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Vaccines against SARS-CoV-2 that induce mucosal immunity capable of preventing infection and disease remain urgently needed. In this study, we demonstrate the efficacy of Bordetella colonization factor A (BcfA), a novel bacteria-derived protein adjuvant, in SARS-CoV-2 spike-based prime-pull immunizations. We show that i.m. priming of mice with an aluminum hydroxide– and BcfA-adjuvanted spike subunit vaccine, followed by a BcfA-adjuvanted mucosal booster, generated Th17-polarized CD4+ tissue-resident memory T cells and neutralizing Abs. Immunization with this heterologous vaccine prevented weight loss following challenge with mouse-adapted SARS-CoV-2 (MA10) and reduced viral replication in the respiratory tract. Histopathology showed a strong leukocyte and polymorphonuclear cell infiltrate without epithelial damage in mice immunized with BcfA-containing vaccines. Importantly, neutralizing Abs and tissue-resident memory T cells were maintained until 3 mo postbooster. Viral load in the nose of mice challenged with the MA10 virus at this time point was significantly reduced compared with naive challenged mice and mice immunized with an aluminum hydroxide–adjuvanted vaccine. We show that vaccines adjuvanted with alum and BcfA, delivered through a heterologous prime-pull regimen, provide sustained protection against SARS-CoV-2 infection.

Influenza virus activates cellular inflammasome pathways, which can be either beneficial or detrimental to infection outcomes. Here, we investigated the role of the inflammasome-activated pore-forming protein gasdermin D (GSDMD) during infection. Ablation of GSDMD in knockout (KO) mice significantly attenuated virus-induced weight loss, lung dysfunction, lung histopathology, and mortality compared with wild type (WT) mice, despite similar viral loads. Infected GSDMD KO mice exhibited decreased inflammatory gene signatures revealed by lung transcriptomics, which also implicated a diminished neutrophil response. Importantly, neutrophil depletion in infected WT mice recapitulated the reduced mortality and lung inflammation observed in GSDMD KO animals, while having no additional protective effects in GSDMD KOs. These findings reveal a new function for GSDMD in promoting lung neutrophil responses that amplify influenza virus-induced inflammation and pathogenesis. Targeting the GSDMD/neutrophil axis may provide a new therapeutic avenue for treating severe influenza.

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the main target for neutralizing antibodies (NAbs). The S protein trimer is anchored in the virion membrane in its prefusion (preS) but metastable form. The preS protein has been stabilized by introducing two or six proline substitutions, to generate stabilized, soluble 2P or HexaPro (6P) preS proteins. Currently, it is not known which form is the most immunogenic. Here, we generated recombinant vesicular stomatitis virus (rVSV) expressing preS-2P, preS-HexaPro, and native full-length S, and compared their immunogenicity in mice and hamsters. The rVSV-preS-HexaPro produced and secreted significantly more preS protein compared to rVSV-preS-2P. Importantly, rVSV-preS-HexaPro triggered significantly more preS-specific serum IgG antibody than rVSV-preS-2P in both mice and hamsters. Antibodies induced by preS-HexaPro neutralized the B.1.1.7, B.1.351, P.1, B.1.427, and B.1.617.2 variants approximately two to four times better than those induced by preS-2P. Furthermore, preS-HexaPro induced a more robust Th1-biased cellular immune response than preS-2P. A single dose (10 4 pfu) immunization with rVSV-preS-HexaPro and rVSV-preS-2P provided complete protection against challenge with mouse-adapted SARS-CoV-2 and B.1.617.2 variant, whereas rVSV-S only conferred partial protection. When the immunization dose was lowered to 10 3 pfu, rVSV-preS-HexaPro induced two- to sixfold higher antibody responses than rVSV-preS-2P in hamsters. In addition, rVSV-preS-HexaPro conferred 70% protection against lung infection whereas only 30% protection was observed in the rVSV-preS-2P. Collectively, our data demonstrate that both preS-2P and preS-HexaPro are highly efficacious but preS-HexaPro is more immunogenic and protective, highlighting the advantages of using preS-HexaPro in the next generation of SARS-CoV-2 vaccines.

Cardiovascular sequelae of severe acute respiratory syndrome (SARS) coronavirus-2 (CoV-2) disease 2019 (COVID-19) contribute to the complications of the disease. One potential complication is lung vascular remodeling, but the exact cause is still unknown. We hypothesized that endothelial TLR3 insufficiency contributes to lung vascular remodeling induced by SARS-CoV-2. In the lungs of COVID-19 patients and SARS-CoV-2 infected Syrian hamsters, we discovered thickening of the pulmonary artery media and microvascular rarefaction, which were associated with decreased TLR3 expression in lung tissue and pulmonary artery endothelial cells (ECs).In vitro, SARS-CoV-2 infection reduced endothelial TLR3 expression. Following infection with mouse-adapted (MA) SARS-CoV-2, TLR3 knockout mice displayed heightened pulmonary artery remodeling and endothelial apoptosis. Treatment with the TLR3 agonist polyinosinic:polycytidylic acid reduced lung tissue damage, lung vascular remodeling, and endothelial apoptosis associated with MA SARS-CoV-2 infection. In conclusion, repression of endothelial TLR3 is a potential mechanism of SARS-CoV-2 infection associated lung vascular remodeling and enhancing TLR3 signaling is a potential strategy for treatment.

