with host cells for virus binding to protect cells from SARS-CoV-2 infection. The hACE2-containing NCs, derived from the cellular membrane of genetically engineered cells stably expressing hACE2, exhibited excellent neutralization ability against pseudoviruses of both wild-type SARS-CoV-2 and the D614G variant. To prevent SARS-CoV-2 infections in the lung, the most vulnerable organ for COVID-19, we develop an inhalable formulation by mixing hACE2-containing NCs with mucoadhesive excipient hyaluronic acid, the latter of which could significantly prolong the retention of NCs in the lung after inhalation. Excitingly, inhalation of our formulation could lead to potent pseudovirus inhibition ability in hACE2-expressing mouse model, without imposing any appreciable side effects. Importantly, our inhalable hACE2-containing NCs in the lyophilized formulation would allow long-term storage, facilitating their future clinical use. Thus, this work may provide an alternative tactic to inhibit SARS-CoV-2 infections even with different mutations, exhibiting great potential for treatment of the ongoing COVID-19 epidemic.

The expanding coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected more than 200 million people and killed over 3 million, and the numbers are still rapidly rising in April 2021 (1, 2). Like other zoonotic coronaviruses, SARS-CoV-2 with the surface spike (S) glycoprotein binds with the receptor human angiotensin-converting enzyme II (hACE2) for cell entry and infection (3–6). Moreover, the S protein also undergoes mutations all the time to optimize its binding affinity and binding mode with ACE2 receptors, which may alter pathogenesis, virulence, and transmissibility (7–9). The spike aspartic acid–614 to glycine (D614G), the dominant SARS-CoV-2 mutational form globally, showed obviously increased binding efficiency with ACE2 receptor during the virus infection process (10, 11). Moreover, a new variant strain of SARS-CoV-2, B.1.1.7 (also known as VOC 202012/01), with a high number of genetic mutations has been found in London and is spreading worldwide (12, 13). The binding affinity of S protein of B.1.1.7 to hACE2 receptor is increased by 1,000 times, and it exhibits 70% more transmissible ability than the previously discovered SARS-CoV-2 (14).Vaccines, one of the effective strategies to prevent the spread of infectious diseases by reducing morbidity and mortality, have attracted wide attention since the outbreak of COVID-19. At present, there are more than 314 SARS-CoV-2 vaccines in the research and development stage in the world, 89 of which have entered the clinical trial stage, and multiple research and development technologies are being promoted in parallel to promote vaccine development (15). Excitingly, thirteen vaccines have been approved for clinical use in different countries, including mRNA vaccines, virus-vectored vaccines, inactivated virus vaccines, and protein subunit vaccines (https://vac-lshtm.shinyapps.io/ncov_vaccine_landscape/#https://vac-lshtm.shinyapps.io/ncov_vaccine_landscape/). The current vaccines primarily protect the host against infection by producing neutralizing antibodies specific for the surface S protein (16–18). However, mutation of the S protein may possibly limit the efficiency of these vaccines (19–21). For example, the neutralizing activity of the serum of volunteers who have received either the Moderna (mRNA-1273) or Pfizer-BioNTech (BNT162b2) vaccines against the South Africa mutant strain (B.1.351) have been proven to be reduced (22, 23). Thus, novel strategies that could effectively and rapidly prevent the infection of SARS-CoV-2 with different mutations take on a renewed urgency in this period for COVID-19.Considering that the infectivity of SARS-CoV-2 depends on the binding with the entry receptor hACE2, recent studies have investigated the potential of cellular nanovesicles (NVs) containing hACE2 to compete with host cells for SARS-CoV-2 binding to protect host cells from the infection of SARS-CoV-2 (24, 25). How to improve the therapeutic efficiency of such neutralizing cellular NVs is still an important issue for COVID-19 treatment. Herein, the hACE2 nanocatchers (NCs) were fabricated from genetically engineering human embryonic kidney 293T cells with hACE2 as the neutralizing NCs to protect the host from the infection of SARS-CoV-2. As expected, such hACE2-containing NCs displayed excellent binding affinity to the coronavirus and its mutant and inhibited their usual infection ability in vitro. Moreover, in view of the fact that the effective retention of cellular NVs in the lungs after inhalation may be a prerequisite for inhibiting SARS-CoV-2 infection of the lung, we try to seek effective mucoadhesives to increase epithelial contact, decrease mucociliary transport rate, and finally, maintain the retention of NCs in the lungs for a longer duration after pulmonary drug delivery. Excitingly, it was found that the mucoadhesive excipient hyaluronic acid (HA) with high biocompatibility introduced here was able to significantly improve the retention of NCs in the lung, exhibiting potent SARS-CoV-2 pseudovirus inhibition ability in the mouse model with replication defective adenovirus encoding for hACE2. More importantly, NCs in the lyophilized formulation were fabricated with the assistance of cryoprotectant sucrose, increasing the feasibility of clinical use including transport and long-term storage.     

Vaccine-induced systemic and mucosal T cell immunity to SARS-CoV-2 viral variants

