A Multitrait Locus Regulates Sarbecovirus Pathogenesis.

Infectious diseases have shaped the human population genetic structure, and genetic variation influences the susceptibility to many viral diseases. However, a variety of challenges have made the implementation of traditional human Genome-wide Association Studies (GWAS) approaches to study these infectious outcomes challenging. In contrast, mouse models of infectious diseases provide an experimental control and precision, which facilitates analyses and mechanistic studies of the role of genetic variation on infection. Here we use a genetic mapping cross between two distinct Collaborative Cross mouse strains with respect to severe acute respiratory syndrome coronavirus (SARS-CoV) disease outcomes. We find several loci control differential disease outcome for a variety of traits in the context of SARS-CoV infection. Importantly, we identify a locus on mouse chromosome 9 that shows conserved synteny with a human GWAS locus for SARS-CoV-2 severe disease. We follow-up and confirm a role for this locus, and identify two candidate genes, CCR9 and CXCR6, that both play a key role in regulating the severity of SARS-CoV, SARS-CoV-2, and a distantly related bat sarbecovirus disease outcomes. As such we provide a template for using experimental mouse crosses to identify and characterize multitrait loci that regulate pathogenic infectious outcomes across species. IMPORTANCE Host genetic variation is an important determinant that predicts disease outcomes following infection. In the setting of highly pathogenic coronavirus infections genetic determinants underlying host susceptibility and mortality remain unclear. To elucidate the role of host genetic variation on sarbecovirus pathogenesis and disease outcomes, we utilized the Collaborative Cross (CC) mouse genetic reference population as a model to identify susceptibility alleles to SARS-CoV and SARS-CoV-2 infections. Our findings reveal that a multitrait loci found in chromosome 9 is an important regulator of sarbecovirus pathogenesis in mice. Within this locus, we identified and validated CCR9 and CXCR6 as important regulators of host disease outcomes. Specifically, both CCR9 and CXCR6 are protective against severe SARS-CoV, SARS-CoV-2, and SARS-related HKU3 virus disease in mice. This chromosome 9 multitrait locus may be important to help identify genes that regulate coronavirus disease outcomes in humans.

A Multitrait Locus Regulates Sarbecovirus Pathogenesis.

Infectious diseases have shaped the human population genetic structure, and genetic variation influences the susceptibility to many viral diseases. However, a variety of challenges have made the implementation of traditional human Genome-wide Association Studies (GWAS) approaches to study these infectious outcomes challenging. In contrast, mouse models of infectious diseases provide an experimental control and precision, which facilitates analyses and mechanistic studies of the role of genetic variation on infection. Here we use a genetic mapping cross between two distinct Collaborative Cross mouse strains with respect to SARS-CoV disease outcomes. We find several loci control differential disease outcome for a variety of traits in the context of SARS-CoV infection. Importantly, we identify a locus on mouse Chromosome 9 that shows conserved synteny with a human GWAS locus for SARS-CoV-2 severe disease. We follow-up and confirm a role for this locus, and identify two candidate genes, CCR9 and CXCR6 that both play a key role in regulating the severity of SARS-CoV, SARS-CoV-2 and a distantly related bat sarbecovirus disease outcomes. As such we provide a template for using experimental mouse crosses to identify and characterize multitrait loci that regulate pathogenic infectious outcomes across species.

Nasally delivered interferon-λ protects mice against infection by SARS-CoV-2 variants including Omicron.

Although vaccines and monoclonal antibody countermeasures have reduced the morbidity and mortality associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, variants with constellations of mutations in the spike gene jeopardize their efficacy. Accordingly, antiviral interventions that are resistant to further virus evolution are needed. The host-derived cytokine interferon lambda (IFN-λ) has been proposed as a possible treatment based on studies in human coronavirus 2019 (COVID-19) patients. Here, we show that IFN-λ protects against SARS-CoV-2 B.1.351 (Beta) and B.1.1.529 (Omicron) variants in three strains of conventional and human ACE2 transgenic mice. Prophylaxis or therapy with nasally delivered IFN-λ2 limits infection of historical or variant SARS-CoV-2 strains in the upper and lower respiratory tracts without causing excessive inflammation. In the lung, IFN-λ is produced preferentially in epithelial cells and acts on radio-resistant cells to protect against SARS-CoV-2 infection. Thus, inhaled IFN-λ may have promise as a treatment for evolving SARS-CoV-2 variants that develop resistance to antibody-based countermeasures.