With the rapid increase in SARS-CoV-2 cases in children, a safe and effective vaccine for this population is urgently needed. The MMR (measles/mumps/rubella) vaccine has been one of the safest and most effective human vaccines used in infants and children since the 1960s. Here, we developed live attenuated recombinant mumps virus (rMuV)–based SARS-CoV-2 vaccine candidates using the MuV Jeryl Lynn (JL2) vaccine strain backbone. The soluble prefusion SARS-CoV-2 spike protein (preS) gene, stablized by two prolines (preS-2P) or six prolines (preS-6P), was inserted into the MuV genome at the P–M or F–SH gene junctions in the MuV genome. preS-6P was more efficiently expressed than preS-2P, and preS-6P expression from the P–M gene junction was more efficient than from the F–SH gene junction. In mice, the rMuV-preS-6P vaccine was more immunogenic than the rMuV-preS-2P vaccine, eliciting stronger neutralizing antibodies and mucosal immunity. Sera raised in response to the rMuV-preS-6P vaccine neutralized SARS-CoV-2 variants of concern, including the Delta variant equivalently. Intranasal and/or subcutaneous immunization of IFNAR1 −/− mice and golden Syrian hamsters with the rMuV-preS-6P vaccine induced high levels of neutralizing antibodies, mucosal immunoglobulin A antibody, and T cell immune responses, and were completely protected from challenge by both SARS-CoV-2 USA-WA1/2020 and Delta variants. Therefore, rMuV-preS-6P is a highly promising COVID-19 vaccine candidate, warranting further development as a tetravalent MMR vaccine, which may include protection against SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS–CoV-2) is a worldwide health concern, and new treatment strategies are needed. Targeting inflammatory innate immunity pathways holds therapeutic promise, but effective molecular targets remain elusive. Here, we show that human caspase-4 (CASP4) and its mouse homolog, caspase-11 (CASP11), are up-regulated in SARS–CoV-2 infections and that CASP4 expression correlates with severity of SARS–CoV-2 infection in humans. SARS–CoV-2–infected Casp11−/− mice were protected from severe weight loss and lung pathology, including blood vessel damage, compared to wild-type (WT) mice and mice lacking the caspase downstream effector gasdermin-D (Gsdmd−/−). Notably, viral titers were similar regardless of CASP11 knockout. Global transcriptomics of SARS–CoV-2–infected WT, Casp11−/−, and Gsdmd−/− lungs identified restrained expression of inflammatory molecules and altered neutrophil gene signatures in Casp11−/− mice. We confirmed that protein levels of inflammatory mediators interleukin (IL)-1β, IL-6, and CXCL1, as well as neutrophil functions, were reduced in Casp11−/− lungs. Additionally, Casp11−/− lungs accumulated less von Willebrand factor, a marker for endothelial damage, but expressed more Kruppel-Like Factor 2, a transcription factor that maintains vascular integrity. Overall, our results demonstrate that CASP4/11 promotes detrimental SARS–CoV-2–induced inflammation and coagulopathy, largely independently of GSDMD, identifying CASP4/11 as a promising drug target for treatment and prevention of severe COVID-19.

Vaccines against SARS-CoV-2 that induce mucosal immunity capable of preventing infection and disease remain urgently needed. We show that intramuscular priming of mice with an alum and BcfA-adjuvanted Spike subunit vaccine, followed by a BcfA-adjuvanted mucosal booster, generated Th17 polarized tissue resident CD4+ T cells, and mucosal and serum antibodies. The serum antibodies efficiently neutralized SARS-CoV-2 and its Delta variant, suggesting cross-protection against a recent variant of concern (VOC). Immunization with this heterologous vaccine prevented weight loss following challenge with mouse-adapted SARS-CoV-2 and reduced viral replication in the nose and lungs. Histopathology showed a strong leukocyte and polymorphonuclear (PMN) cell infiltrate without epithelial damage in mice immunized with BcfA-containing vaccines. In contrast, viral load was not reduced in the upper respiratory tract of IL-17 knockout mice immunized with the same formulation, suggesting that the Th17 polarized T cell responses are critical for protection. We show that vaccines adjuvanted with alum and BcfA, delivered through a heterologous prime-pull regimen, protect against SARS-CoV-2 infection without causing enhanced respiratory disease.SIGNIFICANCEThere remains a need for SARS CoV-2 booster vaccines that generate mucosal immunity and prevent transmission. We show that systemic priming followed by a mucosal booster with a BcfA-adjuvanted subunit vaccine generates neutralizing antibodies and Th17 polarized systemic and tissue-resident immune responses that provide sterilizing immunity against wildtype SARS CoV-2, and a variant of concern. Importantly, in contrast to alum alone, the addition of BcfA prevents respiratory pathology. These results suggest that a BcfA-adjuvanted mucosal booster may elicit mucosal immunity in individuals previously immunized systemically with approved vaccines. This foundational study in mice sets the stage for testing our vaccine regimen in larger animal models as a booster vaccine.

Interferon-induced transmembrane protein 3 (IFITM3) is a host antiviral protein that alters cell membranes to block fusion of viruses. Published reports have identified conflicting pro- and antiviral effects of IFITM3 on SARS-CoV-2 in cultured cells, and its impact on viral pathogenesis in vivo remains unclear. Here, we show that IFITM3 knockout (KO) mice infected with mouse-adapted SARS-CoV-2 experienced extreme weight loss and lethality, while wild type (WT) mice lost minimal weight and recovered. KO mice had higher lung viral titers and increases in lung inflammatory cytokine levels, CD45-positive immune cell infiltration, and histopathology, compared to WT mice. Mechanistically, we observed disseminated viral antigen staining throughout the lung tissue and pulmonary vasculature in KO mice, while staining was observed in confined regions in WT lungs. Global transcriptomic analysis of infected lungs identified upregulation of gene signatures associated with interferons, inflammation, and angiogenesis in KO versus WT animals, highlighting changes in lung gene expression programs that precede severe lung pathology and fatality. Corroborating the protective effect of IFITM3 in vivo, K18-hACE2/IFITM3 KO mice infected with non-adapted SARS-CoV-2 showed enhanced, rapid weight loss and early death compared to control mice. Increased heart infection was observed in both mouse models in the absence of IFITM3, indicating that IFITM3 constrains extrapulmonary dissemination of SARS-CoV-2. Our results establish IFITM3 KO mice as a new animal model for studying severe SARS-CoV-2 infection of the lung and cardiovascular system, and overall demonstrate that IFITM3 is protective in SARS-CoV-2 infections of mice.