The first-generation COVID-19 vaccines have been effective in mitigating severe illness and hospitalization, but recurring waves of infections are associated with the emergence of SARS-CoV-2 variants that display progressive abilities to evade antibodies, leading to diminished vaccine effectiveness. The lack of clarity on the extent to which vaccine-elicited mucosal or systemic memory T cells protect against such antibody-evasive SARS-CoV-2 variants remains a critical knowledge gap in our quest for broadly protective vaccines. Using adjuvanted spike protein–based vaccines that elicit potent T cell responses, we assessed whether systemic or lung-resident CD4 and CD8 T cells protected against SARS-CoV-2 variants in the presence or absence of virus-neutralizing antibodies. We found that 1) mucosal or parenteral immunization led to effective viral control and protected against lung pathology with or without neutralizing antibodies, 2) protection afforded by mucosal memory CD8 T cells was largely redundant in the presence of antibodies that effectively neutralized the challenge virus, and 3) “unhelped” mucosal memory CD8 T cells provided no protection against the homologous SARS-CoV-2 without CD4 T cells and neutralizing antibodies. Significantly, however, in the absence of detectable virus-neutralizing antibodies, systemic or lung-resident memory CD4 and “helped” CD8 T cells provided effective protection against the relatively antibody-resistant B1.351 (β) variant, without lung immunopathology. Thus, induction of systemic and mucosal memory T cells directed against conserved epitopes might be an effective strategy to protect against SARS-CoV-2 variants that evade neutralizing antibodies. Mechanistic insights from this work have significant implications in the development of T cell–targeted immunomodulation or broadly protective SARS-CoV-2 vaccines.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continued to exert devastating impacts on the human life, with >280 million infections and over 5.4 million deaths to date. Although there are millions of convalescent people with some measure of immunity and 8.8 billion doses of vaccine administered to date, further threats of widespread severe COVID-19 disease looms heavily as immunity induced by infection or the first-generation vaccines may not provide effective and durable protection, either due to waning immunity or due to poor antibody cross-reactivity to new variants (1–5).It is clear that virus-neutralizing antibodies provide the most effective protection to SARS-CoV-2, following vaccination or recovery from infection (6). However, T cell–based protection against SARS-CoV-2 has become a central focus because T cells recognize short amino acid sequences that can be conserved across viral variants (7–9). Indeed, T cells in convalescent COVID-19 patients have shown robust responses that are directed at multiple viral proteins, and depletion of these T cells delayed SARS-CoV-2 control in mice (10–12). These data suggest a protective role for T cells in COVID-19 infection. In effect, what constitutes an effective, an ineffective, or a perilous T cell response to SARS-CoV-2 in lungs remains poorly defined. Controlled studies in laboratory animals are of critical importance to elucidate the role and nature of T cells in lungs during SARS-CoV-2 virus infection and in protective immunity.Based on the differentiation state, anatomical localization and traffic patterns, memory T cells are classified into effector memory (T_EM), central memory (T_CM), and tissue-resident memory (T_RM) (13, 14). There is accumulating evidence that airway/lung-resident T_RMs, and not migratory memory T cells (T_EMs) are critical for protective immunity to respiratory mucosal infections with viruses, such as influenza A virus (IAV) and respiratory syncytial virus (15–21). Development of T_RMs from effector T cells in the respiratory tract requires local antigen recognition and exposure to critical factors, such as transforming growth factor (TGF)-β and interleukin (IL)-15 (15). Therefore, mucosal vaccines are more likely to elicit T_RMs in lungs than parenteral vaccines (22, 23). A subset of effector T cells in airways of COVID-19 patients display T_RM-like features (24), but the development of T_RMs or their importance in protective immunity to reinfection are yet to be determined. Furthermore, all SARS-CoV-2 vaccines in use are administered parenterally and less likely to induce lung T_RMs. While depletion of CD8 T cells compromised protection against COVID-19 in vaccinated rhesus macaques (25), the relative effectiveness of vaccine-induced systemic/migratory CD8 T cell memory vs. lung/airway T_RMs in protective immunity to COVID-19 is yet to be defined.In this study, using the K18-hACE2 transgenic (tg) mouse model of SARS-CoV-2 infection, we have interrogated two key aspects of T cell immunity: 1) the requirements for lung-resident vs. migratory T cell memory in vaccine-induced immunity to SARS-CoV-2; and 2) the role of lung-resident memory CD4 vs. CD8 T cells in protection against viral variants in the presence or absence of virus-neutralizing antibodies. Studies of mucosal versus systemic T cell–based vaccine immunity using a subunit protein-based adjuvant system that elicits neutralizing antibodies and T cell immunity, demonstrated that: 1) both mucosal and parenteral vaccinations provide effective immunity to SARS-CoV-2 variants; 2) CD4 T cell–dependent immune mechanisms exert primacy in protection against homologous SARS-CoV-2 strain; and 3) the development of spike (S) protein-specific “unhelped” memory CD8 T cells in the respiratory mucosa are insufficient to protect against a lethal challenge with the homologous Washington (WA) strain of SARS-CoV-2. Unexpectedly, we found that systemic or mucosal lung-resident memory CD4 and “helped” CD8 T cells engendered effective immunity to the South African B1.351 β-variant in the apparent absence of detectable mucosal or circulating virus-neutralizing antibodies. Taken together, mechanistic insights from this study have advanced our understanding of viral pathogenesis and might drive rational development of next-generation broadly protective SARS-CoV-2 vaccines that induce humoral and T cell memory.     

Nonmuscle myosin heavy chain IIA facilitates SARS-CoV-2 infection in human pulmonary cells

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), binds to host receptor angiotensin-converting enzyme 2 (ACE2) through its spike (S) glycoprotein, which mediates membrane fusion and viral entry. However, the expression of ACE2 is extremely low in a variety of human tissues, especially in the airways. Thus, other coreceptors and/or cofactors on the surface of host cells may contribute to SARS-CoV-2 infection. Here, we identified nonmuscle myosin heavy chain IIA (MYH9) as an important host factor for SARS-CoV-2 infection of human pulmonary cells by using APEX2 proximity-labeling techniques. Genetic ablation of MYH9 significantly reduced SARS-CoV-2 pseudovirus infection in wild type (WT) A549 and Calu-3 cells, and overexpression of MYH9 enhanced the pseudovirus infection in WT A549 and H1299 cells. MYH9 was colocalized with the SARS-CoV-2 S and directly interacted with SARS-CoV-2 S through the S2 subunit and S1-NTD (N-terminal domain) by its C-terminal domain (designated as PRA). Further experiments suggested that endosomal or myosin inhibitors effectively block the viral entry of SARS-CoV-2 into PRA-A549 cells, while transmembrane protease serine 2 (TMPRSS2) and cathepsin B and L (CatB/L) inhibitors do not, indicating that MYH9 promotes SARS-CoV-2 endocytosis and bypasses TMPRSS2 and CatB/L pathway. Finally, we demonstrated that loss of MYH9 reduces authentic SARS-CoV-2 infection in Calu-3, ACE2-A549, and ACE2-H1299 cells. Together, our results suggest that MYH9 is a candidate host factor for SARS-CoV-2, which mediates the virus entering host cells by endocytosis in an ACE2-dependent manner, and may serve as a potential target for future clinical intervention strategies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered a pandemic coronavirus disease (COVID-19) (1–3), resulting in more than 209 million infections worldwide since December 2019. SARS-CoV-2 is the seventh coronavirus (CoV) infecting humans beyond endemic human CoVs (HCoV-HKU1, -NL63, -OC43, and -229E), SARS-CoV, and Middle East respiratory syndrome CoV (MERS-CoV). SARS-CoV-2 preferentially infects the airway epithelial cells (1, 4), although viral infection has been detected in a variety of human organs, including the lungs, pharynx, heart, liver, brain, kidneys, and digestive system organs (5–7), causing upper respiratory diseases, fever, and severe pneumonia in humans.CoVs were not considered to be highly pathogenic to humans until the outbreak of SARS-CoV started in 2002 in Guangdong province, China (8–10). The primary determinant of CoV tropism is their spike (S) glycoproteins, which mediate the viral infection by binding to membrane receptors on the host cells and could be cleaved to the N-terminal S1 and C-terminal S2 subunit by the host proteases such as transmembrane protease serine 2 (TMPRSS2) and Furin (11). Studies have shown that ACE2, a cellular receptor for SARS-CoV, also binds SARS-CoV-2 S and serves as the entry point for SARS-CoV-2 (3, 12). However, ACE2 cannot fully explain the tissue tropism of SARS-CoV-2 because of the virus detection in tissues with little ACE2 expression, such as the liver, brain, and blood, and even in the lung, only a small subset of cells expresses ACE2 (SI Appendix, Fig. 1B) (13–16). Recently, neuropilin-1 (NRP1) (17, 18) and heparan sulfate (19, 20) have been identified as cofactors implicated in enhancing ACE2-dependent SARS-CoV-2 infection, while tyrosine-protein kinase receptor (AXL) (21) and CD147 (22) were identified as receptors involved in SARS-CoV-2 cell entry independently of ACE2. These highlight the multiple routes of SARS-CoV-2 infection, likely contributing to high infectivity and spread of COVID-19. Hence, SARS-CoV-2 is assumed to be dependent on other potential receptors or coreceptors to facilitate its infection in humans.Here, we applied engineered ascorbate peroxidase APEX2-based subcellular proteomics (23–25) to capture spatiotemporal membrane protein complexes interacting with SARS-CoV-2 S in living cells. We found that the nonmuscle myosin heavy chain IIA (MYH9) specifically interacts with SARS-CoV-2 S protein. The C-terminal domain (designated as PRA) of MYH9 interacts with the S2 subunit and the N-terminal domain (NTD) of the S1 subunit, and PRA overexpression facilitates a pan-CoV entry into host cells. In addition, depletion of MYH9 significantly reduced authentic SARS-CoV-2 infection in human lung cell lines. Mechanistically, a myosin inhibitor but not TMPRSS2 and CatB/L inhibitors blocked SARS-CoV-2 infection in PRA-A549 cells, and the presence of ACE2 is required for MYH9 mediated SARS-CoV-2 entry. Collectively, our results suggest that MYH9 is a coreceptor of ACE2 for SARS-CoV-2 infection in human pulmonary cells.     

