Aspergillus fumigatus Supernatants Disrupt Bronchial Epithelial Monolayers: Potential Role for Enhanced Invasion in Cystic Fibrosis.

Aspergillus fumigatus is the most commonly isolated fungus in chronic lung diseases, with a prevalence of up to 60% in cystic fibrosis patients. Despite this, the impact of A. fumigatus colonisation on lung epithelia has not been thoroughly explored. We investigated the influence of A. fumigatus supernatants and the secondary metabolite, gliotoxin, on human bronchial epithelial cells (HBE) and CF bronchial epithelial (CFBE) cells. CFBE (F508del CFBE41o-) and HBE (16HBE14o-) trans-epithelial electrical resistance (TEER) was measured following exposure to A. fumigatus reference and clinical isolates, a gliotoxin-deficient mutant (ΔgliG) and pure gliotoxin. The impact on tight junction (TJ) proteins, zonula occludens-1 (ZO-1) and junctional adhesion molecule-A (JAM-A) were determined by western blot analysis and confocal microscopy. A. fumigatus conidia and supernatants caused significant disruption to CFBE and HBE TJs within 24 h. Supernatants from later cultures (72 h) caused the greatest disruption while ΔgliG mutant supernatants caused no disruption to TJ integrity. The ZO-1 and JAM-A distribution in epithelial monolayers were altered by A. fumigatus supernatants but not by ΔgliG supernatants, suggesting that gliotoxin is involved in this process. The fact that ΔgliG conidia were still capable of disrupting epithelial monolayers indicates that direct cell-cell contact also plays a role, independently of gliotoxin production. Gliotoxin is capable of disrupting TJ integrity which has the potential to contribute to airway damage, and enhance microbial invasion and sensitisation in CF.

Targeted protein S-nitrosylation of ACE2 inhibits SARS-CoV-2 infection.

Prevention of infection and propagation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a high priority in the Coronavirus Disease 2019 (COVID-19) pandemic. Here we describe S-nitrosylation of multiple proteins involved in SARS-CoV-2 infection, including angiotensin-converting enzyme 2 (ACE2), the receptor for viral entry. This reaction prevents binding of ACE2 to the SARS-CoV-2 spike protein, thereby inhibiting viral entry, infectivity and cytotoxicity. Aminoadamantane compounds also inhibit coronavirus ion channels formed by envelope (E) protein. Accordingly, we developed dual-mechanism aminoadamantane nitrate compounds that inhibit viral entry and, thus, the spread of infection by S-nitrosylating ACE2 via targeted delivery of the drug after E protein channel blockade. These non-toxic compounds are active in vitro and in vivo in the Syrian hamster COVID-19 model and, thus, provide a novel avenue to pursue therapy.

Broadly neutralizing anti-S2 antibodies protect against all three human betacoronaviruses that cause deadly disease.

Pan-betacoronavirus neutralizing antibodies may hold the key to developing broadly protective vaccines against novel pandemic coronaviruses and to more effectively respond to SARS-CoV-2 variants. The emergence of Omicron and subvariants of SARS-CoV-2 illustrates the limitations of solely targeting the receptor-binding domain (RBD) of the spike (S) protein. Here, we isolated a large panel of broadly neutralizing antibodies (bnAbs) from SARS-CoV-2 recovered-vaccinated donors, which targets a conserved S2 region in the betacoronavirus spike fusion machinery. Select bnAbs showed broad in vivo protection against all three deadly betacoronaviruses, SARS-CoV-1, SARS-CoV-2, and MERS-CoV, which have spilled over into humans in the past two decades. Structural studies of these bnAbs delineated the molecular basis for their broad reactivity and revealed common antibody features targetable by broad vaccination strategies. These bnAbs provide new insights and opportunities for antibody-based interventions and for developing pan-betacoronavirus vaccines.

A single-administration therapeutic interfering particle reduces SARS-CoV-2 viral shedding and pathogenesis in hamsters.