Cardiac dysfunction is a common extrapulmonary complication of severe influenza virus infection. Prevailing models propose that influenza-associated heart dysfunction is indirectly triggered by cytokine mediated cardiotoxicity downstream of the inflamed lung, rather than by direct infection of cardiac tissue. To test the etiology of cardiac dysfunction resulting from influenza virus infection, we generated a novel recombinant H1N1 influenza A virus that was attenuated in cardiomyocytes by incorporation of target sequences for miRNAs expressed specifically in that cell type (miR133b and miR206). Compared with control virus, mice infected with the miR-targeted virus had significantly reduced heart viral titers, confirming cardiac attenuation of viral replication. The miR-targeted virus, however, was fully replicative and inflammatory in lungs when compared to control virus, and induced similar systemic weight loss. The miR-targeted virus induced considerably lower levels of cardiac arrhythmia, fibrosis, and inflammation, compared with control virus, in mice lacking interferon induced transmembrane protein 3 (IFITM3), which serve as the only available model for severe influenza-associated cardiac pathology. We conclude that robust replication of virus in the heart is required for pathology even when lung inflammation is severe. Indeed, we show that human stem cell-derived cardiomyocytes are susceptible to influenza virus infection. This work establishes a fundamental new paradigm in which influenza virus damages the heart through direct infection of cardiomyocytes.

The current pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to dramatic economic and health burdens. Although the worldwide SARS-CoV-2 vaccination campaign has begun, exploration of other vaccine candidates is needed due to uncertainties with the current approved vaccines, such as durability of protection, cross-protection against variant strains, and costs of long-term production and storage. In this study, we developed a methyltransferase-defective recombinant vesicular stomatitis virus (mtdVSV)-based SARS-CoV-2 vaccine candidate. We generated mtdVSVs expressing SARS-CoV-2 full-length spike (S) protein, S1, or its receptor-binding domain (RBD). All of these recombinant viruses grew to high titers in mammalian cells despite high attenuation in cell culture. The SARS-CoV-2 S protein and its truncations were highly expressed by the mtdVSV vector. These mtdVSV-based vaccine candidates were completely attenuated in both immunocompetent and immunocompromised mice. Among these constructs, mtdVSV-S induced high levels of SARS-CoV-2-specific neutralizing antibodies (NAbs) and Th1-biased T-cell immune responses in mice. In Syrian golden hamsters, the serum levels of SARS-CoV-2-specific NAbs triggered by mtdVSV-S were higher than the levels of NAbs in convalescent plasma from recovered COVID-19 patients. In addition, hamsters immunized with mtdVSV-S were completely protected against SARS-CoV-2 replication in lung and nasal turbinate tissues, cytokine storm, and lung pathology. Collectively, our data demonstrate that mtdVSV expressing SARS-CoV-2 S protein is a safe and highly efficacious vaccine candidate against SARS-CoV-2 infection. IMPORTANCE Viral mRNA cap methyltransferase (MTase) is essential for mRNA stability, protein translation, and innate immune evasion. Thus, viral mRNA cap MTase activity is an excellent target for development of live attenuated or live vectored vaccine candidates. Here, we developed a panel of MTase-defective recombinant vesicular stomatitis virus (mtdVSV)-based SARS-CoV-2 vaccine candidates expressing full-length S, S1, or several versions of the RBD. These mtdVSV-based vaccine candidates grew to high titers in cell culture and were completely attenuated in both immunocompetent and immunocompromised mice. Among these vaccine candidates, mtdVSV-S induces high levels of SARS-CoV-2-specific neutralizing antibodies (Nabs) and Th1-biased immune responses in mice. Syrian golden hamsters immunized with mtdVSV-S triggered SARS-CoV-2-specific NAbs at higher levels than those in convalescent plasma from recovered COVID-19 patients. Furthermore, hamsters immunized with mtdVSV-S were completely protected against SARS-CoV-2 challenge. Thus, mtdVSV is a safe and highly effective vector to deliver SARS-CoV-2 vaccine.

SARS-CoV-2 is a worldwide health concern, and new treatment strategies are needed 1. Targeting inflammatory innate immunity pathways holds therapeutic promise, but effective molecular targets remain elusive. Here, we show that human caspase-4 (CASP4), and its mouse homologue, caspase-11 (CASP11), are upregulated in SARS-CoV-2 infections, and that CASP4 expression correlates with severity of SARS-CoV-2 infection in humans. SARS-CoV-2-infected Casp11-/- mice were protected from severe weight loss and lung pathology, including blood vessel damage, compared to wild-type (WT) and gasdermin-D knock out (Gsdmd-/-) mice. GSDMD is a downstream effector of CASP11 and CASP1. Notably, viral titers were similar in the three genotypes. Global transcriptomics of SARS-CoV-2-infected WT, Casp11-/- and Gsdmd-/- lungs identified restrained expression of inflammatory molecules and altered neutrophil gene signatures in Casp11-/- mice. We confirmed that protein levels of inflammatory mediators IL-1β, IL6, and CXCL1, and neutrophil functions, were reduced in Casp11-/- lungs. Additionally, Casp11-/- lungs accumulated less von Willebrand factor, a marker for endothelial damage, but expressed more Kruppel-Like Factor 2, a transcription factor that maintains vascular integrity. Overall, our results demonstrate that CASP4/11, promotes detrimental SARS-CoV-2-associated inflammation and coagulopathy, largely independently of GSDMD, identifying CASP4/11 as a promising drug target for treatment and prevention of severe COVID-19.

The SERINC (serine incorporator) proteins are host restriction factors that inhibit infection by HIV through their incorporation into virions. Here, we found that SERINC3 and SERINC5 exhibited additional antiviral activities by enhancing the expression of genes encoding type I interferons (IFNs) and nuclear factor κB (NF-κB) signaling. SERINC5 interacted with the outer mitochondrial membrane protein MAVS (mitochondrial antiviral signaling) and the E3 ubiquitin ligase and adaptor protein TRAF6, resulting in MAVS aggregation and polyubiquitylation of TRAF6. Knockdown of SERINC5 in target cells increased single-round HIV-1 infectivity, as well as infection by recombinant vesicular stomatitis virus (rVSV) bearing VSV-G or Ebola virus (EBOV) glycoproteins. Infection by an endemic Asian strain of Zika virus (ZIKV), FSS13025, was also enhanced by SERINC5 knockdown, suggesting that SERINC5 has direct antiviral activities in host cells in addition to the indirect inhibition mediated by its incorporation into virions. Further experiments suggested that the antiviral activity of SERINC5 was type I IFN–dependent. Together, these results highlight a previously uncharacterized function of SERINC proteins in promoting NF-κB inflammatory signaling and type I IFN production, thus contributing to its antiviral activities.