Nasally-delivered interferon-λ protects mice against upper and lower respiratory tract infection of SARS-CoV-2 variants including Omicron.

Although vaccines and monoclonal antibody countermeasures have reduced the morbidity and mortality associated with SARS-CoV-2 infection, variants with constellations of mutations in the spike gene threaten their efficacy. Accordingly, antiviral interventions that are resistant to further virus evolution are needed. The host-derived cytokine IFN-λ has been proposed as a possible treatment based on correlative studies in human COVID-19 patients. Here, we show IFN-λ protects against SARS-CoV-2 B.1.351 (Beta) and B.1.1.529 (Omicron)variants in three strains of conventional and human ACE2 transgenic mice. Prophylaxis or therapy with nasally-delivered IFN-λ2 limited infection of historical or variant (B.1.351 and B.1.1.529) SARS-CoV-2 strains in the upper and lower respiratory tracts without causing excessive inflammation. In the lung, IFN-λ was produced preferentially in epithelial cells and acted on radio-resistant cells to protect against of SARS-CoV-2 infection. Thus, inhaled IFN-λ may have promise as a treatment for evolving SARS-CoV-2 variants that develop resistance to antibody-based countermeasures.

SARS-CoV-2 Causes Lung Infection without Severe Disease in Human ACE2 Knock-In Mice.

The development of mouse models for coronavirus disease 2019 (COVID-19) has enabled testing of vaccines and therapeutics and defining aspects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis. SARS-CoV-2 disease is severe in K18 transgenic mice (K18-hACE2 Tg) expressing human angiotensin-converting enzyme 2 (hACE2), the SARS-CoV-2 receptor, under an ectopic cytokeratin promoter, with high levels of infection measured in the lung and brain. Here, we evaluated SARS-CoV-2 infection in hACE2 knock-in (KI) mice that express hACE2 under an endogenous promoter in place of murine ACE2 (mACE2). Intranasal inoculation of hACE2 KI mice with SARS-CoV-2 WA1/2020 resulted in substantial viral replication within the upper and lower respiratory tracts with limited spread to extrapulmonary organs. However, SARS-CoV-2-infected hACE2 KI mice did not lose weight and developed limited pathology. Moreover, no significant differences in viral burden were observed in hACE2 KI mice infected with B.1.1.7 or B.1.351 variants compared to the WA1/2020 strain. Because the entry mechanisms of SARS-CoV-2 in mice remain uncertain, we evaluated the impact of the naturally occurring, mouse-adapting N501Y mutation by comparing infection of hACE2 KI, K18-hACE2 Tg, ACE2-deficient, and wild-type C57BL/6 mice. The N501Y mutation minimally affected SARS-CoV-2 infection in hACE2 KI mice but was required for viral replication in wild-type C57BL/6 mice in a mACE2-dependent manner and augmented pathogenesis in the K18-hACE2 Tg mice. Thus, the N501Y mutation likely enhances interactions with mACE2 or hACE2 in vivo. Overall, our study highlights the hACE2 KI mice as a model of mild SARS-CoV-2 infection and disease and clarifies the requirement of the N501Y mutation in mice. IMPORTANCE Mouse models of SARS-CoV-2 pathogenesis have facilitated the rapid evaluation of countermeasures. While the first generation of models developed pneumonia and severe disease after SARS-CoV-2 infection, they relied on ectopic expression of supraphysiological levels of human ACE2 (hACE2). This has raised issues with their relevance to humans, as the hACE2 receptor shows a more restricted expression pattern in the respiratory tract. Here, we evaluated SARS-CoV-2 infection and disease with viruses containing or lacking a key mouse-adapting mutation in the spike gene in hACE2 KI mice, which express hACE2 under an endogenous promoter in place of murine ACE2. While infection of hACE2 KI mice with multiple strains of SARS-CoV-2 including variants of concern resulted in viral replication within the upper and lower respiratory tracts, the animals did not sustain severe lung injury. Thus, hACE2 KI mice serve as a model of mild infection with both ancestral and emerging SARS-CoV-2 variant strains.

