Efficacy of a stable broadly protective subunit vaccine platform against SARS-CoV-2 variants of concern.

The emergence and ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the need for rapid vaccine development platforms that can be updated to counteract emerging variants of currently circulating and future emerging coronaviruses. Here we report the development of a "train model" subunit vaccine platform that contains a SARS-CoV-2 Wuhan S1 protein (the "engine") linked to a series of flexible receptor binding domains (RBDs; the "cars") derived from SARS-CoV-2 variants of concern (VOCs). We demonstrate that these linked subunit vaccines when combined with Sepivac SWE™, a squalene in water emulsion (SWE) adjuvant, are immunogenic in Syrian hamsters and subsequently provide protection from infection with SARS-CoV-2 VOCs Omicron (BA.1), Delta, and Beta. Importantly, the bivalent and trivalent vaccine candidates offered protection against some heterologous SARS-CoV-2 VOCs that were not included in the vaccine design, demonstrating the potential for broad protection against a range of different VOCs. Furthermore, these formulated vaccine candidates were stable at 2-8 °C for up to 13 months post-formulation, highlighting their utility in low-resource settings. Indeed, our vaccine platform will enable the development of safe and broadly protective vaccines against emerging betacoronaviruses that pose a significant health risk for humans and agricultural animals.

A Significant Contribution of the Classical Pathway of Complement in SARS-CoV-2 Neutralization of Convalescent and Vaccinee Sera.

Although high titers of neutralizing Abs in human serum are associated with protection from reinfection by SARS-CoV-2, there is considerable heterogeneity in human serum-neutralizing Abs against SARS-CoV-2 during convalescence between individuals. Standard human serum live virus neutralization assays require inactivation of serum/plasma prior to testing. In this study, we report that the SARS-CoV-2 neutralization titers of human convalescent sera were relatively consistent across all disease states except for severe COVID-19, which yielded significantly higher neutralization titers. Furthermore, we show that heat inactivation of human serum significantly lowered neutralization activity in a live virus SARS-CoV-2 neutralization assay. Heat inactivation of human convalescent serum was shown to inactivate complement proteins, and the contribution of complement in SARS-CoV-2 neutralization was often >50% of the neutralizing activity of human sera without heat inactivation and could account for neutralizing activity when standard titers were zero after heat inactivation. This effect was also observed in COVID-19 vaccinees and could be abolished in individuals who were undergoing treatment with therapeutic anti-complement Abs. Complement activity was mainly dependent on the classical pathway with little contributions from mannose-binding lectin and alternative pathways. Our study demonstrates the importance of the complement pathway in significantly increasing viral neutralization activity against SARS-CoV-2 in spike seropositive individuals.

The translational potential of studying bat immunity.

Molecular studies in bats have led to the discovery of antiviral adaptations that may explain how some bat species have evolved enhanced immune tolerance towards viruses. Accumulating data suggest that some bat species have also evolved remarkable features of longevity and low rates of cancer. Furthermore, recent research strongly suggests that discovering immune adaptations in bat models can be translated to develop immune modulators and recognize alternate therapeutic strategies for diseases affecting humans. We posit that research in bat immunology will lead to discoveries that can potentially be translated to improve health outcomes in humans.

Virology-the path forward.

In the United States (US), biosafety and biosecurity oversight of research on viruses is being reappraised. Safety in virology research is paramount and oversight frameworks should be reviewed periodically. Changes should be made with care, however, to avoid impeding science that is essential for rapidly reducing and responding to pandemic threats as well as addressing more common challenges caused by infectious diseases. Decades of research uniquely positioned the US to be able to respond to the COVID-19 crisis with astounding speed, delivering life-saving vaccines within a year of identifying the virus. We should embolden and empower this strength, which is a vital part of protecting the health, economy, and security of US citizens. Herein, we offer our perspectives on priorities for revised rules governing virology research in the US.

Genomic and transcriptomic characterization of delta SARS-CoV-2 infection in free-ranging white-tailed deer (Odocoileus virginianus).

White-tailed deer (WTD) are susceptible to SARS-CoV-2 and represent an important species for surveillance. Samples from WTD (n = 258) collected in November 2021 from Québec, Canada were analyzed for SARS-CoV-2 RNA. We employed viral genomics and host transcriptomics to further characterize infection and investigate host response. We detected Delta SARS-CoV-2 (B.1.617.2) in WTD from the Estrie region; sequences clustered with human sequences from October 2021 from Vermont, USA, which borders this region. Mutations in the S-gene and a deletion in ORF8 were detected. Host expression patterns in SARS-CoV-2 infected WTD were associated with the innate immune response, including signaling pathways related to anti-viral, pro- and anti-inflammatory signaling, and host damage. We found limited correlation between genes associated with innate immune response from human and WTD nasal samples, suggesting differences in responses to SARS-CoV-2 infection. Our findings provide preliminary insights into host response to SARS-CoV-2 infection in naturally infected WTD.