The authors have withdrawn their manuscript. While attempting to reproduce the data on the alternative splicing of exon 2 of U2AF2, they observed that the proposed splicing mechanism could not give rise to a functional U2AF2 protein. In addition, they observed evidence of manipulation in the electropherogram of the splicing junction between exons 1 and 3 and in the primary data on which this electropherogram was based, which were deposited in Mendeley by the first author. These observations raise questions on the integrity of the reported results. In light of this information, the authors have no confidence in the key findings of the paper, and therefore, do not wish it to be cited. If you have any questions, please contact the corresponding author.

Viral infection of the heart is a common but underappreciated cause of heart failure. Viruses can cause direct cardiac damage by lysing infected cardiomyocytes. Inflammatory immune responses that limit viral replication can also indirectly cause damage during infection, making regulatory factors that fine-tune these responses particularly important. Identifying and understanding these factors that regulate cardiac immune responses during infection will be essential for developing targeted treatments for virus-associated heart failure. Our laboratory has discovered Brain Expressed X-linked protein 1 (BEX1) as a novel stress-regulated pro-inflammatory factor in the heart. Here we report that BEX1 plays a cardioprotective role in the heart during viral infection. Specifically, we adopted genetic gain- and loss-of-function strategies to modulate BEX1 expression in the heart in the context of coxsackievirus B3 (CVB3)-induced cardiomyopathy and found that BEX1 limits viral replication in cardiomyocytes. Interestingly, despite the greater viral load observed in mice lacking BEX1, inflammatory immune cell recruitment in the mouse heart was profoundly impaired in the absence of BEX1. Overall, the absence of BEX1 accelerated CVB3-driven heart failure and pathologic heart remodeling. This result suggests that limiting inflammatory cell recruitment has detrimental consequences for the heart during viral infections. Conversely, transgenic mice overexpressing BEX1 in cardiomyocytes revealed the efficacy of BEX1 for counteracting viral replication in the heart in vivo. We also found that BEX1 retains its antiviral role in isolated cells. Indeed, BEX1 was necessary and sufficient to counteract viral replication in both isolated primary cardiomyocytes and mouse embryonic fibroblasts suggesting a broader applicability of BEX1 as antiviral agent that extended to viruses other than CVB3, including Influenza A and Sendai virus. Mechanistically, BEX1 regulated interferon beta (IFN-β) expression in infected cells. Overall, our study suggests a multifaceted role of BEX1 in the cardiac antiviral immune response.

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 is an enveloped virus responsible for the COVID-19 respiratory disease pandemic. While induction of adaptive antiviral immunity via vaccination holds promise for combatting the pandemic, the emergence of new potentially more transmissible and vaccine-resistant variants of SARS-CoV-2 is an ever-present threat. Thus, it remains essential to better understand innate immune mechanisms that are active against the virus. One component of the innate immune system with broad anti-pathogen, including antiviral, activity is a group of cationic immune peptides termed defensins. The defensins’ ability to neutralize enveloped and non-enveloped viruses and to inactivate numerous bacterial toxins correlate with their ability to promote the unfolding of thermodynamically pliable proteins. Accordingly, we found that human neutrophil a-defensin HNP1 and retrocyclin RC-101 destabilize SARS-CoV-2 Spike protein and interfere with Spike-mediated membrane fusion and SARS-CoV-2 infection in cell culture. We show that HNP1 binds to Spike with submicromolar affinity. Although binding of HNP1 to serum albumin is more than 20-fold weaker, serum reduces the anti-SARS-CoV-2 activity of HNP1. At high concentrations of HNP1, its ability to inactivate the virus was preserved even in the presence of serum. These results suggest that specific a- and 8-defensins may be valuable tools in developing SARS-CoV-2 infection prevention strategies.

SummarySevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leads to NF-κB activation and induction of pro-inflammatory cytokines, though the underlying mechanism for this activation is not fully understood. Our results reveal that the SARS-CoV-2 Nsp14 protein contributes to the viral activation of NF-κB signaling. Nsp14 caused the nuclear translocation of NF-κB p65. Nsp14 induced the upregulation of IL-6 and IL-8, which also occurred in SARS-CoV-2 infected cells. IL-8 upregulation was further confirmed in lung tissue samples from COVID-19 patients. A previous proteomic screen identified the putative interaction of Nsp14 with host Inosine-5’-monophosphate dehydrogenase 2 (IMPDH2) protein, which is known to regulate NF-κB signaling. We confirmed the Nsp14-IMPDH2 protein interaction and found that IMPDH2 knockdown or chemical inhibition using ribavirin (RIB) and mycophenolic acid (MPA) abolishes Nsp14-mediated NF-κB activation and cytokine induction. Furthermore, IMDPH2 inhibitors (RIB, MPA) efficiently blocked SARS-CoV-2 infection, indicating that IMDPH2, and possibly NF-κB signaling, is beneficial to viral replication. Overall, our results identify a novel role of SARS-CoV-2 Nsp14 in causing the activation of NF-κB.

SummaryCells acquire fatty acids from dietary sources or via de novo palmitate production by fatty acid synthase (FASN). Although most cells express FASN at low levels, it is upregulated in cancers and during replication of many viruses. The precise role of FASN in disease pathogenesis is poorly understood, and whether de novo fatty acid synthesis contributes to host or viral protein acylation has been traditionally difficult to study. We describe a cell permeable, click-chemistry compatible alkynyl-acetate analog (Alk-4) that functions as a reporter of FASN-dependent protein acylation. In a FASN-dependent manner, Alk-4 selectively labeled the cellular protein interferon-induced transmembrane protein 3 (IFITM3) at its palmitoylation sites, and the HIV-1 matrix protein at its myristoylation site. Alk-4 metabolic labeling also enabled biotin-based purification and identification of more than 200 FASN-dependent acylated cellular proteins. Thus, Alk-4 is a useful bioorthogonal tool to selectively probe FASN-mediated protein acylation in normal and diseased states.