Implications of a highly divergent dengue virus strain for cross-neutralization, protection, and vaccine immunity.

Although divergent dengue viruses (DENVs) have been isolated in insects, nonhuman primates, and humans, their relationships to the four canonical serotypes (DENV 1-4) are poorly understood. One virus isolated from a dengue patient, DKE-121, falls between genotype and serotype levels of sequence divergence to DENV-4. To examine its antigenic relationship to DENV-4, we assessed serum neutralizing and protective activity. Whereas DENV-4-immune mouse sera neutralize DKE-121 infection, DKE-121-immune sera inhibit DENV-4 less efficiently. Passive transfer of DENV-4 or DKE-121-immune sera protects mice against homologous, but not heterologous, DENV-4 or DKE-121 challenge. Antigenic cartography suggests that DENV-4 and DKE-121 are related but antigenically distinct. However, DENV-4 vaccination confers protection against DKE-121 in nonhuman primates, and serum from humans immunized with a tetravalent vaccine neutralize DENV-4 and DKE-121 infection equivalently. As divergent DENV strains, such as DKE-121, may meet criteria for serotype distinction, monitoring their capacity to impact dengue disease and vaccine efficacy appears warranted.

A potently neutralizing SARS-CoV-2 antibody inhibits variants of concern by utilizing unique binding residues in a highly conserved epitope.

With the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with increased transmissibility and potential resistance, antibodies and vaccines with broadly inhibitory activity are needed. Here, we developed a panel of neutralizing anti-SARS-CoV-2 monoclonal antibodies (mAbs) that bound the receptor binding domain of the spike protein at distinct epitopes and blocked virus attachment to its host receptor, human angiotensin converting enzyme-2 (hACE2). Although several potently neutralizing mAbs protected K18-hACE2 transgenic mice against infection caused by ancestral SARS-CoV-2 strains, others induced escape variants in vivo or lost neutralizing activity against emerging strains. One mAb, SARS2-38, potently neutralized all tested SARS-CoV-2 variants of concern and protected mice against challenge by multiple SARS-CoV-2 strains. Structural analysis showed that SARS2-38 engaged a conserved epitope proximal to the receptor binding motif. Thus, treatment with or induction of neutralizing antibodies that bind conserved spike epitopes may limit the loss of potency of therapies or vaccines against emerging SARS-CoV-2 variants.

Convergent antibody responses to the SARS-CoV-2 spike protein in convalescent and vaccinated individuals.

Unrelated individuals can produce genetically similar clones of antibodies, known as public clonotypes, which have been seen in responses to different infectious diseases, as well as healthy individuals. Here we identify 37 public clonotypes in memory B cells from convalescent survivors of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or in plasmablasts from an individual after vaccination with mRNA-encoded spike protein. We identify 29 public clonotypes, including clones recognizing the receptor-binding domain (RBD) in the spike protein S1 subunit (including a neutralizing, angiotensin-converting enzyme 2 [ACE2]-blocking clone that protects in vivo) and others recognizing non-RBD epitopes that bind the S2 domain. Germline-revertant forms of some public clonotypes bind efficiently to spike protein, suggesting these common germline-encoded antibodies are preconfigured for avid recognition. Identification of large numbers of public clonotypes provides insight into the molecular basis of efficacy of SARS-CoV-2 vaccines and sheds light on the immune pressures driving the selection of common viral escape mutants.

Pan-protective anti-alphavirus human antibodies target a conserved E1 protein epitope.

Alphaviruses are emerging, mosquito-transmitted pathogens that cause musculoskeletal and neurological disease in humans. Although neutralizing antibodies that inhibit individual alphaviruses have been described, broadly reactive antibodies that protect against both arthritogenic and encephalitic alphaviruses have not been reported. Here, we identify DC2.112 and DC2.315, two pan-protective yet poorly neutralizing human monoclonal antibodies (mAbs) that avidly bind to viral antigen on the surface of cells infected with arthritogenic and encephalitic alphaviruses. These mAbs engage a conserved epitope in domain II of the E1 protein proximal to and within the fusion peptide. Treatment with DC2.112 or DC2.315 protects mice against infection by both arthritogenic (chikungunya and Mayaro) and encephalitic (Venezuelan, Eastern, and Western equine encephalitis) alphaviruses through multiple mechanisms, including inhibition of viral egress and monocyte-dependent Fc effector functions. These findings define a conserved epitope recognized by weakly neutralizing yet protective antibodies that could be targeted for pan-alphavirus immunotherapy and vaccine design.