In vivo kinetics of SARS-CoV-2 infection and its relationship with a person’s infectiousness

The within-host viral kinetics of SARS-CoV-2 infection and how they relate to a person’s infectiousness are not well understood. This limits our ability to quantify the impact of interventions on viral transmission. Here, we develop viral dynamic models of SARS-CoV-2 infection and fit them to data to estimate key within-host parameters such as the infected cell half-life and the within-host reproductive number. We then develop a model linking viral load (VL) to infectiousness and show a person’s infectiousness increases sublinearly with VL and that the logarithm of the VL in the upper respiratory tract is a better surrogate of infectiousness than the VL itself. Using data on VL and the predicted infectiousness, we further incorporated data on antigen and RT-PCR tests and compared their usefulness in detecting infection and preventing transmission. We found that RT-PCR tests perform better than antigen tests assuming equal testing frequency; however, more frequent antigen testing may perform equally well with RT-PCR tests at a lower cost but with many more false-negative tests. Overall, our models provide a quantitative framework for inferring the impact of therapeutics and vaccines that lower VL on the infectiousness of individuals and for evaluating rapid testing strategies.

SARS-CoV-2 is a new human pathogen that causes COVID-19 (1). It is highly contagious, spread rapidly across the globe and has caused 5 million deaths worldwide as of the end of October 2021. At the molecular level, SARS-CoV-2 enters host cells via the angiotensin converting enzyme 2 (ACE-2) receptor. It infects cells in the upper respiratory tract (URT), can rapidly reach a high viral load (VL) and be effectively transmitted (2–4). However, it is not clear how VL, symptom onset, and infectiousness are quantitatively related.Previously, both VL and log₁₀ VL have been used as surrogates for infectiousness of influenza (5) and SARS-CoV-2 (6, 7). A quantitative understanding of the relationship is critical for both nonpharmaceutical and pharmaceutical interventions. First, it would allow for more precise prediction of the infectiousness of infected individuals, including children and pre- or asymptomatic individuals, based on their VL measurements (8, 9). This could in turn lead to quantification of their contribution to the overall transmission in a community and help to better inform public health policy decisions. Second, as administration of vaccines may lead to lowered VLs in breakthrough infections (10–12), a quantitative understanding will inform how these reductions in VL impact infectiousness and thus allow better predictions of how much transmission vaccinated individuals with breakthrough infection cause. Third, it would provide better insight into a person’s infectiousness throughout the course of infection and thus inform testing strategies for work/school reopening, travel, etc. The effectiveness of test, trace, and quarantine as control strategies heavily depends on the sensitivity and specificity of the tests and rate of testing being implemented (13). It was recently proposed that antigen tests with low sensitivity are preferred over highly sensitive RT-PCR tests because of their potential for wide coverage and short turnaround time (6). However, the effectiveness of this strategy has not been evaluated based on VL and infectiousness dynamics inferred from data.Here, we construct viral dynamic models of SARS-CoV-2 URT infection and a model linking VL to infectiousness. Mathematical modeling has been applied, by us and others, to understand SARS-CoV-2 infection and the potential impact of therapy (14–18). However, there were large uncertainties in model parameter estimates because in almost all studies, viral dynamic models were fit to data that were taken after symptom onset without knowledge of the patients’ infection dates and early VL dynamics. We resolve this issue by using two unique datasets and by using clinical and epidemiological data to inform the quantitative relationship between VL and infectiousness. Using this relationship, we further evaluate the effectiveness of testing strategies using either antigen or RT-PCR tests at different testing frequencies.     

A serum-stable RNA aptamer specific for SARS-CoV-2 neutralizes viral entry

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has created an urgent need for new technologies to treat COVID-19. Here we report a 2′-fluoro protected RNA aptamer that binds with high affinity to the receptor binding domain (RBD) of SARS-CoV-2 spike protein, thereby preventing its interaction with the host receptor ACE2. A trimerized version of the RNA aptamer matching the three RBDs in each spike complex enhances binding affinity down to the low picomolar range. Binding mode and specificity for the aptamer–spike interaction is supported by biolayer interferometry, single-molecule fluorescence microscopy, and flow-induced dispersion analysis in vitro. Cell culture experiments using virus-like particles and live SARS-CoV-2 show that the aptamer and, to a larger extent, the trimeric aptamer can efficiently block viral infection at low concentration. Finally, the aptamer maintains its high binding affinity to spike from other circulating SARS-CoV-2 strains, suggesting that it could find widespread use for the detection and treatment of SARS-CoV-2 and emerging variants.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic in 2020–2021 has launched a global quest to find new molecular tools for the detection of the virus and treatment of the potential deadly disease it causes, COVID-19. Despite the exceptional efforts worldwide for containment and unprecedented technological progress in vaccine development, the challenge to find an effective cure remains, due to the limited access to SARS-CoV-2 vaccines, particularly in developing countries, and the emergence of new viral strains that can evade immune responses and potentially compromise the efficacy of current vaccines. Therefore, it is of utmost importance to focus efforts on developing affordable and easy-to-produce antiviral molecules against SARS-CoV-2 infection.Like other coronaviruses, SARS-CoV-2 expresses a surface spike (S) glycoprotein which is composed of two domains (S1 and S2) (1) and forms a trimeric structure capable of interacting with human cells (2, 3). In particular, the receptor binding domain (RBD) located on the S1 subunit of the spike protein binds with high affinity to human angiotensin-converting enzyme 2 (ACE2) (4, 5), which, in conjunction with the associated transmembrane protease, serine 2 (TMPRSS2), facilitates viral uptake. Efforts to neutralize viral infection have therefore mainly focused on inhibiting the spike–ACE2 interaction. Antibodies (Abs) have been developed and are currently used for SARS-CoV-2 detection, and some, primarily those targeting RBD, show therapeutic potential due to their potent neutralizing effect (6–13). However, the high costs of Ab production, the use of animals to generate them, and their poor stability at ambient temperatures remain a disadvantage. Moreover, Ab immunogenicity and the risk of Ab-dependent enhancement of infection associated with Fc-containing Abs put their therapeutic potential at risk (14).V_HH Abs or nanobodies raised to the spike protein may overcome some of these drawbacks (15–20) but are more prone to immunological response (21, 22). Interesting alternatives such as de novo proteins based on the host ACE2 receptor (23) and other synthetic molecules (24) have been investigated and may, if potential immunogenicity and stability problems are solved, help develop efficient detection methods and drugs.Nucleic acid-based aptamers have gained increased attention as alternatives to Abs due to their ease of production, low immunogenicity, high thermal and chemical stability, and smaller size, while they still retain comparable target binding and specificity. Aptamers are short single-stranded oligonucleotides, developed through an in vitro selection process termed SELEX (systematic evolution of ligands by exponential enrichment), that bind with high affinity and selectivity to cognate targets (25–27). During the last few decades, a wide variety of aptamers binding to diverse biologically relevant targets (28), including viruses (29, 30), have been identified. However, selection of aptamers targeting spike protein has proven difficult. An explanation for this may be that highly glycosylated proteins such as SARS-CoV-2 spike are challenging to target with nucleic acid-based binders. Indeed, to date, there are only a few reports on DNA aptamers targeting SARS-CoV-2 spike where the authors report leading aptamers with affinities in the nanomolar range (31–35).Here we report the selection and characterization of a serum-stable RNA aptamer, RBD-PB6, that binds with nanomolar affinity to the RBD of SARS-CoV-2 spike protein and neutralizes viral infectivity. The aptamer contains 2′-fluoro pyrimidine modifications to increase its chemical stability and resistance to nucleases (36, 37), and it shows high selectivity to SARS-CoV-2 and related strains, including alpha and beta. Aptamer multimerization strongly enhances its affinity to the picomolar range as well as its SARS-CoV-2 neutralizing potency. These unique features open avenues for developing inexpensive, fast, and reliable detection platforms for SARS-CoV-2 and therapeutic application for COVID-19.     