Infection by SARS-CoV-2 generally causes mild symptoms but can lead to severe disease and death in certain populations, including the immunocompromised. Drug repurposing efforts are underway to identify compounds that interfere with SARS-CoV-2 replication or the immunopathology it can elicit. Rapamycin is among those being currently tested in clinical trials for impacts on COVID-19 severity. While rapamycin and rapamycin analogs (rapalogs) are FDA-approved for use as mTOR inhibitors in multiple clinical settings, including cancer, we previously found that rapamycin can increase the susceptibility of cells to infection by Influenza A virus. In this study, we tested the impact of rapalogs on cellular susceptibility to SARS-CoV-2 infection. We report that rapamycin and rapalogs increased SARS-CoV-2 titers in human cervical epithelial and lung epithelial cell lines to different extents, and a similar pattern of enhancement was observed using pseudovirus incorporating viral fusion proteins from SARS-CoV-2, SARS-CoV, MERS, and Influenza A Virus. Rapalogs also promoted cell entry driven by SARS-CoV-2 Spike in nasal cells and primary small airway cells, representing proximal and distal ends of the human respiratory tract, respectively. Interestingly, cell entry enhancement by the rapalog ridaforolimus was cell type-dependent, revealing a previously unrecognized functional divergence between rapalogs. The differential activity of rapalogs was associated with their capacity to induce the degradation of interferon-inducible transmembrane (IFITM) proteins, restriction factors that broadly inhibit virus infection. Our findings will spur the development of mTOR inhibitors that do not suppress the cells first line of antiviral defense. SignificanceDue to a lack of effective antivirals available to combat SARS-CoV-2 infection and the disease it causes (COVID-19), existing drugs that are clinically-approved for use in humans are being considered for deployment during this protracted pandemic. Rapamycin is an immunosuppressant that is administered to humans suffering from cancer, autoimmunity, atherosclerosis, and organ transplant rejection. While generally recognized for its inhibitory effects on the adaptive immune response, we previously showed that rapamycin can also impair intrinsic immunity, the immune system present within all cells of the body. Here, we show that rapamycin and rapamycin analogs (rapalogs) promote the first step of the SARS-CoV-2 infection cycle--entry into cells--by inducing the destruction of antiviral proteins that stand guard during virus invasion.

Significance Measles virus (MeV) vaccine is one of the safest and most efficient vaccines with a track record in children. Here, we generated a panel of rMeV-based vaccines with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S antigens inserted near 3′ of the MeV genome. The rMeV expressing a soluble stabilized, prefusion spike (preS) is much more potent in triggering SARS-CoV-2–specific neutralizing antibody than rMeV-based full-length S vaccine candidate. A single dose of rMeV-preS is sufficient to induce high levels of SARS-CoV-2 antibody in animals. Furthermore, rMeV-preS induces high levels of Th1-biased immunity. Hamsters immunized with rMeV-preS were completely protected against SARS-CoV-2 challenge. Our results demonstrate rMeV-preS is a safe and highly efficacious bivalent vaccine candidate for SARS-CoV-2 and MeV., The current pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlights an urgent need to develop a safe, efficacious, and durable vaccine. Using a measles virus (rMeV) vaccine strain as the backbone, we developed a series of recombinant attenuated vaccine candidates expressing various forms of the SARS-CoV-2 spike (S) protein and its receptor binding domain (RBD) and evaluated their efficacy in cotton rat, IFNAR−/−mice, IFNAR−/−-hCD46 mice, and golden Syrian hamsters. We found that rMeV expressing stabilized prefusion S protein (rMeV-preS) was more potent in inducing SARS-CoV-2–specific neutralizing antibodies than rMeV expressing full-length S protein (rMeV-S), while the rMeVs expressing different lengths of RBD (rMeV-RBD) were the least potent. Animals immunized with rMeV-preS produced higher levels of neutralizing antibody than found in convalescent sera from COVID-19 patients and a strong Th1-biased T cell response. The rMeV-preS also provided complete protection of hamsters from challenge with SARS-CoV-2, preventing replication in lungs and nasal turbinates, body weight loss, cytokine storm, and lung pathology. These data demonstrate that rMeV-preS is a safe and highly efficacious vaccine candidate, supporting its further development as a SARS-CoV-2 vaccine.

Maternal infection during pregnancy is a known risk factor for offspring mental health disorders. Animal models of maternal immune activation (MIA) have implicated specific cellular and molecular etiologies of psychiatric illness, but most rely on pathogen mimetics. Here, we developed a mouse model of live H3N2 influenza A virus (IAV) infection during pregnancy that induces a robust inflammatory response but is sublethal to both dams and offspring. We observed lung inflammatory cytokine production and severely diminished weight gain in IAV-infected dams. This was accompanied by immune cell infiltration in the placenta and partial breakdown of placental integrity. However, indications of IL-17A signaling and fetal neuroinflammation, which are hallmarks of mimetic-induced MIA, were not detected. Our results suggest that mild or moderately pathogenic IAV infection during pregnancy does not inflame the developing fetal brain, and highlight the importance of live pathogen infection models for the study of MIA. HighlightsO_LIA mouse model of influenza A virus (IAV) infection during pregnancy was established C_LIO_LIModerate IAV infection induced lung inflammation and blunted weight gain in dams C_LIO_LIMaternal IAV infection caused mild pathology in the placenta without pup loss C_LIO_LIModerate gestational IAV infection did not induce fetal brain inflammation C_LIO_LIAn IAV infection severity threshold may exist for inducing fetal neuroinflammation C_LI