The high transmissibility of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a primary driver of the COVID-19 pandemic. While existing interventions prevent severe disease, they exhibit mixed efficacy in preventing transmission, presumably due to their limited antiviral effects in the respiratory mucosa, whereas interventions targeting the sites of viral replication might more effectively limit respiratory virus transmission. Recently, intranasally administered RNA-based therapeutic interfering particles (TIPs) were reported to suppress SARS-CoV-2 replication, exhibit a high barrier to resistance, and prevent serious disease in hamsters. Since TIPs intrinsically target the tissues with the highest viral replication burden (i.e., respiratory tissues for SARS-CoV-2), we tested the potential of TIP intervention to reduce SARS-CoV-2 shedding. Here, we report that a single, postexposure TIP dose lowers SARS-CoV-2 nasal shedding, and at 5 days postinfection, infectious virus shed is below detection limits in 4 out of 5 infected animals. Furthermore, TIPs reduce shedding of Delta variant or WA-1 from infected to uninfected hamsters. Cohoused "contact" animals exposed to infected, TIP-treated animals exhibited significantly lower viral loads, reduced inflammatory cytokines, no severe lung pathology, and shortened shedding duration compared to animals cohoused with untreated infected animals. TIPs may represent an effective countermeasure to limit SARS-CoV-2 transmission.

Broadly neutralizing antibodies to SARS-related viruses can be readily induced in rhesus macaques.

To prepare for future coronavirus (CoV) pandemics, it is desirable to generate vaccines capable of eliciting broadly neutralizing antibody responses to CoVs. Here, we show that immunization of macaques with SARS-CoV-2 spike (S) protein with a two-shot protocol generated potent serum receptor binding domain cross-neutralizing antibody responses to both SARS-CoV-2 and SARS-CoV-1. Furthermore, responses were equally effective against most SARS-CoV-2 variants of concern (VOCs) and some were highly effective against Omicron. This result contrasts with human infection or many two-shot vaccination protocols where responses were typically more SARS-CoV-2 specific and where VOCs were less well neutralized. Structural studies showed that cloned macaque neutralizing antibodies, particularly using a given heavy chain germline gene, recognized a relatively conserved region proximal to the angiotensin converting enzyme 2 receptor binding site (RBS), whereas many frequently elicited human neutralizing antibodies targeted more variable epitopes overlapping the RBS. B cell repertoire differences between humans and macaques appeared to influence the vaccine response. The macaque neutralizing antibodies identified a pan-SARS-related virus epitope region less well targeted by human antibodies that could be exploited in rational vaccine design.

Targeted isolation of diverse human protective broadly neutralizing antibodies against SARS-like viruses.

The emergence of current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) and potential future spillovers of SARS-like coronaviruses into humans pose a major threat to human health and the global economy. Development of broadly effective coronavirus vaccines that can mitigate these threats is needed. Here, we utilized a targeted donor selection strategy to isolate a large panel of human broadly neutralizing antibodies (bnAbs) to sarbecoviruses. Many of these bnAbs are remarkably effective in neutralizing a diversity of sarbecoviruses and against most SARS-CoV-2 VOCs, including the Omicron variant. Neutralization breadth is achieved by bnAb binding to epitopes on a relatively conserved face of the receptor-binding domain (RBD). Consistent with targeting of conserved sites, select RBD bnAbs exhibited protective efficacy against diverse SARS-like coronaviruses in a prophylaxis challenge model in vivo. These bnAbs provide new opportunities and choices for next-generation antibody prophylactic and therapeutic applications and provide a molecular basis for effective design of pan-sarbecovirus vaccines.

A human antibody reveals a conserved site on beta-coronavirus spike proteins and confers protection against SARS-CoV-2 infection.

Broadly neutralizing antibodies (bnAbs) to coronaviruses (CoVs) are valuable in their own right as prophylactic and therapeutic reagents to treat diverse CoVs and as templates for rational pan-CoV vaccine design. We recently described a bnAb, CC40.8, from a CoV disease 2019 (COVID-19) convalescent donor that exhibits broad reactivity with human ß-CoVs. Here, we showed that CC40.8 targets the conserved S2 stem helix region of the CoV spike fusion machinery. We determined a crystal structure of CC40.8 Fab with a SARS-CoV-2 S2 stem peptide at 1.6-Å resolution and found that the peptide adopted a mainly helical structure. Conserved residues in ß-CoVs interacted with CC40.8 antibody, thereby providing a molecular basis for its broad reactivity. CC40.8 exhibited in vivo protective efficacy against SARS-CoV-2 challenge in two animal models. In both models, CC40.8-treated animals exhibited less weight loss and reduced lung viral titers compared to controls. Furthermore, we noted that CC40.8-like bnAbs are relatively rare in human COVID-19 infection, and therefore, their elicitation may require rational structure-based vaccine design strategies. Overall, our study describes a target on ß-CoV spike proteins for protective antibodies that may facilitate the development of pan-ß-CoV vaccines.