A single intranasal dose of a live-attenuated parainfluenza virus-vectored SARS-CoV-2 vaccine is protective in hamsters

Single-dose vaccines with the ability to restrict SARS-CoV-2 replication in the respiratory tract are needed for all age groups, aiding efforts toward control of COVID-19. We developed a live intranasal vector vaccine for infants and children against COVID-19 based on replication-competent chimeric bovine/human parainfluenza virus type 3 (B/HPIV3) that express the native (S) or prefusion-stabilized (S-2P) SARS-CoV-2 S spike protein, the major protective and neutralization antigen of SARS-CoV-2. B/HPIV3/S and B/HPIV3/S-2P replicated as efficiently as B/HPIV3 in vitro and stably expressed SARS-CoV-2 S. Prefusion stabilization increased S expression by B/HPIV3 in vitro. In hamsters, a single intranasal dose of B/HPIV3/S-2P induced significantly higher titers compared to B/HPIV3/S of serum SARS-CoV-2–neutralizing antibodies (12-fold higher), serum IgA and IgG to SARS-CoV-2 S protein (5-fold and 13-fold), and IgG to the receptor binding domain (10-fold). Antibodies exhibited broad neutralizing activity against SARS-CoV-2 of lineages A, B.1.1.7, and B.1.351. Four weeks after immunization, hamsters were challenged intranasally with 10^4.5 50% tissue-culture infectious-dose (TCID₅₀) of SARS-CoV-2. In B/HPIV3 empty vector-immunized hamsters, SARS-CoV-2 replicated to mean titers of 10^6.6 TCID₅₀/g in lungs and 10⁷ TCID₅₀/g in nasal tissues and induced moderate weight loss. In B/HPIV3/S-immunized hamsters, SARS-CoV-2 challenge virus was reduced 20-fold in nasal tissues and undetectable in lungs. In B/HPIV3/S-2P–immunized hamsters, infectious challenge virus was undetectable in nasal tissues and lungs; B/HPIV3/S and B/HPIV3/S-2P completely protected against weight loss after SARS-CoV-2 challenge. B/HPIV3/S-2P is a promising vaccine candidate to protect infants and young children against HPIV3 and SARS-CoV-2.

The betacoronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in 2019 and rapidly spread globally (1). In the first year of the pandemic, over 105 million infections and 2.3 million deaths have been reported worldwide, including over 27 million cases and 500,000 deaths in the United States (https://covid19.who.int/). Vaccines are being rapidly deployed in a race to control ongoing infections and the emergence of variants of concern (2), with increased virulence and altered antigenicity.SARS-CoV-2 infects and spreads primarily via the respiratory route (3, 4), and mucosal surfaces of the respiratory tract represent the primary site of infection. COVID-19, the disease caused by SARS-CoV-2, is characterized by upper and lower respiratory tract symptoms, fever, chills, body aches, and fatigue, and in some cases gastrointestinal and other symptoms with involvement of additional tissues (5, 6).SARS-CoV-2 infection is initiated by the spike (S) surface glycoprotein, the main target for SARS-CoV-2–neutralizing antibodies. The S protein is a trimeric class I fusion glycoprotein. Each protomer consists of two functionally distinct subunits, S1 and S2, linked by a furin cleavage site; S2 contains an additional proteolytic cleavage site S2′. S2/S2′ cleavage is mediated by the transmembrane protease serine 2 (TMPRSS2) (1, 7–9). The S1 subunit contains the receptor-binding domain (RBD). The S2 subunit contains the membrane fusion machinery, including the hydrophobic fusion peptide and α-helical heptad repeats (7, 9).Binding of the S RBD to its receptor, human angiotensin converting enzyme 2, triggers a change, from the closed and metastable prefusion conformation to the open and stable postfusion form that drives membrane fusion enabling viral entry (1). Stabilization of the S protein in its native prefusion state should preserve antibody epitopes, including immunodominant sites of the RBD, required to elicit high-quality neutralizing antibody responses (9–13). Thus, a prefusion-stabilized version of the S protein is the optimal vaccine immunogen (13–15).Vaccines for SARS-CoV-2 are available, but currently are limited to individuals 12 y of age or older. They are administered intramuscularly, which does not directly stimulate mucosal immunity in the respiratory tract, the primary site of SARS-CoV-2 infection and shedding. While the major burden of COVID-19 disease is in adults, infection and disease also occurs in infants and young children, contributing to viral transmission. Therefore, the development of safe and effective pediatric COVID-19 vaccines is critical for worldwide control of COVID-19. The ideal vaccine should be effective at a single dose, inducing durable and broad systemic immunity, as well as T and B cell respiratory mucosal immunity that completely blocks SARS-CoV-2 infection and transmission.Here we describe a vectored SARS-CoV-2 vaccine candidate for intranasal immunization of infants and young children. The vaccine is based on an attenuated, replication-competent parainfluenza virus type 3 (PIV3) vector called B/HPIV3 (16) expressing the SARS-CoV-2 S protein. B/HPIV3 consists of bovine PIV3 (BPIV3) strain Kansas in which the BPIV3 hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins (the two PIV3 neutralization antigens) have been replaced by those of human PIV3 strain JS (16, 17). The BPIV3 backbone provides host range restriction of replication in humans, serving as the basis for strong and stable attenuation (17, 18). B/HPIV3 originally was developed as a live vaccine candidate against HPIV3, and was well-tolerated in young children (17). Moreover, B/HPIV3 has been used to express the F glycoprotein of another human respiratory pathogen, human respiratory syncytial virus (HRSV), as a bivalent HPIV3/HRSV vaccine candidate. This vaccine candidate was well-tolerated in children >2 mo of age (18) (Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00686075","term_id":"NCT00686075"}}NCT00686075), and optimized versions are in further clinical development as pediatric vaccines (19, 20). In the present study, we used B/HPIV3 to express wild-type (S) or prefusion-stabilized (S-2P) versions of the SARS-CoV-2 S protein, creating the vaccine candidates B/HPIV3/S and B/HPIV3/S-2P. These were evaluated in vitro and in hamsters as live-attenuated SARS-CoV-2 intranasal vaccine candidates.     