Rationale: Influenza A virus (IAV) and SARS-CoV-2 are both acute respiratory viruses currently circulating in the human population. Prior IAV infection enhances SARS-CoV-2 infectivity and lung pathogenesis in mice; however, underlying mechanisms and the extent that co-infection leads to involvement of other organs in disease severity remains unknown. Objective: Herein, we investigated the impact of prior IAV infection on SARS-CoV-2 pathogenesis and cardiomyocyte function. Methods and Results: IAV infection in human primary lung epithelial cells, lung fibroblasts, macrophages, cardiac fibroblasts, and hiPSC-Cardiomyocytes enhances the expression of, the SARS-CoV-2 receptor ACE2. Also, we found that IAV infection enhances levels of ACE2 in the lungs of mice and humans. Interestingly, we detected poorly glycosylated ACE2 in A549 lung cells, primary lung epithelial cells, and cardiac fibroblasts. In contrast, expression of a heavily glycosylated form of ACE2 is induced by IAV infection in cardiomyocytes. In all cell types, IAV infection enhances SARS-CoV-2 spike protein binding and viral entry. However, efficient SARS-CoV-2 replication was uniquely inhibited in cardiomyocytes. Glycosylation of ACE2 correlated with enzymatic conversion of its substrate Ang II, induction of eNOS and nitric oxide production, providing a mechanistic underpinning for restricted viral replication in cardiomyocytes. Conclusions: Our results indicate that IAV-mediated induction of ACE2 is double-edged, providing increased risk for SARS-CoV-2 coinfection in human lung epithelial cells and cardiac fibroblasts , while limiting SARS-CoV-2 replication in cardiomyocytes. We conclude that differential glycosylation of ACE2 may be a molecular determinant- not of SARS-CoV-2 infection, but of replication. Funding: The work was supported by NIH grants AI 146252 and AI 146690. Declaration of Interests: The authors declare that they have no conflict of interest. Ethics Approval Statement: All procedures were approved by the OSU IACUC.We have obtained ethics approval from the Ohio State University, The Institutional Animal Care and Use Committee (#2016A00000100-R1), Institutional Biosafety Committee (#2020R00000135) for animal work and Institutional Review Board (#2020H0222) for human subjects.

Type I IFNs orchestrate the antiviral response. Interestingly, IFNA1 and IFNB1 genes are naturally intronless. Based on previous work, the splicing factor U2 Associated Factor 65 (U2AF65), encoded by U2AF2, and pre-mRNA Processing factor 19 (Prp19) function on the Cytoplasmic Accumulation Region Elements (CAR-E), affecting the nuclear export of intronless genes. We have previously shown that the loss of IWS1 phosphorylation by AKT3, promotes the alternative RNA splicing of U2AF2, resulting in novel transcripts lacking exon 2. This exon encodes part of the Serine-Rich (RS) domain of U2AF65, which is responsible for its binding with Prp19. Here, we show that IWS1 phosphorylation and the U2AF2 RNA splicing pattern affect the nuclear export of introless mRNAs. We also demonstrate that the same axis is required for the proper function of the CAR-Es. Mechanistically, whereas both U2AF65 isoforms bind CAR-E, the recruitment of Prp19 occurs only in cells expressing phosphorylated IWS1, promoting intronless genes export. Moreover, analysis of Lung adenocarcinoma patients showed that high p-IWS1 activity correlates with the assembly of the U2AF65/Prp19 complex and export of intronless genes, in vivo. Accordingly, the expression of type I IFNs was decreased in cells deficient in IWS1 phosphorylation and the viral infection was increased. Furthermore, following infection with oncolytic virus, we observed reduced activation of p-STAT1 and expression of Interferon Stimulated Genes (ISG), in cells stimulated by shIWS1-derived supernatant, or cells treated with the pan-AKT inhibitor, MK2206. Consistently, killing curves and apoptosis assays after infection with oncolytic viruses, revealed increased susceptibility upon the loss of IWS1, with subsequent activation of Caspase-mediated death. The treatment of the lung adenocarcinoma cells with MK2206, phenocopied the loss of IWS1 phosphorylation. These data identify a novel mechanism by which the AKT/p-IWS1 axis, by hijacking the epigenetic regulation of RNA splicing and processing, contributes to the resistance to oncolytic viral infection, suggesting that combined inhibition of the splicing machinery and AKT/p-IWS1 signals would sensitize tumors to oncolytic viral treatment.