ASK1 inhibitors are potential pan-antiviral drugs, which dampen replication of diverse viruses including SARS-CoV2.

Apoptosis signal-regulating kinase 1 (ASK1)/MAP3K5 is a stress response kinase that is activated by various stimuli. It is known as an upstream activator of p38- Mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase (JNK) that are reactive oxygen species (ROS)-induced kinases. Accumulating evidence show that ROS accumulate in virus-infected cells. Here, we investigated the relationship between viruses and ASK1/p38MAPK or ASK1/JNK pathways. Our findings suggest that virus infection activates ASK1 related pathways. In parallel, ASK1 inhibition led to a remarkable reduction in the replication of a broad range of viruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), vaccinia virus (VV), vesicular stomatitis virus (VSV), Herpes Simplex Virus (HSV), and Human Immunodeficiency virus (HIV) in different human cell lines. Our work demonstrates the potential therapeutic use of Selonsertib, an ASK1 inhibitor, as a pan-antiviral drug in humans. Surprisingly, we observed differential effects of Selonsertib in in vitro and in vivo hamster models, suggesting caution in using rodent models to predict clinical and therapeutic outcomes in humans.

Sensitivity to Neutralizing Antibodies and Resistance to Type I Interferons in SARS-CoV-2 R.1 Lineage Variants, Canada.

Isolating and characterizing emerging SARS-CoV-2 variants is key to understanding virus pathogenesis. In this study, we isolated samples of the SARS-CoV-2 R.1 lineage, categorized as a variant under monitoring by the World Health Organization, and evaluated their sensitivity to neutralizing antibodies and type I interferons. We used convalescent serum samples from persons in Canada infected either with ancestral virus (wave 1) or the B.1.1.7 (Alpha) variant of concern (wave 3) for testing neutralization sensitivity. The R.1 isolates were potently neutralized by both the wave 1 and wave 3 convalescent serum samples, unlike the B.1.351 (Beta) variant of concern. Of note, the R.1 variant was significantly more resistant to type I interferons (IFN-α/ß) than was the ancestral isolate. Our study demonstrates that the R.1 variant retained sensitivity to neutralizing antibodies but evolved resistance to type I interferons. This critical driving force will influence the trajectory of the pandemic.

Mycobacterium tuberculosis and SARS-CoV-2 co-infections: The knowns and unknowns.

Health impacts of Mycobacterium tuberculosis (Mtb) and SARS-CoV-2 co-infections are not fully understood. Both pathogens modulate host responses and induce immunopathology with extensive lung damage. With a quarter of the world's population harboring latent TB, exploring the relationship between SARS-CoV-2 infection and its effect on the transition of Mtb from latent to active form is paramount to control this pathogen. The effects of active Mtb infection on establishment and severity of COVID-19 are also unknown, despite the ability of TB to orchestrate profound long-lasting immunopathologies in the lungs. Absence of mechanistic studies and co-infection models hinder the development of effective interventions to reduce the health impacts of SARS-CoV-2 and Mtb co-infection. Here, we highlight dysregulated immune responses induced by SARS-CoV-2 and Mtb, their potential interplay, and implications for co-infection in the lungs. As both pathogens master immunomodulation, we discuss relevant converging and diverging immune-related pathways underlying SARS-CoV-2 and Mtb co-infections.

Regulatory dissection of the severe COVID-19 risk locus introgressed by Neanderthals.

Individuals infected with the SARS-CoV-2 virus present with a wide variety of symptoms ranging from asymptomatic to severe and even lethal outcomes. Past research has revealed a genetic haplotype on chromosome 3 that entered the human population via introgression from Neanderthals as the strongest genetic risk factor for the severe response to COVID-19. However, the specific variants along this introgressed haplotype that contribute to this risk and the biological mechanisms that are involved remain unclear. Here, we assess the variants present on the risk haplotype for their likelihood of driving the genetic predisposition to severe COVID-19 outcomes. We do this by first exploring their impact on the regulation of genes involved in COVID-19 infection using a variety of population genetics and functional genomics tools. We then perform a locus-specific massively parallel reporter assay to individually assess the regulatory potential of each allele on the haplotype in a multipotent immune-related cell line. We ultimately reduce the set of over 600 linked genetic variants to identify four introgressed alleles that are strong functional candidates for driving the association between this locus and severe COVID-19. Using reporter assays in the presence/absence of SARS-CoV-2, we find evidence that these variants respond to viral infection. These variants likely drive the locus' impact on severity by modulating the regulation of two critical chemokine receptor genes: CCR1 and CCR5. These alleles are ideal targets for future functional investigations into the interaction between host genomics and COVID-19 outcomes.