Immunogenicity and efficacy of the COVID-19 candidate vector vaccine MVA-SARS-2-S in preclinical vaccination

Severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) has emerged as the infectious agent causing the pandemic coronavirus disease 2019 (COVID-19) with dramatic consequences for global human health and economics. Previously, we reached clinical evaluation with our vector vaccine based on modified vaccinia virus Ankara (MVA) against the Middle East respiratory syndrome coronavirus (MERS-CoV), which causes an infection in humans similar to SARS and COVID-19. Here, we describe the construction and preclinical characterization of a recombinant MVA expressing full-length SARS-CoV-2 spike (S) protein (MVA-SARS-2-S). Genetic stability and growth characteristics of MVA-SARS-2-S, plus its robust expression of S protein as antigen, make it a suitable candidate vaccine for industrial-scale production. Vaccinated mice produced S-specific CD8⁺ T cells and serum antibodies binding to S protein that neutralized SARS-CoV-2. Prime-boost vaccination with MVA-SARS-2-S protected mice sensitized with a human ACE2-expressing adenovirus from SARS-CoV-2 infection. MVA-SARS-2-S is currently being investigated in a phase I clinical trial as aspirant for developing a safe and efficacious vaccine against COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of coronavirus disease 2019 (COVID-19), first emerged in late 2019 in China (1). SARS-CoV-2 exhibits extremely efficient human-to-human transmission, the new pathogen rapidly spread worldwide, and within months it caused a global pandemic, changing daily life for billions of people. The COVID-19 case fatality rate of ∼2–5% makes the development of countermeasures a global priority. In fact, the development of COVID-19 vaccine candidates is advancing at an international level with unprecedented speed. About 1 y after the first known cases of COVID-19, we can account for >80 SARS-CoV-2–specific vaccines in clinical evaluations and >10 candidate vaccines already in phase III trials (2–4). However, we still lack information on the key immune mechanisms needed for protection against COVID-19. A better understanding of the types of immune response elicited upon natural SARS-CoV-2 infections has become an essential component to assess the promise of various vaccination strategies (5).The SARS-CoV-2 spike (S) protein serves as the most important target antigen for vaccine development based on preclinical research on candidate vaccines against SARS-CoV or Middle East respiratory syndrome coronavirus (MERS-CoV). The trimeric S protein is a prominent structure at the virion surface and essential for SARS-CoV-2 cell entry. As a class I viral fusion protein, it mediates virus interaction with the cellular receptor angiotensin-converting enzyme 2 (ACE2), and fusion with the host cell membrane, both key steps in infection. Thus, infection can be prevented by S-specific antibodies neutralizing the virus (6–9).Among the front-runner vaccines are new technologies such as messenger RNA (mRNA)-based vaccines and nonreplicating adenovirus vector vaccines (10–13). First reports from these SARS-CoV-2-S–specific vaccines in phase 1/2 clinical studies demonstrated acceptable safety and promising immunogenicity profiles, and by now data from large phase 3 clinical trials show promising levels of protective efficacy (4, 12–14). In December 2020, the first mRNA-based COVID-19 vaccines received emergency use authorization or conditional licensing by the US Food and Drug Administration and European Medicines Agency (11, 15, 16). By March 2021, two adenovirus vector-based COVID-10 vaccines had been approved by regulatory authorities (17, 18). This is good news because efficacious vaccines will provide a strategy to change SARS-CoV-2 transmission dynamics. In addition, multiple vaccine types will be advantageous to meet specific demands across different target populations. This includes the possibility of using heterologous immunization strategies depending on an individual’s health status, boosting capacities, and the need for balanced humoral and Th1-directed cellular immune responses.MVA, a highly attenuated strain of vaccinia virus originating from growth selection on chicken embryo tissue cultures, shows a characteristic replication defect in mammalian cells but allows unimpaired production of heterologous proteins (19). At present, MVA serves as an advanced vaccine technology platform for developing new vector vaccines against infectious disease including emerging viruses and cancer (20). In response to the ongoing pandemic, the MVA vector vaccine platform allows rapid generation of experimental SARS-CoV-2–specific vaccines (21). Previous work from our laboratory addressed the development of an MVA candidate vaccine against MERS with immunizations in animal models demonstrating the safety, immunogenicity, and protective efficacy of MVA-induced MERS-CoV S-antigen–specific immunity (22–25). Clinical safety and immunogenicity of the MVA-MERS-S candidate vaccine was established in a first-in-human phase I clinical study under funding from the German Center for Infection Research (DZIF) (26).Here, we show that a recombinant MVA produces the full-length S protein of SARS-CoV-2 as ∼190- to 200-kDa N-glycosylated protein. Our studies confirmed cleavage of the mature full-length S protein into an amino-terminal domain (S1) and a ∼80- to 100-kDa carboxyl-terminal domain (S2) that is anchored to the membrane. When tested as a vaccine in BALB/c mice, recombinant MVA expressing the S protein induced SARS-CoV-2–specific T cells and antibodies, and robustly protected vaccinated animals against lung infection upon SARS-CoV-2 challenge.     

How immunity from and interaction with seasonal coronaviruses can shape SARS-CoV-2 epidemiology

We hypothesized that cross-protection from seasonal epidemics of human coronaviruses (HCoVs) could have affected severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission, including generating reduced susceptibility in children. To determine what the prepandemic distribution of immunity to HCoVs was, we fitted a mathematical model to 6 y of seasonal coronavirus surveillance data from England and Wales. We estimated a duration of immunity to seasonal HCoVs of 7.8 y (95% CI 6.3 to 8.1) and show that, while cross-protection between HCoV and SARS-CoV-2 may contribute to the age distribution, it is insufficient to explain the age pattern of SARS-CoV-2 infections in the first wave of the pandemic in England and Wales. Projections from our model illustrate how different strengths of cross-protection between circulating coronaviruses could determine the frequency and magnitude of SARS-CoV-2 epidemics over the coming decade, as well as the potential impact of cross-protection on future seasonal coronavirus transmission.