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 is a novel and highly pathogenic coronavirus and is the causative agent of the coronavirus disease 2019 (COVID-19). The high morbidity and mortality associated with COVID-19 and the lack of an approved drug or vaccine for SARS-CoV-2 underscores the urgent need for developing effective antiviral therapies. Therapeutics that target essential viral proteins are effective at controlling virus replication and spread. Coronavirus Spike glycoproteins mediate viral entry and fusion with the host cell, and thus are essential for viral replication. To enter host cells, the Spike proteins of SARS-CoV-2 and related coronavirus, SARS-CoV, bind the host angiotensin-converting enzyme 2 (ACE2) receptor through their receptor binding domains (RBDs). Here, we rationally designed a panel of ACE2-derived peptides based on the RBD-ACE2 binding interfaces of SARS-CoV-2 and SARS-CoV. Using SARS-CoV-2 and SARS-CoV Spike-pseudotyped viruses, we found that a subset of peptides inhibits Spike-mediated infection with IC50 values in the low millimolar range. We identified two peptides that bound Spike RBD in affinity precipitation assays and inhibited infection with genuine SARS-CoV-2. Moreover, these peptides inhibited the replication of a common cold causing coronavirus, which also uses ACE2 as its entry receptor. Results from the infection experiments and modeling of the peptides with Spike RBD identified a 6-amino-acid (Glu37-Gln42) ACE2 motif that is important for SARS-CoV-2 inhibition. Our work demonstrates the feasibility of inhibiting SARS-CoV-2 with peptide-based inhibitors. These findings will allow for the successful development of engineered peptides and peptidomimetic-based compounds for the treatment of COVID-19.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus known to cause epidemics resulting in predominantly symptomatic infections, which in rare cases cause long term debilitating arthritis and arthralgia. Significant progress has been made in understanding the roles of canonical RNA sensing pathways in the host recognition of CHIKV; however, less is known regarding antagonism of CHIKV by cytosolic DNA sensing pathways like that of cyclic GMP-AMP synthase (cGAS) and Stimulator of Interferon Genes (STING). With the use of cGAS or STING null cells we demonstrate that the pathway restricts CHIKV replication in fibroblasts and immune cells. We show that DNA accumulates in the cytoplasm of infected cells and that CHIKV blocks DNA dependent IFN-β transcription. This antagonism of DNA sensing is via an early autophagy-mediated degradation of cGAS and expression of the CHIKV capsid protein is sufficient to induce cGAS degradation. Furthermore, we identify an interaction of CHIKV nsP1 with STING and map the interaction to 23 residues in the cytosolic loop of the adaptor protein. This interaction stabilizes the viral protein and increases the level of palmitoylated nsP1 in cells. Together, this work supports previous publications highlighting the relevance of the cGAS-STING pathway in the early detection of (+)ssRNA viruses and provides direct evidence that CHIKV interacts with and antagonizes cGAS-STING signaling., Author summary Chikungunya virus (CHIKV) is an emerging vector-borne virus which causes outbreaks that spread within populations quickly. The virus has been shown to be highly susceptible to type-I interferon signaling and can block this antiviral pathway. No work has identified if CHIKV is able to block the induction of type-I IFN by antagonizing cytosolic DNA sensing. Here we show that CHIKV induces the accumulation of DNA in the cytoplasm of infected cells and that the virus blocks cGAS-STING dependent innate immune sensing via degradation of cGAS. This work provides the first description of CHIKV mediated inhibition of DNA dependent innate immune sensing and expands upon our previous works by identifying and characterizing another RNA virus family able to counteract cGAS-STING mediated DNA sensing.

Rapid and specific antibody testing is crucial for improved understanding, control, and treatment of COVID-19 pathogenesis. Herein, we describe and apply a rapid, sensitive, and accurate virus neutralization assay for SARS-CoV-2 antibodies. The assay is based on an HIV-1 lentiviral vector that contains a secreted intron Gaussia luciferase (Gluc) or secreted nano-luciferase reporter cassette, pseudotyped with the SARS-CoV-2 spike (S) glycoprotein, and is validated with a plaque-reduction assay using an authentic, infectious SARS-CoV-2 strain. The assay was used to evaluate SARS-CoV-2 antibodies in serum from individuals with a broad range of COVID-19 symptoms; patients included those in the intensive care unit (ICU), health care workers (HCWs), and convalescent plasma donors. The highest neutralizing antibody titers were observed among ICU patients, followed by general hospitalized patients, HCWs, and convalescent plasma donors. Our study highlights a wide phenotypic variation in human antibody responses against SARS-CoV-2 and demonstrates the efficacy of a potentially novel lentivirus pseudotype assay for high-throughput serological surveys of neutralizing antibody titers in large cohorts.

Rapid and specific antibody testing is crucial for improved understanding, control, and treatment of COVID-19 pathogenesis. Herein, we describe and apply a rapid, sensitive, and accurate virus neutralization assay for SARS-CoV-2 antibodies. The assay is based on an HIV-1 lentiviral vector that contains a secreted intron Gaussia luciferase (Gluc) or secreted nano-luciferase reporter cassette, pseudotyped with the SARS-CoV-2 spike (S) glycoprotein, and is validated with a plaque-reduction assay using an authentic, infectious SARS-CoV-2 strain. The assay was used to evaluate SARS-CoV-2 antibodies in serum from individuals with a broad range of COVID-19 symptoms; patients included those in the intensive care unit (ICU), health care workers (HCWs), and convalescent plasma donors. The highest neutralizing antibody titers were observed among ICU patients, followed by general hospitalized patients, HCWs, and convalescent plasma donors. Our study highlights a wide phenotypic variation in human antibody responses against SARS-CoV-2 and demonstrates the efficacy of a potentially novel lentivirus pseudotype assay for high-throughput serological surveys of neutralizing antibody titers in large cohorts., A rapid and sensitive high-throughput neutralization assay for evaluating the neutralizing antibody levels in COVID-19-exposed individuals.

Interferon-induced transmembrane proteins (IFITMs) restrict infections by many viruses, but a subset of IFITMs enhance infections by specific coronaviruses through currently unknown mechanisms. Here we show that SARS-CoV-2 Spike-pseudotyped virus and genuine SARS-CoV-2 infections are generally restricted by expression of human IFITM1, IFITM2, and IFITM3, using both gain- and loss-of-function approaches. Mechanistically, restriction of SARS-CoV-2 occurred independently of IFITM3S-palmitoylation sites, indicating a restrictive capacity that is distinct from reported inhibition of other viruses. In contrast, the IFITM3 amphipathic helix and its amphipathic properties were required for virus restriction. Mutation of residues within the human IFITM3 endocytosis-promoting YxxΦ motif converted human IFITM3 into an enhancer of SARS-CoV-2 infection, and cell-to-cell fusion assays confirmed the ability of endocytic mutants to enhance Spike-mediated fusion with the plasma membrane. Overexpression of TMPRSS2, which reportedly increases plasma membrane fusion versus endosome fusion of SARS-CoV-2, attenuated IFITM3 restriction and converted amphipathic helix mutants into strong enhancers of infection. In sum, these data uncover new pro- and anti-viral mechanisms of IFITM3, with clear distinctions drawn between enhancement of viral infection at the plasma membrane and amphipathicity-based mechanisms used for endosomal virus restriction. Indeed, the net effect of IFITM3 on SARS-CoV-2 infections may be a result of these opposing activities, suggesting that shifts in the balance of these activities could be coopted by viruses to escape this important first line innate defense mechanism.