Identification of a therapeutic interfering particle-A single-dose SARS-CoV-2 antiviral intervention with a high barrier to resistance.

Viral-deletion mutants that conditionally replicate and inhibit the wild-type virus (i.e., defective interfering particles, DIPs) have long been proposed as single-administration interventions with high genetic barriers to resistance. However, theories predict that robust, therapeutic DIPs (i.e., therapeutic interfering particles, TIPs) must conditionally spread between cells with R0 >1. Here, we report engineering of TIPs that conditionally replicate with SARS-CoV-2, exhibit R0 >1, and inhibit viral replication 10- to 100-fold. Inhibition occurs via competition for viral replication machinery, and a single administration of TIP RNA inhibits SARS-CoV-2 sustainably in continuous cultures. Strikingly, TIPs maintain efficacy against neutralization-resistant variants (e.g., B.1.351). In hamsters, both prophylactic and therapeutic intranasal administration of lipid-nanoparticle TIPs durably suppressed SARS-CoV-2 by 100-fold in the lungs, reduced pro-inflammatory cytokine expression, and prevented severe pulmonary edema. These data provide proof of concept for a class of single-administration antivirals that may circumvent current requirements to continually update medical countermeasures against new variants.

Development of a Synthetic Poxvirus-Based SARS-CoV-2 Vaccine

Modified Vaccinia Ankara (MVA) is a highly attenuated poxvirus vector that is widely used to develop vaccines for infectious diseases and cancer. We developed a novel vaccine platform based on a unique three-plasmid system to efficiently generate recombinant MVA vectors from chemically synthesized DNA. In response to the ongoing global pandemic caused by SARS coronavirus-2 (SARS-CoV-2), we used this novel vaccine platform to rapidly produce fully synthetic MVA (sMVA) vectors co-expressing SARS-CoV-2 spike and nucleocapsid antigens, two immunodominant antigens implicated in protective immunity. Mice immunized with these sMVA vectors developed robust SARS-CoV-2 antigen-specific humoral and cellular immune responses, including potent neutralizing antibodies. These results demonstrate the potential of a novel vaccine platform based on synthetic DNA to efficiently generate recombinant MVA vectors and to rapidly develop a multi-antigenic poxvirus-based SARS-CoV-2 vaccine candidate.

Preclinical characterization of an intravenous coronavirus 3CL protease inhibitor for the potential treatment of COVID19.

COVID-19 caused by the SARS-CoV-2 virus has become a global pandemic. 3CL protease is a virally encoded protein that is essential across a broad spectrum of coronaviruses with no close human analogs. PF-00835231, a 3CL protease inhibitor, has exhibited potent in vitro antiviral activity against SARS-CoV-2 as a single agent. Here we report, the design and characterization of a phosphate prodrug PF-07304814 to enable the delivery and projected sustained systemic exposure in human of PF-00835231 to inhibit coronavirus family 3CL protease activity with selectivity over human host protease targets. Furthermore, we show that PF-00835231 has additive/synergistic activity in combination with remdesivir. We present the ADME, safety, in vitro, and in vivo antiviral activity data that supports the clinical evaluation of PF-07304814 as a potential COVID-19 treatment.