Due to the relatively short time since severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged, little is yet known about the duration of infection-induced immunity. While instances of confirmed reinfection of SARS-CoV-2 have been identified (1), these are rare (2), indicating protection lasts for at least 6 mo to 8 mo, which concurs with estimates from prospective studies (3, 4). Cross-protection from seasonal human coronaviruses (HCoVs) could have impacted the transmission dynamics of SARS-CoV-2, and explain the relatively low SARS-CoV-2 infection rate in children (5–8). Since children likely have a higher annual attack rate of endemic HCoVs due to their higher contact rates (9), they may be less susceptible to SARS-CoV-2 due to cross-protection.In order to evaluate the impacts of cross-immunity, we first need to quantify the immune protection from seasonal coronaviruses. Four coronavirus strains from two different genera are endemic in humans: Two are alphacoronaviruses (HCoV-229E and HCoV-NL63), and two are betacoronaviruses (HCoV-HKU1 and HCoV-OC43); SARS-CoV-2 is a member of the latter genera, as are SARS-CoV-1 and Middle East respiratory syndrome coronavirus (MERS-CoV). In the United Kingdom, seasonal human coronavirus (HCoV) case incidence peaks January–February each year. The first infection with seasonal HCoVs typically occurs in childhood (10), and reinfection with the same strain has been observed within a year (11, 12). However, there are also indications that immunity lasts longer, with few reinfections in a 3-y cohort study (13) and sterilizing immunity to homologous strains of HCoV-229E after 1 y in a challenge study (14).There may also be cross-protective immunity between seasonal HCoVs and SARS-family coronaviruses following infection. Human sera collected before the SARS-CoV-2 pandemic showed high IgG reactivity to seasonal HCoVs, but also low reactivity to SARS-CoV-2 (15), and SARS-CoV-1 infection induced antibody production against seasonal HCoVs (16, 17). Cross-reactive T cells to SARS-CoV-2 have been found in 20% (18) to 50% (38) of unexposed individuals, with evidence that these responses stem from seasonal coronavirus infection (20). It has also been noted that these are more prevalent in children and adolescents (21).Cross-protection from seasonal HCoVs may have, therefore, partially shaped the observed epidemiology of SARS-CoV-2. Using England and Wales as a case study, we use dynamic models to estimate 1) the duration of infection-induced immunity to seasonal HCoVs, 2) the ability of potential cross-protection from seasonal HCoVs to explain the age patterns in the first wave of the SARS-CoV-2 pandemic, and 3) the implications of the duration of immunity and potential cross-protection on future dynamics of SARS-CoV-2.     

Efficacy and breadth of adjuvanted SARS-CoV-2 receptor-binding domain nanoparticle vaccine in macaques

Emergence of novel variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underscores the need for next-generation vaccines able to elicit broad and durable immunity. Here we report the evaluation of a ferritin nanoparticle vaccine displaying the receptor-binding domain of the SARS-CoV-2 spike protein (RFN) adjuvanted with Army Liposomal Formulation QS-21 (ALFQ). RFN vaccination of macaques using a two-dose regimen resulted in robust, predominantly Th1 CD4+ T cell responses and reciprocal peak mean serum neutralizing antibody titers of 14,000 to 21,000. Rapid control of viral replication was achieved in the upper and lower airways of animals after high-dose SARS-CoV-2 respiratory challenge, with undetectable replication within 4 d in seven of eight animals receiving 50 µg of RFN. Cross-neutralization activity against SARS-CoV-2 variant B.1.351 decreased only approximately twofold relative to WA1/2020. In addition, neutralizing, effector antibody and cellular responses targeted the heterotypic SARS-CoV-1, highlighting the broad immunogenicity of RFN-ALFQ for SARS-CoV−like Sarbecovirus vaccine development.

The COVID-19 pandemic, precipitated by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), continues to threaten global public health and economies. Threats of future outbreaks also loom, as evidenced by three emergent SARS-like diseases caused by zoonotic Betacoronaviruses in the last two decades. While several emergency use authorized (EUA) vaccines currently in use are expected to curb both disease and transmission of SARS-CoV-2 (1–6), the emergence of circulating variants of concern (VOCs) less sensitive to vaccine-elicited immunity has raised concerns for sustained vaccine efficacy (7). Logistical challenges of vaccine production, distribution, storage, and access for these vaccines must be resolved to achieve resolution to the pandemic (8, 9). The rapid and unparalleled spread of SARS-CoV-2 has driven an urgent need to deploy scalable vaccine platforms to combat the ongoing pandemic and mitigate future outbreaks.Current vaccines primarily focus the immune response on the spike glycoprotein (S) as it mediates host cell viral fusion and entry. The receptor-binding domain (RBD) of S engages the primary host cell receptor, angiotensin-converting enzyme 2 (ACE2), for both SARS-CoV-2 and SARS-CoV-1, making RBD a promising domain for vaccine-elicited immune focus (10–12). Moreover, many of the potently neutralizing monoclonal antibodies isolated against SARS-CoV-2 target the RBD (13, 14). Vaccination of nonhuman primates (NHPs) with RBD-encoding RNA or DNA protects against respiratory tract challenge, indicating that immune responses to the RBD can prevent viral replication (15, 16). RBD vaccination also elicits cross-reactive responses to circulating SARS-CoV-2 VOCs in both animals and humans (17, 18), with decrements against the B.1.351 variant similar to that seen with S immunogens (19). The breadth of RBD immunogenicity is further supported by the ability of RBD-specific monoclonal antibodies isolated from SARS-CoV-1 convalescent individuals to cross-neutralize SARS-CoV-2 (20, 21). These findings suggest potential for RBD-based vaccines being efficacious against SARS-CoV-2 variants and other related coronavirus species.Approaches to improve immunogenicity of S or RBD protein vaccines include optimizing antigen presentation and coformulating with adjuvants to enhance the protective immunity. One common approach to enhance the induction of adaptive immune responses is the multimeric presentation of antigen, for example, on the surface of nanoparticles or virus-like particles (22). Presenting RBD in ordered, multivalent arrays on the surface of self-assembling protein nanoparticles is immunogenic and efficacious in animals (23–28), with improved immunogenicity relative to monomeric soluble RBD and cross-reactive responses to variants (17, 24, 26). However, it is unknown whether RBD nanoparticle vaccines protect against infection in primates, which have become a standard model for benchmarking performance of vaccine candidates by virological and immunologic endpoints. Liposomal adjuvants incorporating QS-21, such as that used in the efficacious varicella zoster vaccine, SHINGRIX, may augment protective immunity to SARS-CoV-2 vaccines. Such adjuvants have superior humoral and cellular immunogenicity relative to conventional adjuvants (29, 30).Here, we evaluate the use of a ferritin nanoparticle vaccine presenting the SARS-CoV-2 RBD (RFN) adjuvanted with the Army Liposomal Formulation QS-21 (ALFQ) (31). Both ferritin nanoparticles and ALFQ have been evaluated for vaccination against multiple pathogens in humans in phase 1 clinical trials (32–34). We demonstrate, in an NHP model, that immunization with RFN induces robust and broad antibody and T cell responses, as well as protection against viral replication and lung pathology following high-dose respiratory tract challenge with SARS-CoV-2.     