Butyrate is an abundant metabolite produced by gut microbiota. While butyrate is a known histone deacetylase inhibitor that activates expression of many genes involved in immune system pathways, its effects on virus infections and on the antiviral type I interferon (IFN) response have not been adequately investigated. We found that butyrate increases cellular infection with viruses relevant to human and animal health, including influenza virus, reovirus, HIV-1, human metapneumovirus, and vesicular stomatitis virus. Mechanistically, butyrate suppresses levels of specific antiviral IFN-stimulated gene (ISG) products, such as RIG-I and IFITM3, in human and mouse cells without inhibiting IFN-induced phosphorylation or nuclear translocation of the STAT1 and STAT2 transcription factors. Accordingly, we discovered that although butyrate globally increases baseline expression of more than 800 cellular genes, it strongly represses IFN-induced expression of 60% of ISGs and upregulates 3% of ISGs. Our findings reveal that there are differences in the IFN responsiveness of major subsets of ISGs depending on the presence of butyrate in the cell environment, and overall, they identify a new mechanism by which butyrate influences virus infection of cells. IMPORTANCE Butyrate is a lipid produced by intestinal bacteria. Here, we newly show that butyrate reprograms the innate antiviral immune response mediated by type I interferons (IFNs). Many of the antiviral genes induced by type I IFNs are repressed in the presence of butyrate, resulting in increased virus infection and replication. Our research demonstrates that metabolites produced by the gut microbiome, such as butyrate, can have complex effects on cellular physiology, including dampening of an inflammatory innate immune pathway resulting in a proviral cellular environment. Our work further suggests that butyrate could be broadly used as a tool to increase growth of virus stocks for research and for the generation of vaccines.

Antiviral restriction factors are cellular proteins that inhibit the entry, replication, or spread of viruses. These proteins are critical components of the innate immune system and function to limit the severity and host range of virus infections. Here we review the current knowledge on the mechanisms of action of several restriction factors that affect multiple viruses at distinct stages of their life cycles. For example, APOBEC3G deaminates cytosines to hypermutate reverse transcribed viral DNA; IFITM3 alters membranes to inhibit virus membrane fusion; MXA/B oligomerize on viral protein complexes to inhibit virus replication; SAMHD1 decreases dNTP intracellular concentrations to prevent reverse transcription of retrovirus genomes; tetherin prevents release of budding virions from cells; Viperin catalyzes formation of a nucleoside analogue that inhibits viral RNA polymerases; and ZAP binds virus RNAs to target them for degradation. We also discuss countermeasures employed by specific viruses against these restriction factors, and mention secondary functions of several of these factors in modulating immune responses. These important examples highlight the diverse strategies cells have evolved to combat virus infections.

Interferon-induced transmembrane protein 3 (IFITM3) is a host antiviral protein that alters cell membranes to block fusion of viruses. Published reports have identified conflicting pro- and anti-viral effects of IFITM3 on SARS-CoV-2 in cultured cells, and its impact on viral pathology in vivo remains unclear. Here, we show that IFITM3 knockout (KO) mice infected with mouse-adapted SARS-CoV-2 experienced extreme weight loss and lethality, while wild type (WT) mice lost only 10% of their body weight and recovered. KO mice had higher lung viral titers and increases in lung inflammatory cytokine levels, CD45-positive immune cell infiltration, and histopathology, compared to WT mice. Mechanistically, we observed disseminated viral antigen staining throughout the lung tissue and pulmonary vasculature in KO mice, while staining was observed in confined regions in WT lungs. Global transcriptomic analysis of infected lungs identified upregulation of gene signatures associated with interferons, inflammation, and angiogenesis in KO versus WT animals, highlighting changes in lung gene expression programs that precede severe lung pathology and fatality. Corroborating the protective effect of IFITM3 in vivo, K18-hACE2/IFITM3 KO mice infected with non-adapted SARS-CoV-2 showed enhanced, rapid weight loss and early death compared to control mice. Increased heart infection was observed in both mouse models in the absence of IFITM3, indicating that IFITM3 constrains extrapulmonary dissemination of SARS-CoV-2. Our results establish IFITM3 KO mice as a new animal model for studying severe SARS-CoV-2 infection, and overall demonstrate that IFITM3 is protective in SARS-CoV-2 infections of mice. Supported by grants from the NIH (R01 AI130110, R21 AI151230, R01 HL154001, R21 AI142256, and U54 CA260582)

Interferon-lambda (IFN-λ) is currently being investigated in a phase III clinical trial as a therapeutic against COVID-19. Exogenous IFN-λ restricts SARS-CoV-2 in vitro and in Balb/c and C57Bl/6 (WT) murine models of infection. However, roles of endogenously produced IFN-λ in SARS-CoV-2 pathogenesis are not currently known, and the overall mechanisms by which IFN-λ modulates the induction of protective immune responses in SARS-CoV-2 infections remains to be elucidated. We find that IFN-λ receptor deficient mice (Ifnlr1−/−) infected with mouse-adapted SARS-CoV-2 lose significantly more weight and have increased SARS-CoV-2 viral replication compared to WT through day 5 post infection. Intriguingly, Ifit3 and Ifitm3 are increased in the lungs of Ifnlr1−/− mice compared to WT following infection, despite similar IFN-α/β and IFN-λ mRNA levels, suggesting compensatory increases in type I IFN signaling are not driving the increased weight loss or ISG induction observed in Ifnlr1−/− mice. Global transcriptomics revealed induction of a suppressive immunoregulatory signature with increased IL-10 as a hallmark in Ifnlr1−/− lungs, suggesting IFN-λ is critically involved in regulating appropriate immune activation to limit SARS-CoV-2 pathogenesis. Histological analysis revealed a significant increase in CD45+ cells (but not neutrophils) in the lungs of Ifnlr1−/− mice compared to WT on day 5 post infection, and we identified increases in pathways associated with myeloid cell function and T cell activation in Ifnlr1−/− by comparative transcriptomics. Overall, broadening the understanding of how IFN-λ regulates SARS-CoV-2 infection, pathogenesis, and immunity will inform the utilization of IFN-λ as an immunotherapy and adjuvant. Supported by NIH Award K22 AI146141 to EAH.