Bispecific antibodies targeting distinct regions of the spike protein potently neutralize SARS-CoV-2 variants of concern.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern threatens the efficacy of existing vaccines and therapeutic antibodies and underscores the need for additional antibody-based tools that potently neutralize variants by targeting multiple sites of the spike protein. We isolated 216 monoclonal antibodies targeting SARS-CoV-2 from plasmablasts and memory B cells collected from patients with coronavirus disease 2019. The three most potent antibodies targeted distinct regions of the receptor binding domain (RBD), and all three neutralized the SARS-CoV-2 Alpha and Beta variants. The crystal structure of the most potent antibody, CV503, revealed that it binds to the ridge region of SARS-CoV-2 RBD, competes with the angiotensin-converting enzyme 2 receptor, and has limited contact with key variant residues K417, E484, and N501. We designed bispecific antibodies by combining nonoverlapping specificities and identified five bispecific antibodies that inhibit SARS-CoV-2 infection at concentrations of less than 1 ng/ml. Through a distinct mode of action, three bispecific antibodies cross-linked adjacent spike proteins using dual N-terminal domain­RBD specificities. One bispecific antibody was greater than 100-fold more potent than a cocktail of its parent monoclonals in vitro and prevented clinical disease in a hamster model at a dose of 2.5 mg/kg. Two bispecific antibodies in our panel comparably neutralized the Alpha, Beta, Gamma, and Delta variants and wild-type virus. Furthermore, a bispecific antibody that neutralized the Beta variant protected hamsters against SARS-CoV-2 expressing the E484K mutation. Thus, bispecific antibodies represent a promising next-generation countermeasure against SARS-CoV-2 variants of concern.

Adult stem cell-derived complete lung organoid models emulate lung disease in COVID-19.

Background: SARS-CoV-2, the virus responsible for COVID-19, causes widespread damage in the lungs in the setting of an overzealous immune response whose origin remains unclear. Methods: We present a scalable, propagable, personalized, cost-effective adult stem cell-derived human lung organoid model that is complete with both proximal and distal airway epithelia. Monolayers derived from adult lung organoids (ALOs), primary airway cells, or hiPSC-derived alveolar type II (AT2) pneumocytes were infected with SARS-CoV-2 to create in vitro lung models of COVID-19. Results: Infected ALO monolayers best recapitulated the transcriptomic signatures in diverse cohorts of COVID-19 patient-derived respiratory samples. The airway (proximal) cells were critical for sustained viral infection, whereas distal alveolar differentiation (AT2→AT1) was critical for mounting the overzealous host immune response in fatal disease; ALO monolayers with well-mixed proximodistal airway components recapitulated both. Conclusions: Findings validate a human lung model of COVID-19, which can be immediately utilized to investigate COVID-19 pathogenesis and vet new therapies and vaccines. Funding: This work was supported by the National Institutes for Health (NIH) grants 1R01DK107585-01A1, 3R01DK107585-05S1 (to SD); R01-AI141630, CA100768 and CA160911 (to PG) and R01-AI 155696 (to PG, DS and SD); R00-CA151673 and R01-GM138385 (to DS), R01- HL32225 (to PT), UCOP-R00RG2642 (to SD and PG), UCOP-R01RG3780 (to P.G. and D.S) and a pilot award from the Sanford Stem Cell Clinical Center at UC San Diego Health (P.G, S.D, D.S). GDK was supported through The American Association of Immunologists Intersect Fellowship Program for Computational Scientists and Immunologists. L.C.A's salary was supported in part by the VA San Diego Healthcare System. This manuscript includes data generated at the UC San Diego Institute of Genomic Medicine (IGC) using an Illumina NovaSeq 6000 that was purchased with funding from a National Institutes of Health SIG grant (#S10 OD026929).

Taskforce report on the diagnosis and clinical management of COVID-19 associated pulmonary aspergillosis.