Versatile use of bat ACE2 for cellular entry by MERS-CoV-like viruses.

Cellular entry receptors for bat MERS-CoV-like viruses NeoCoV and PDF-2180 were unknown, leaving their zoonotic potential ambiguous. A recent study by Xiong et al. published in Nature identified bat ACE2 as the cellular entry receptor for both viruses, highlighting the ability of coronaviruses to utilize a range of entry receptors.

Survival-based CRISPR genetic screens across a panel of permissive cell lines identify common and cell-specific SARS-CoV-2 host factors.

SARS-CoV-2 depends on host cell components for infection and replication. Identification of virus-host dependencies offers an effective way to elucidate mechanisms involved in viral infection and replication. If druggable, host factor dependencies may present an attractive strategy for anti-viral therapy. In this study, we performed genome wide CRISPR knockout screens in Vero E6 cells and four human cell lines including Calu-3, UM-UC-4, HEK-293 and HuH-7 to identify genetic regulators of SARS-CoV-2 infection. Our findings identified only ACE2, the cognate SARS-CoV-2 entry receptor, as a common host dependency factor across all cell lines, while other host genes identified were largely cell line specific, including known factors TMPRSS2 and CTSL. Several of the discovered host-dependency factors converged on pathways involved in cell signalling, immune-related pathways, and chromatin modification. Notably, the chromatin modifier gene KMT2C in Calu-3 cells had the strongest impact in preventing SARS-CoV-2 infection when perturbed.

Laying the foundation for single-cell studies in bats.

Bats serve as hosts of viruses that can cause disease in humans. In this issue of Immunity, Gamage et al. characterize the immune cell repertoire in Eonycteris spelaea bat lung tissue using single-cell transcriptomics, providing insight into the in vivo immune response to infection with a Pteropine orthoreovirus (PRV3M) and establishing a paradigm for future comparative immunology studies.

SARS-CoV-2 mitochondriopathy in COVID-19 pneumonia exacerbates hypoxemia.

RATIONALE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19 pneumonia. We hypothesize that SARS-CoV-2 causes alveolar injury and hypoxemia by damaging mitochondria in airway epithelial cells (AEC) and pulmonary artery smooth muscle cells (PASMC), triggering apoptosis and bioenergetic impairment, and impairing hypoxic pulmonary vasoconstriction (HPV), respectively. OBJECTIVES: We examined the effects of: A) human betacoronaviruses, SARS-CoV-2 and HCoV-OC43, and individual SARS-CoV-2 proteins on apoptosis, mitochondrial fission, and bioenergetics in AEC; and B) SARS-CoV-2 proteins and mouse hepatitis virus (MHV-1) infection on HPV. METHODS: We used transcriptomic data to identify temporal changes in mitochondrial-relevant gene ontology (GO) pathways post-SARS-CoV-2 infection. We also transduced AECs with SARS-CoV-2 proteins (M, Nsp7 or Nsp9) and determined effects on mitochondrial permeability transition pore (mPTP) activity, relative membrane potential, apoptosis, mitochondrial fission, and oxygen consumption rates (OCR). In human PASMC, we assessed the effects of SARS-CoV-2 proteins on hypoxic increases in cytosolic calcium, an HPV proxy. In MHV-1 pneumonia, we assessed HPV via cardiac catheterization and apoptosis using the TUNEL assay. RESULTS: SARS-CoV-2 regulated mitochondrial apoptosis, mitochondrial membrane permeabilization and electron transport chain (ETC) GO pathways within 2 hours of infection. SARS-CoV-2 downregulated ETC Complex I and ATP synthase genes, and upregulated apoptosis-inducing genes. SARS-CoV-2 and HCoV-OC43 upregulated and activated dynamin-related protein 1 (Drp1) and increased mitochondrial fission. SARS-CoV-2 and transduced SARS-CoV-2 proteins increased apoptosis inducing factor (AIF) expression and activated caspase 7, resulting in apoptosis. Coronaviruses also reduced OCR, decreased ETC Complex I activity and lowered ATP levels in AEC. M protein transduction also increased mPTP opening. In human PASMC, M and Nsp9 proteins inhibited HPV. In MHV-1 pneumonia, infected AEC displayed apoptosis and HPV was suppressed. BAY K8644, a calcium channel agonist, increased HPV and improved SpO2. CONCLUSIONS: Coronaviruses, including SARS-CoV-2, cause AEC apoptosis, mitochondrial fission, and bioenergetic impairment. SARS-CoV-2 also suppresses HPV by targeting mitochondria. This mitochondriopathy is replicated by transduction with SARS-CoV-2 proteins, indicating a mechanistic role for viral-host mitochondrial protein interactions. Mitochondriopathy is a conserved feature of coronaviral pneumonia that may exacerbate hypoxemia and constitutes a therapeutic target.