SARS-CoV-2 escape from a highly neutralizing COVID-19 convalescent plasma

To investigate the evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the immune population, we coincupi bated the authentic virus with a highly neutralizing plasma from a COVID-19 convalescent patient. The plasma fully neutralized the virus for seven passages, but, after 45 d, the deletion of F140 in the spike N-terminal domain (NTD) N3 loop led to partial breakthrough. At day 73, an E484K substitution in the receptor-binding domain (RBD) occurred, followed, at day 80, by an insertion in the NTD N5 loop containing a new glycan sequon, which generated a variant completely resistant to plasma neutralization. Computational modeling predicts that the deletion and insertion in loops N3 and N5 prevent binding of neutralizing antibodies. The recent emergence in the United Kingdom, South Africa, Brazil, and Japan of natural variants with similar changes suggests that SARS-CoV-2 has the potential to escape an effective immune response and that vaccines and antibodies able to control emerging variants should be developed.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causative agent of COVID-19, accounts for over 105 million cases of infections and more than 2.3 million deaths worldwide. Thanks to an incredible scientific and financial effort, several prophylactic and therapeutic tools, such as vaccines and monoclonal antibodies (mAbs), have been developed in less than 1 y to combat this pandemic (1–4). The main target of vaccines and mAbs is the SARS-CoV-2 spike protein (S protein), a large class I trimeric fusion protein which plays a key role in viral pathogenesis (3, 5, 6). The SARS-CoV-2 S protein is composed of two subunits: S1, which contains the receptor-binding domain (RBD) responsible for the interaction with receptors on the host cells, and S2, which mediates membrane fusion and viral entry (7, 8). The S1 subunit presents two highly immunogenic domains, the N-terminal domain (NTD) and the RBD, which are the major targets of polyclonal and monoclonal neutralizing antibodies (4, 9, 10). The continued spread in immune-competent populations has led to adaptations of the virus to the host and generation of new SARS-CoV-2 variants. Indeed, S-protein variants have been recently described in the United Kingdom, South Africa, Brazil, and Japan (11–13), and the Global Initiative on Sharing All Influenza Data (GISAID) database reports more than 1,100 amino acid changes in the S protein (14, 15).An important question for vaccine development is whether the authentic virus, under the selective pressure of the polyclonal immune response in convalescent or vaccinated people, can evolve to fully escape immunity and antibody treatment. To address this question, we incubated the authentic SARS-CoV-2 wild-type (WT) virus for more than 90 d in the presence of a potent neutralizing plasma.     

Engineered SARS-CoV-2 receptor binding domain improves manufacturability in yeast and immunogenicity in mice

Global containment of COVID-19 still requires accessible and affordable vaccines for low- and middle-income countries (LMICs). Recently approved vaccines provide needed interventions, albeit at prices that may limit their global access. Subunit vaccines based on recombinant proteins are suited for large-volume microbial manufacturing to yield billions of doses annually, minimizing their manufacturing cost. These types of vaccines are well-established, proven interventions with multiple safe and efficacious commercial examples. Many vaccine candidates of this type for SARS-CoV-2 rely on sequences containing the receptor-binding domain (RBD), which mediates viral entry to cells via ACE2. Here we report an engineered sequence variant of RBD that exhibits high-yield manufacturability, high-affinity binding to ACE2, and enhanced immunogenicity after a single dose in mice compared to the Wuhan-Hu-1 variant used in current vaccines. Antibodies raised against the engineered protein exhibited heterotypic binding to the RBD from two recently reported SARS-CoV-2 variants of concern (501Y.V1/V2). Presentation of the engineered RBD on a designed virus-like particle (VLP) also reduced weight loss in hamsters upon viral challenge.

Prevention of COVID-19 on a global scale will require >10 billion doses of vaccines for SARS-CoV-2; most of which are needed in low- and middle-income countries (LMICs) (1). To ensure adequate supply and global access, vaccine manufacturers must select highly immunogenic vaccine antigens that offer broad protection against emerging variants and are compatible with large-volume production in existing manufacturing facilities (2, 3). Vaccines using mRNA have established the efficacy of vaccines for SARS-CoV-2 based on full-length trimeric spike (S) protein (4, 5). Recombinant S protein produced in mammalian or insect cells has also shown immunogenicity and efficacy in nonhuman primates (6). Protein-based vaccines hold promise for large-volume, low-cost production, and are safe and efficacious (7–9). The receptor-binding domain (RBD) subunit, which mediates viral entry to cells via ACE2 (10–12), has emerged as an important alternative antigen (13). Antibodies to RBD account for most of the neutralizing activity elicited in natural infections, and several potent monoclonal antibodies have been discovered from convalescent patients (14, 15). A His-tagged SARS-CoV-2 RBD construct based on SARS-CoV-2 Wuhan-Hu-1 and produced in insect cells has elicited neutralizing antibodies in mice and protective immunity in nonhuman primates (16). Similar tagged constructs have also been adapted for production in yeast like Komagataella phaffii (Pichia pastoris) (17, 18), establishing the RBD domain as a prominent candidate for large-volume manufacturing of COVID-19 vaccines.Despite its significance for low-cost vaccine candidates, recombinant RBD based on the original SARS-CoV-2 clade 19A sequence has shown limited immunogenicity to date. Reported candidates would require as many as three doses or large doses to elicit strong neutralizing antibody responses in mice when formulated with adjuvants (16, 18). Increasing the number of doses or amounts required could limit its benefits for affordable and accessible vaccines. An engineered design for the RBD, therefore, could enhance the potency of many subunit-based vaccine candidates using this domain.     

Targeting stem-loop 1 of the SARS-CoV-2 5′ UTR to suppress viral translation and Nsp1 evasion

SARS-CoV-2 is a highly pathogenic virus that evades antiviral immunity by interfering with host protein synthesis, mRNA stability, and protein trafficking. The SARS-CoV-2 nonstructural protein 1 (Nsp1) uses its C-terminal domain to block the messenger RNA (mRNA) entry channel of the 40S ribosome to inhibit host protein synthesis. However, how SARS-CoV-2 circumvents Nsp1-mediated suppression for viral protein synthesis and if the mechanism can be targeted therapeutically remain unclear. Here, we show that N- and C-terminal domains of Nsp1 coordinate to drive a tuned ratio of viral to host translation, likely to maintain a certain level of host fitness while maximizing replication. We reveal that the stem-loop 1 (SL1) region of the SARS-CoV-2 5′ untranslated region (5′ UTR) is necessary and sufficient to evade Nsp1-mediated translational suppression. Targeting SL1 with locked nucleic acid antisense oligonucleotides inhibits viral translation and makes SARS-CoV-2 5′ UTR vulnerable to Nsp1 suppression, hindering viral replication in vitro at a nanomolar concentration, as well as providing protection against SARS-CoV-2–induced lethality in transgenic mice expressing human ACE2. Thus, SL1 allows Nsp1 to switch infected cells from host to SARS-CoV-2 translation, presenting a therapeutic target against COVID-19 that is conserved among immune-evasive variants. This unique strategy of unleashing a virus’ own virulence mechanism against itself could force a critical trade-off between drug resistance and pathogenicity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the infectious disease COVID-19, is a highly contagious and deadly virus with fast person-to-person transmission and potent pathogenicity (1, 2). It is an enveloped, single-stranded betacoronavirus that contains a positive-sense RNA genome of about 29.9 kb (3–5). The SARS-CoV-2 genome codes for two large overlapping open reading frames (ORF1a and ORF1b) and a variety of structural and nonstructural accessory proteins (6). Upon infection, the polyproteins ORF1a and ORF1b are synthesized by host machinery and proteolytically cleaved into 16 mature nonstructural proteins, namely Nsp1 to Nsp16 (1, 6, 7).Nsp1 is a critical virulence factor of coronaviruses and plays key roles in suppressing host gene expression, which facilitates viral replication and immune evasion, presumably by repurposing the host translational machinery for viral production and preventing the induction of type I interferons (IFNs) (8–11). It has been shown that SARS-CoV Nsp1 effectively suppresses the translation of host messenger RNAs (mRNAs) by directly binding to the 40S small ribosomal subunit (12, 13). Recent cryoelectron microscopy (cryo-EM) structures of SARS-CoV-2 Nsp1 indeed reveal the binding of its C-terminal domain (CT) to the mRNA entry channel of the 40S subunit (14–17), which contributes to blocking translation. These structural data are further supported by experiments demonstrating that Nsp1 binding to the 40S ribosome requires an open-head conformation induced by core elongation initiation factors and that Nsp1 cannot bind to a 40S with an mRNA already occupying the entry channel (18).Besides directly inhibiting mRNA translation, Nsp1 has also been shown to reduce the available pool of host cytosolic mRNAs by both promoting their degradation and inhibiting their nuclear export (19–21). Mutants of Nsp1 that disrupt ribosome binding also abolish mRNA degradation, suggesting that the degradation is likely downstream of the Nsp1 translational block, and these two processes likely synergize to shut off host protein expression (12, 22).Previous studies on SARS-CoV and SARS-CoV-2 have implicated stem-loop 1 (SL1) in the leader region of the 5′ untranslated region (5′ UTR) in protecting the virus against Nsp1-mediated mRNA translation inhibition (9, 17, 22, 23). However, how SARS-CoV-2 overcomes Nsp1-mediated translation suppression for its replication and whether this mechanism can be targeted for therapeutic intervention remain open questions. Here, we show that SARS-CoV-2 depends on SL1 to escape Nsp1 suppression to effectively switch the translational machinery from synthesizing host proteins to making viral proteins, and that both the CT and the N-terminal domain (NT) are required for the transition from host to viral translation. The latter is supported by complementary experiments in a study released while this paper was in preparation (24). We further show that SL1 can be targeted by locked nucleic acid (LNA) antisense oligonucleotides to prevent the SARS-CoV-2 5′ UTR from evading its own translational suppression to potently inhibit viral replication.     