PURPOSE: Invasive pulmonary aspergillosis (IPA) is increasingly reported in patients with severe coronavirus disease 2019 (COVID-19) admitted to the intensive care unit (ICU). Diagnosis and management of COVID-19 associated pulmonary aspergillosis (CAPA) are challenging and our aim was to develop practical guidance. METHODS: A group of 28 international experts reviewed current insights in the epidemiology, diagnosis and management of CAPA and developed recommendations using GRADE methodology. RESULTS: The prevalence of CAPA varied between 0 and 33%, which may be partly due to variable case definitions, but likely represents true variation. Bronchoscopy and bronchoalveolar lavage (BAL) remain the cornerstone of CAPA diagnosis, allowing for diagnosis of invasive Aspergillus tracheobronchitis and collection of the best validated specimen for Aspergillus diagnostics. Most patients diagnosed with CAPA lack traditional host factors, but pre-existing structural lung disease and immunomodulating therapy may predispose to CAPA risk. Computed tomography seems to be of limited value to rule CAPA in or out, and serum biomarkers are negative in 85% of patients. As the mortality of CAPA is around 50%, antifungal therapy is recommended for BAL positive patients, but the decision to treat depends on the patients' clinical condition and the institutional incidence of CAPA. We recommend against routinely stopping concomitant corticosteroid or IL-6 blocking therapy in CAPA patients. CONCLUSION: CAPA is a complex disease involving a continuum of respiratory colonization, tissue invasion and angioinvasive disease. Knowledge gaps including true epidemiology, optimal diagnostic work-up, management strategies and role of host-directed therapy require further study.

AI-guided discovery of the invariant host response to viral pandemics.

BACKGROUND: Coronavirus Disease 2019 (Covid-19) continues to challenge the limits of our knowledge and our healthcare system. Here we sought to define the host immune response, a.k.a, the "cytokine storm" that has been implicated in fatal COVID-19 using an AI-based approach. METHOD: Over 45,000 transcriptomic datasets of viral pandemics were analyzed to extract a 166-gene signature using ACE2 as a 'seed' gene; ACE2 was rationalized because it encodes the receptor that facilitates the entry of SARS-CoV-2 (the virus that causes COVID-19) into host cells. An AI-based approach was used to explore the utility of the signature in navigating the uncharted territory of Covid-19, setting therapeutic goals, and finding therapeutic solutions. FINDINGS: The 166-gene signature was surprisingly conserved across all viral pandemics, including COVID-19, and a subset of 20-genes classified disease severity, inspiring the nomenclatures ViP and severe-ViP signatures, respectively. The ViP signatures pinpointed a paradoxical phenomenon wherein lung epithelial and myeloid cells mount an IL15 cytokine storm, and epithelial and NK cell senescence and apoptosis determine severity/fatality. Precise therapeutic goals could be formulated; these goals were met in high-dose SARS-CoV-2-challenged hamsters using either neutralizing antibodies that abrogate SARS-CoV-2•ACE2 engagement or a directly acting antiviral agent, EIDD-2801. IL15/IL15RA were elevated in the lungs of patients with fatal disease, and plasma levels of the cytokine prognosticated disease severity. INTERPRETATION: The ViP signatures provide a quantitative and qualitative framework for titrating the immune response in viral pandemics and may serve as a powerful unbiased tool to rapidly assess disease severity and vet candidate drugs. FUNDING: This work was supported by the National Institutes for Health (NIH) [grants CA151673 and GM138385 (to DS) and AI141630 (to P.G), DK107585-05S1 (SD) and AI155696 (to P.G, D.S and S.D), U19-AI142742 (to S. C, CCHI: Cooperative Centers for Human Immunology)]; Research Grants Program Office (RGPO) from the University of California Office of the President (UCOP) (R00RG2628 & R00RG2642 to P.G, D.S and S.D); the UC San Diego Sanford Stem Cell Clinical Center (to P.G, D.S and S.D); LJI Institutional Funds (to S.C); the VA San Diego Healthcare System Institutional funds (to L.C.A). GDK was supported through The American Association of Immunologists Intersect Fellowship Program for Computational Scientists and Immunologists. ONE SENTENCE SUMMARY: The host immune response in COVID-19.

Drug repurposing screens identify chemical entities for the development of COVID-19 interventions.