Human coronaviruses disassemble processing bodies.

A dysregulated proinflammatory cytokine response is characteristic of severe coronavirus infections caused by SARS-CoV-2, yet our understanding of the underlying mechanism responsible for this imbalanced immune response remains incomplete. Processing bodies (PBs) are cytoplasmic membraneless ribonucleoprotein granules that control innate immune responses by mediating the constitutive decay or suppression of mRNA transcripts, including many that encode proinflammatory cytokines. PB formation promotes turnover or suppression of cytokine RNAs, whereas PB disassembly corresponds with the increased stability and/or translation of these cytokine RNAs. Many viruses cause PB disassembly, an event that can be viewed as a switch that rapidly relieves cytokine RNA repression and permits the infected cell to respond to viral infection. Prior to this submission, no information was known about how human coronaviruses (CoVs) impacted PBs. Here, we show SARS-CoV-2 and the common cold CoVs, OC43 and 229E, induced PB loss. We screened a SARS-CoV-2 gene library and identified that expression of the viral nucleocapsid (N) protein from SARS-CoV-2 was sufficient to mediate PB disassembly. RNA fluorescent in situ hybridization revealed that transcripts encoding TNF and IL-6 localized to PBs in control cells. PB loss correlated with the increased cytoplasmic localization of these transcripts in SARS-CoV-2 N protein-expressing cells. Ectopic expression of the N proteins from five other human coronaviruses (OC43, MERS, 229E, NL63 and SARS-CoV) did not cause significant PB disassembly, suggesting that this feature is unique to SARS-CoV-2 N protein. These data suggest that SARS-CoV-2-mediated PB disassembly contributes to the dysregulation of proinflammatory cytokine production observed during severe SARS-CoV-2 infection.

Erratum: Probe design for simultaneous, targeted capture of diverse metagenomic targets.

[This corrects the article DOI: 10.1016/j.crmeth.2021.100069.].

Molecular, ecological, and behavioral drivers of the bat-virus relationship.

Bats perform important ecological roles in our ecosystem. However, recent studies have demonstrated that bats are reservoirs of emerging viruses that have spilled over into humans and agricultural animals to cause severe diseases. These viruses include Hendra and Nipah paramyxoviruses, Ebola and Marburg filoviruses, and coronaviruses that are closely related to severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and the recently emerged SARS-CoV-2. Intriguingly, bats that are naturally or experimentally infected with these viruses do not show clinical signs of disease. Here we have reviewed ecological, behavioral, and molecular factors that may influence the ability of bats to harbor viruses. We have summarized known zoonotic potential of bat-borne viruses and stress on the need for further studies to better understand the evolutionary relationship between bats and their viruses, along with discovering the intrinsic and external factors that facilitate the successful spillover of viruses from bats.

Intronic regulation of SARS-CoV-2 receptor (ACE2) expression mediated by immune signaling and oxidative stress pathways.

The angiotensin-converting enzyme 2 (ACE2) protein is a key catalytic regulator of the renin-angiotensin system (RAS), involved in fluid homeostasis and blood pressure modulation. ACE2 also serves as a cell-surface receptor for some coronaviruses such as SARS-CoV and SARS-CoV-2. Improved characterization of ACE2 regulation may help us understand the effects of pre-existing conditions on COVID-19 incidence, as well as pathogenic dysregulation following viral infection. Here, we perform bioinformatic analyses to hypothesize on ACE2 gene regulation in two different physiological contexts, identifying putative regulatory elements of ACE2 expression. We perform functional validation of our computational predictions via targeted CRISPR-Cas9 deletions of these elements in vitro, finding them responsive to immune signaling and oxidative-stress pathways. This contributes to our understanding of ACE2 gene regulation at baseline and immune challenge. Our work supports pursuit of these putative mechanisms in our understanding of infection/disease caused by current, and future, SARS-related viruses such as SARS-CoV-2.