Therapeutic alphavirus cross-reactive E1 human antibodies inhibit viral egress.

Alphaviruses cause severe arthritogenic or encephalitic disease. The E1 structural glycoprotein is highly conserved in these viruses and mediates viral fusion with host cells. However, the role of antibody responses to the E1 protein in immunity is poorly understood. We isolated E1-specific human monoclonal antibodies (mAbs) with diverse patterns of recognition for alphaviruses (ranging from Eastern equine encephalitis virus [EEEV]-specific to alphavirus cross-reactive) from survivors of natural EEEV infection. Antibody binding patterns and epitope mapping experiments identified differences in E1 reactivity based on exposure of epitopes on the glycoprotein through pH-dependent mechanisms or presentation on the cell surface prior to virus egress. Therapeutic efficacy in vivo of these mAbs corresponded with potency of virus egress inhibition in vitro and did not require Fc-mediated effector functions for treatment against subcutaneous EEEV challenge. These studies reveal the molecular basis for broad and protective antibody responses to alphavirus E1 proteins.

In vivo monoclonal antibody efficacy against SARS-CoV-2 variant strains.

Rapidly emerging SARS-CoV-2 variants jeopardize antibody-based countermeasures. Although cell culture experiments have demonstrated a loss of potency of several anti-spike neutralizing antibodies against variant strains of SARS-CoV-21-3, the in vivo importance of these results remains uncertain. Here we report the in vitro and in vivo activity of a panel of monoclonal antibodies (mAbs), which correspond to many in advanced clinical development by Vir Biotechnology, AbbVie, AstraZeneca, Regeneron and Lilly, against SARS-CoV-2 variant viruses. Although some individual mAbs showed reduced or abrogated neutralizing activity in cell culture against B.1.351, B.1.1.28, B.1.617.1 and B.1.526 viruses with mutations at residue E484 of the spike protein, low prophylactic doses of mAb combinations protected against infection by many variants in K18-hACE2 transgenic mice, 129S2 immunocompetent mice and hamsters, without the emergence of resistance. Exceptions were LY-CoV555 monotherapy and LY-CoV555 and LY-CoV016 combination therapy, both of which lost all protective activity, and the combination of AbbVie 2B04 and 47D11, which showed a partial loss of activity. When administered after infection, higher doses of several mAb cocktails protected in vivo against viruses with a B.1.351 spike gene. Therefore, many-but not all-of the antibody products with Emergency Use Authorization should retain substantial efficacy against the prevailing variant strains of SARS-CoV-2.

Ultrapotent miniproteins targeting the SARS-CoV-2 receptor-binding domain protect against infection and disease.

Despite the introduction of public health measures and spike protein-based vaccines to mitigate the COVID-19 pandemic, SARS-CoV-2 infections and deaths continue to have a global impact. Previously, we used a structural design approach to develop picomolar range miniproteins targeting the SARS-CoV-2 spike receptor-binding domain. Here, we investigated the capacity of modified versions of one lead miniprotein, LCB1, to protect against SARS-CoV-2-mediated lung disease in mice. Systemic administration of LCB1-Fc reduced viral burden, diminished immune cell infiltration and inflammation, and completely prevented lung disease and pathology. A single intranasal dose of LCB1v1.3 reduced SARS-CoV-2 infection in the lung when given as many as 5 days before or 2 days after virus inoculation. Importantly, LCB1v1.3 protected in vivo against a historical strain (WA1/2020), an emerging B.1.1.7 strain, and a strain encoding key E484K and N501Y spike protein substitutions. These data support development of LCB1v1.3 for prevention or treatment of SARS-CoV-2 infection.