The ongoing pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), necessitates strategies to identify prophylactic and therapeutic drug candidates for rapid clinical deployment. Here, we describe a screening pipeline for the discovery of efficacious SARS-CoV-2 inhibitors. We screen a best-in-class drug repurposing library, ReFRAME, against two high-throughput, high-content imaging infection assays: one using HeLa cells expressing SARS-CoV-2 receptor ACE2 and the other using lung epithelial Calu-3 cells. From nearly 12,000 compounds, we identify 49 (in HeLa-ACE2) and 41 (in Calu-3) compounds capable of selectively inhibiting SARS-CoV-2 replication. Notably, most screen hits are cell-line specific, likely due to different virus entry mechanisms or host cell-specific sensitivities to modulators. Among these promising hits, the antivirals nelfinavir and the parent of prodrug MK-4482 possess desirable in vitro activity, pharmacokinetic and human safety profiles, and both reduce SARS-CoV-2 replication in an orthogonal human differentiated primary cell model. Furthermore, MK-4482 effectively blocks SARS-CoV-2 infection in a hamster model. Overall, we identify direct-acting antivirals as the most promising compounds for drug repurposing, additional compounds that may have value in combination therapies, and tool compounds for identification of viral host cell targets.

Cross-reactive serum and memory B-cell responses to spike protein in SARS-CoV-2 and endemic coronavirus infection.

Pre-existing immunity to seasonal endemic coronaviruses could have profound consequences for antibody responses to SARS-CoV-2, induced from natural infection or vaccination. A first step to establish whether pre-existing responses can impact SARS-CoV-2 infection is to understand the nature and extent of cross-reactivity in humans to coronaviruses. Here we compare serum antibody and memory B cell responses to coronavirus spike proteins from pre-pandemic and SARS-CoV-2 convalescent donors using binding and functional assays. We show weak evidence of pre-existing SARS-CoV-2 cross-reactive serum antibodies in pre-pandemic donors. However, we find evidence of pre-existing cross-reactive memory B cells that are activated during SARS-CoV-2 infection. Monoclonal antibodies show varying degrees of cross-reactivity with betacoronaviruses, including SARS-CoV-1 and endemic coronaviruses. We identify one cross-reactive neutralizing antibody specific to the S2 subunit of the S protein. Our results suggest that pre-existing immunity to endemic coronaviruses should be considered in evaluating antibody responses to SARS-CoV-2.

Antiviral drug screen identifies DNA-damage response inhibitor as potent blocker of SARS-CoV-2 replication.

SARS-CoV-2 has currently precipitated the COVID-19 global health crisis. We developed a medium-throughput drug-screening system and identified a small-molecule library of 34 of 430 protein kinase inhibitors that were capable of inhibiting the SARS-CoV-2 cytopathic effect in human epithelial cells. These drug inhibitors are in various stages of clinical trials. We detected key proteins involved in cellular signaling pathways mTOR-PI3K-AKT, ABL-BCR/MAPK, and DNA-damage response that are critical for SARS-CoV-2 infection. A drug-protein interaction-based secondary screen confirmed compounds, such as the ATR kinase inhibitor berzosertib and torin2 with anti-SARS-CoV-2 activity. Berzosertib exhibited potent antiviral activity against SARS-CoV-2 in multiple cell types and blocked replication at the post-entry step. Berzosertib inhibited replication of SARS-CoV-1 and the Middle East respiratory syndrome coronavirus (MERS-CoV) as well. Our study highlights key promising kinase inhibitors to constrain coronavirus replication as a host-directed therapy in the treatment of COVID-19 and beyond as well as provides an important mechanism of host-pathogen interactions.

Development of a multi-antigenic SARS-CoV-2 vaccine candidate using a synthetic poxvirus platform.

Modified Vaccinia Ankara (MVA) is a highly attenuated poxvirus vector that is widely used to develop vaccines for infectious diseases and cancer. We demonstrate the construction of a vaccine platform based on a unique three-plasmid system to efficiently generate recombinant MVA vectors from chemically synthesized DNA. In response to the ongoing global pandemic caused by SARS coronavirus-2 (SARS-CoV-2), we use this vaccine platform to rapidly produce fully synthetic MVA (sMVA) vectors co-expressing SARS-CoV-2 spike and nucleocapsid antigens, two immunodominant antigens implicated in protective immunity. We show that mice immunized with these sMVA vectors develop robust SARS-CoV-2 antigen-specific humoral and cellular immune responses, including potent neutralizing antibodies. These results demonstrate the potential of a vaccine platform based on synthetic DNA to efficiently generate recombinant MVA vectors and to rapidly develop a multi-antigenic poxvirus-based SARS-CoV-2 vaccine candidate.