SARS-CoV-2 infection of airway cells causes intense viral and cell shedding,two spreading mechanisms affected by IL-13

Muco-obstructive lung diseases are typically associated with high risks of COVID-19 severity; however, allergic asthma showed reduced susceptibility. To investigate viral spread, primary human airway epithelial (HAE) cell cultures were infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and host–virus interactions were examined via electron microscopy, immunohistochemistry, RNA in situ hybridization, and gene expression analyses. In HAE cell cultures, angiotensin-converting enzyme 2 (ACE2) expression governed cell tropism and viral load and was up-regulated by infection. Electron microscopy identified intense viral egress from infected ciliated cells and severe cytopathogenesis, culminating in the shedding of ciliated cells packed with virions, providing a large viral reservoir for spread and transmission. Intracellular stores of MUC5AC, a major airway mucin involved in asthma, were rapidly depleted, likely to trap viruses. To mimic asthmatic airways, HAE cells were treated with interleukin-13 (IL-13), which reduced viral titers, viral messenger RNA, and cell shedding, and significantly diminished the number of infected cells. Although mucus hyperproduction played a shielding role, IL-13–treated cells maintained a degree of protection despite the removal of mucus. Using Gene Expression Omnibus databases, bulk RNA-sequencing analyses revealed that IL-13 up-regulated genes controlling glycoprotein synthesis, ion transport, and antiviral processes (albeit not the typical interferon-induced genes) and down-regulated genes involved in cilial function and ribosomal processing. More precisely, we showed that IL-13 reduced ACE2 expression, intracellular viral load, and cell-to-cell transmission while increasing the cilial keratan sulfate coating. In conclusion, intense viral and cell shedding caused by SARS-CoV-2 infection was attenuated by IL-13, which affected viral entry, replication, and spread.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing COVID-19, is the third coronavirus outbreak affecting the human population since the beginning of the 21st century and was preceded by SARS-CoV in 2002 and Middle East respiratory syndrome (MERS)–CoV in 2012 (1–3). More than 2 y after the initial outbreak, SARS-CoV-2 continues to be actively transmitted in the human population and has already killed >5 million people. The clinical symptoms of COVID-19 vary greatly between infected individuals, ranging from asymptomatic disease to severe respiratory illness and even death in nearly 2% of cases (4, 5). Despite similarities in transmission, infection, and clinical symptoms with other human coronaviruses, the elevated transmissibility and global burden posed by SARS-CoV-2 warrant further investigation into its tropism, pathogenesis, and spreading mechanism.SARS-CoV-2 utilizes the angiotensin-converting enzyme 2 (ACE2) receptor and a transmembrane serine protease (TMPRSS2) to enter host cells, and both proteins are coexpressed in respiratory epithelia (6, 7). ACE2 expression has been detected in ciliated and goblet cells, two important cell types involved in airway clearance (8, 9). Using human airway epithelial (HAE) cell models, we have shown that SARS-CoV-2 efficiently infected cells from the upper airways, suggesting that the nose and large airways are preferred sites for viral transmission and replication (10, 11). Furthermore, scanning electron microscopy (SEM) confirmed direct interactions between SARS-CoV-2 viruses and ciliated epithelial cells (12).In the lungs, ciliated cells coordinate the movement of secretions to clear inhaled particles, a process referred to as mucociliary transport (MCT). Thus, viral infections targeting this particular cell type can affect airway clearance. Tropism for ciliated cells has been reported for other respiratory viruses (e.g., respiratory syncytial virus, influenza, and other coronaviruses), which was associated with cell shedding (13–15). Another important player in viral infection is the glycocalyx coating the periciliary (PCL) region to provide a barrier while facilitating cilia beating (16, 17). In response to infection, goblet cells increase the secretion of mucins, the large polymeric glycoproteins responsible for the viscoelastic properties of mucus (18, 19). MUC5AC, a major gel-forming mucin expressed in the lungs, is secreted in small amounts in healthy individuals and is up-regulated during respiratory infections, suggesting a protective role against pathogens (20–23). In diseases such as chronic obstructive pulmonary disease (COPD) and asthma, MUC5AC is up-regulated, and has been associated with reduced MCT (24–26). COPD patients infected with SARS-CoV-2 are at higher risk of severe clinical outcomes, but, for asthmatic patients, the data are more contradictory (27–31). The variability within the asthmatic population may originate from pathophysiologic differences based on Th2-low (nonallergic) and Th2-high (allergic) inflammatory profiles, with allergic asthma being protective (32, 33). IL-13, a type 2 cytokine associated with allergic asthma, has been shown to increase MUC5AC secretion (34, 35) and down-regulate ACE2 expression (30, 36), and the effects of IL-13 on SARS-CoV-2 infection are only beginning to be explored (37).In this study, we infected HAE cell cultures with SARS-CoV-2 and examined the fate of infected airway cells using fluorescent labeling, combined with SEM and transmission electron microscopy (TEM). These approaches characterized the fate of infected cells by examining the mode of virion cellular release and the detachment of infected cells. In addition, we investigated whether IL-13 protects against viral spread. The effects of IL-13 on airway cultures were determined by measuring viral titers, viral gene replication, and epithelial cell damage. Mucus hyperproduction provided a physical barrier; however, after removing mucus either by cell washing or using MUC5AC knockout (KO) cells, lower viral loads were maintained in IL-13–treated cells. To investigate how IL-13 modified host antiviral responses, the effects of IL-13 administration on gene expression in HAE cultures were analyzed from three independent bulk RNA-sequencing studies available on the Gene Expression Omnibus (GEO) database. These analyses revealed that critical processes for viral entry, replication, and spread, pertinent to asthma and COVID-19 severity, were affected by IL-13 treatment.