Common Mechanism of SARS-CoV and SARS-CoV-2 Pathogenesis across Species.

Sarbecovirus (CoV) infections, including Severe Acute Respiratory CoV (SARS-CoV) and SARS-CoV-2, are considerable human threats. Human GWAS studies have recently identified loci associated with variation in SARS-CoV-2 susceptibility. However, genetically tractable models that reproduce human CoV disease outcomes are needed to mechanistically evaluate genetic determinants of CoV susceptibility. We used the Collaborative Cross (CC) and human GWAS datasets to elucidate host susceptibility loci that regulate CoV infections and to identify host quantitative trait loci that modulate severe CoV and pan-CoV disease outcomes including a major disease regulating loci including CCR9. CCR9 ablation resulted in enhanced titer, weight loss, respiratory dysfunction, mortality, and inflammation, providing mechanistic support in mitigating protection from severe SARS-CoV-2 pathogenesis across species. This study represents a comprehensive analysis of susceptibility loci for an entire genus of human pathogens conducted, identifies a large collection of susceptibility loci and candidate genes that regulate multiple aspects type-specific and cross-CoV pathogenesis, and also validates the paradigm of using the CC platform to identify common cross-species susceptibility loci and genes for newly emerging and pre-epidemic viruses.

Convergent antibody responses to the SARS-CoV-2 spike protein in convalescent and vaccinated individuals.

Unrelated individuals can produce genetically similar clones of antibodies, known as public clonotypes, which have been seen in responses to different infectious diseases as well as healthy individuals. Here we identify 37 public clonotypes in memory B cells from convalescent survivors of SARS-CoV-2 infection or in plasmablasts from an individual after vaccination with mRNA-encoded spike protein. We identified 29 public clonotypes, including clones recognizing the receptor-binding domain (RBD) in the spike protein S1 subunit (including a neutralizing, ACE2-blocking clone that protects in vivo ), and others recognizing non-RBD epitopes that bound the heptad repeat 1 region of the S2 domain. Germline-revertant forms of some public clonotypes bound efficiently to spike protein, suggesting these common germline-encoded antibodies are preconfigured for avid recognition. Identification of large numbers of public clonotypes provides insight into the molecular basis of efficacy of SARS-CoV-2 vaccines and sheds light on the immune pressures driving the selection of common viral escape mutants.

Profiling B cell immunodominance after SARS-CoV-2 infection reveals antibody evolution to non-neutralizing viral targets.

Dissecting the evolution of memory B cells (MBCs) against SARS-CoV-2 is critical for understanding antibody recall upon secondary exposure. Here, we used single-cell sequencing to profile SARS-CoV-2-reactive B cells in 38 COVID-19 patients. Using oligo-tagged antigen baits, we isolated B cells specific to the SARS-CoV-2 spike, nucleoprotein (NP), open reading frame 8 (ORF8), and endemic human coronavirus (HCoV) spike proteins. SARS-CoV-2 spike-specific cells were enriched in the memory compartment of acutely infected and convalescent patients several months post symptom onset. With severe acute infection, substantial populations of endemic HCoV-reactive antibody-secreting cells were identified and possessed highly mutated variable genes, signifying preexisting immunity. Finally, MBCs exhibited pronounced maturation to NP and ORF8 over time, especially in older patients. Monoclonal antibodies against these targets were non-neutralizing and non-protective in vivo. These findings reveal antibody adaptation to non-neutralizing intracellular antigens during infection, emphasizing the importance of vaccination for inducing neutralizing spike-specific MBCs.

Western diet induces Paneth cell defects through microbiome alterations and farnesoid X receptor and type I interferon activation.

Intestinal Paneth cells modulate innate immunity and infection. In Crohn's disease, genetic mutations together with environmental triggers can disable Paneth cell function. Here, we find that a western diet (WD) similarly leads to Paneth cell dysfunction through mechanisms dependent on the microbiome and farnesoid X receptor (FXR) and type I interferon (IFN) signaling. Analysis of multiple human cohorts suggests that obesity is associated with Paneth cell dysfunction. In mouse models, consumption of a WD for as little as 4 weeks led to Paneth cell dysfunction. WD consumption in conjunction with Clostridium spp. increased the secondary bile acid deoxycholic acid levels in the ileum, which in turn inhibited Paneth cell function. The process required excess signaling of both FXR and IFN within intestinal epithelial cells. Our findings provide a mechanistic link between poor diet and inhibition of gut innate immunity and uncover an effect of FXR activation in gut inflammation.