Cross-reactive serum and memory B cell responses to spike protein in SARS-CoV-2 and endemic coronavirus infection.

Pre-existing immune responses to seasonal endemic coronaviruses could have profound consequences for antibody responses to SARS-CoV-2, either induced in natural infection or through vaccination. Such consequences are well established in the influenza and flavivirus fields. A first step to establish whether pre-existing responses can impact SARS-CoV-2 infection is to understand the nature and extent of cross-reactivity in humans to coronaviruses. We compared serum antibody and memory B cell responses to coronavirus spike (S) proteins from pre-pandemic and SARS-CoV-2 convalescent donors using a series of binding and functional assays. We found weak evidence of pre-existing SARS-CoV-2 cross-reactive serum antibodies in pre-pandemic donors. However, we found stronger evidence of pre-existing cross-reactive memory B cells that were activated on SARS-CoV-2 infection. Monoclonal antibodies (mAbs) isolated from the donors showed varying degrees of cross-reactivity with betacoronaviruses, including SARS and endemic coronaviruses. None of the cross-reactive mAbs were neutralizing except for one that targeted the S2 subunit of the S protein. The results suggest that pre-existing immunity to endemic coronaviruses should be considered in evaluating antibody responses to SARS-CoV-2.

AI-guided discovery of the invariant host response to viral pandemics.

We sought to define the host immune response, a.k.a, the "cytokine storm" that has been implicated in fatal COVID-19 using an AI-based approach. Over 45,000 transcriptomic datasets of viral pandemics were analyzed to extract a 166-gene signature using ACE2 as a 'seed' gene; ACE2 was rationalized because it encodes the receptor that facilitates the entry of SARS-CoV-2 (the virus that causes COVID-19) into host cells. Surprisingly, this 166-gene signature was conserved in all vi ral p andemics, including COVID-19, and a subset of 20-genes classified disease severity, inspiring the nomenclatures ViP and severe-ViP signatures, respectively. The ViP signatures pinpointed a paradoxical phenomenon wherein lung epithelial and myeloid cells mount an IL15 cytokine storm, and epithelial and NK cell senescence and apoptosis determines severity/fatality. Precise therapeutic goals were formulated and subsequently validated in high-dose SARS-CoV-2-challenged hamsters using neutralizing antibodies that abrogate SARS-CoV-2•ACE2 engagement or a directly acting antiviral agent, EIDD-2801. IL15/IL15RA were elevated in the lungs of patients with fatal disease, and plasma levels of the cytokine tracked with disease severity. Thus, the ViP signatures provide a quantitative and qualitative framework for titrating the immune response in viral pandemics and may serve as a powerful unbiased tool to rapidly assess disease severity and vet candidate drugs. ONE SENTENCE SUMMARY: The host immune response in COVID-19. PANEL RESEARCH IN CONTEXT: Evidence before this study: The SARS-CoV-2 pandemic has inspired many groups to find innovative methodologies that can help us understand the host immune response to the virus; unchecked proportions of such immune response have been implicated in fatality. We searched GEO and ArrayExpress that provided many publicly available gene expression data that objectively measure the host immune response in diverse conditions. However, challenges remain in identifying a set of host response events that are common to every condition. There are no studies that provide a reproducible assessment of prognosticators of disease severity, the host response, and therapeutic goals. Consequently, therapeutic trials for COVID-19 have seen many more 'misses' than 'hits'. This work used multiple (> 45,000) gene expression datasets from GEO and ArrayExpress and analyzed them using an unbiased computational approach that relies upon fundamentals of gene expression patterns and mathematical precision when assessing them.Added value of this study: This work identifies a signature that is surprisingly conserved in all viral pandemics, including Covid-19, inspiring the nomenclature ViP-signature. A subset of 20-genes classified disease severity in respiratory pandemics. The ViP signatures pinpointed the nature and source of the 'cytokine storm' mounted by the host. They also helped formulate precise therapeutic goals and rationalized the repurposing of FDA-approved drugs.Implications of all the available evidence: The ViP signatures provide a quantitative and qualitative framework for assessing the immune response in viral pandemics when creating pre-clinical models; they serve as a powerful unbiased tool to rapidly assess disease severity and vet candidate drugs.