A potently neutralizing anti-SARS-CoV-2 antibody inhibits variants of concern by binding a highly conserved epitope.

With the emergence of SARS-CoV-2 variants with increased transmissibility and potential resistance, antibodies and vaccines with broadly inhibitory activity are needed. Here we developed a panel of neutralizing anti-SARS-CoV-2 mAbs that bind the receptor binding domain of the spike protein at distinct epitopes and block virus attachment to cells and its receptor, human angiotensin converting enzyme-2 (hACE2). While several potently neutralizing mAbs protected K18-hACE2 transgenic mice against infection caused by historical SARS-CoV-2 strains, others induced escape variants in vivo and lost activity against emerging strains. We identified one mAb, SARS2-38, that potently neutralizes all SARS-CoV-2 variants of concern tested and protects mice against challenge by multiple SARS-CoV-2 strains. Structural analysis showed that SARS2-38 engages a conserved epitope proximal to the receptor binding motif. Thus, treatment with or induction of inhibitory antibodies that bind conserved spike epitopes may limit the loss of potency of therapies or vaccines against emerging SARS-CoV-2 variants.

Resistance of SARS-CoV-2 variants to neutralization by monoclonal and serum-derived polyclonal antibodies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the global COVID-19 pandemic. Rapidly spreading SARS-CoV-2 variants may jeopardize newly introduced antibody and vaccine countermeasures. Here, using monoclonal antibodies (mAbs), animal immune sera, human convalescent sera and human sera from recipients of the BNT162b2 mRNA vaccine, we report the impact on antibody neutralization of a panel of authentic SARS-CoV-2 variants including a B.1.1.7 isolate, chimeric strains with South African or Brazilian spike genes and isogenic recombinant viral variants. Many highly neutralizing mAbs engaging the receptor-binding domain or N-terminal domain and most convalescent sera and mRNA vaccine-induced immune sera showed reduced inhibitory activity against viruses containing an E484K spike mutation. As antibodies binding to spike receptor-binding domain and N-terminal domain demonstrate diminished neutralization potency in vitro against some emerging variants, updated mAb cocktails targeting highly conserved regions, enhancement of mAb potency or adjustments to the spike sequences of vaccines may be needed to prevent loss of protection in vivo.

On the road to ending the COVID-19 pandemic: Are we there yet?

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged into the human population in late 2019 and caused the global COVID-19 pandemic. SARS-CoV-2 has spread to more than 215 countries and infected many millions of people. Despite the introduction of numerous governmental and public health measures to control disease spread, infections continue at an unabated pace, suggesting that effective vaccines and antiviral drugs will be required to curtail disease, end the pandemic, and restore societal norms. Here, we review the current developments in antibody and vaccine countermeasures to limit or prevent disease.

Ultrapotent miniproteins targeting the receptor-binding domain protect against SARS-CoV-2 infection and disease in mice.

Despite the introduction of public health measures and spike protein-based vaccines to mitigate the COVID-19 pandemic, SARS-CoV-2 infections and deaths continue to rise. Previously, we used a structural design approach to develop picomolar range miniproteins targeting the SARS-CoV-2 receptor binding domain. Here, we investigated the capacity of modified versions of one lead binder, LCB1, to protect against SARS-CoV-2-mediated lung disease in human ACE2-expressing transgenic mice. Systemic administration of LCB1-Fc reduced viral burden, diminished immune cell infiltration and inflammation, and completely prevented lung disease and pathology. A single intranasal dose of LCB1v1.3 reduced SARS-CoV-2 infection in the lung even when given as many as five days before or two days after virus inoculation. Importantly, LCB1v1.3 protected in vivo against a historical strain (WA1/2020), an emerging B.1.1.7 strain, and a strain encoding key E484K and N501Y spike protein substitutions. These data support development of LCB1v1.3 for prevention or treatment of SARS-CoV-2 infection.