Viral replication in human macrophages enhances an inflammatory cascade and interferon driven chronic COVID-19 in humanized mice.

Severe COVID-19 is characterized by persistent lung inflammation, inflammatory cytokine production, viral RNA, and sustained interferon (IFN) response all of which are recapitulated and required for pathology in the SARS-CoV-2 infected MISTRG6-hACE2 humanized mouse model of COVID-19 with a human immune system1-20. Blocking either viral replication with Remdesivir21-23 or the downstream IFN stimulated cascade with anti-IFNAR2 in vivo in the chronic stages of disease attenuated the overactive immune-inflammatory response, especially inflammatory macrophages. Here, we show SARS-CoV-2 infection and replication in lung-resident human macrophages is a critical driver of disease. In response to infection mediated by CD16 and ACE2 receptors, human macrophages activate inflammasomes, release IL-1 and IL-18 and undergo pyroptosis thereby contributing to the hyperinflammatory state of the lungs. Inflammasome activation and its accompanying inflammatory response is necessary for lung inflammation, as inhibition of the NLRP3 inflammasome pathway reverses chronic lung pathology. Remarkably, this same blockade of inflammasome activation leads to the release of infectious virus by the infected macrophages. Thus, inflammasomes oppose host infection by SARS-CoV-2 by production of inflammatory cytokines and suicide by pyroptosis to prevent a productive viral cycle.

A stem-loop RNA RIG-I agonist confers prophylactic and therapeutic protection against acute and chronic SARS-CoV-2 infection in mice

As SARS-CoV-2 continues to cause morbidity and mortality around the world, there is an urgent need for the development of effective medical countermeasures. Here, we assessed the antiviral capacity of a minimal RIG-I agonist, stem-loop RNA 14 (SLR14), in viral control, disease prevention, post-infection therapy, and cross-variant protection in mouse models of SARS-CoV-2 infection. A single dose of SLR14 prevented viral replication in the lower respiratory tract and development of severe disease in a type I interferon (IFN-I) dependent manner. SLR14 demonstrated remarkable protective capacity against lethal SARS-CoV-2 infection when used prophylactically and retained considerable efficacy as a therapeutic agent. In immunodeficient mice carrying chronic SARS-CoV-2 infection, SLR14 elicited near-sterilizing innate immunity by inducing IFN-I responses in the absence of the adaptive immune system. In the context of infection with variants of concern (VOC), SLR14 conferred broad protection and uncovered an IFN-I resistance gradient across emerging VOC. These findings demonstrate the therapeutic potential of SLR14 as a host-directed, broad-spectrum antiviral for early post-exposure treatment and for treatment of chronically infected immunosuppressed patients.

High-affinity, neutralizing antibodies to SARS-CoV-2 can be made in the absence of T follicular helper cells

T follicular helper (Tfh) cells are the conventional drivers of protective, germinal center (GC)-based antiviral antibody responses. However, loss of Tfh cells and GCs has been observed in patients with severe COVID-19. As T cell-B cell interactions and immunoglobulin class switching still occur in these patients, non-canonical pathways of antibody production may be operative during SARS-CoV-2 infection. We found that both Tfh-dependent and -independent antibodies were induced against SARS-CoV-2 as well as influenza A virus. Tfh-independent responses were mediated by a population we call lymph node (LN)-Th1 cells, which remain in the LN and interact with B cells outside of GCs to promote high-affinity but broad-spectrum antibodies. Strikingly, antibodies generated in the presence and absence of Tfh cells displayed similar neutralization potency against homologous SARS-CoV-2 as well as the B.1.351 variant of concern. These data support a new paradigm for the induction of B cell responses during viral infection that enables effective, neutralizing antibody production to complement traditional GCs and even compensate for GCs damaged by viral inflammation. One-Sentence SummaryComplementary pathways of antibody production mediate neutralizing responses to SARS-CoV-2.

Adaptive immune determinants of viral clearance and protection in mouse models of SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused more than 160 million infections and more than 3 million deaths worldwide. While effective vaccines are currently being deployed, the adaptive immune determinants which promote viral clearance and confer protection remain poorly defined. Using mouse models of SARS-CoV-2, we demonstrate that both humoral and cellular adaptive immunity contributes to viral clearance in the setting of primary infection. Furthermore, we find that either convalescent mice, or mice that receive mRNA vaccination are protected from both homologous infection and infection with a variant of concern, B.1.351. Additionally, we find this protection to be largely mediated by antibody response and not cellular immunity. These results highlight the in vivo protective capacity of antibodies generated to both vaccine and natural infection. One-Sentence SummaryDefining the roles of humoral and cellular adaptive immunity in viral clearance and protection from SARS-CoV-2 and a variant of concern.

Longitudinal immune profiling of a SARS-CoV-2 reinfection in a solid organ transplant recipient

Prior to the emergence of antigenically distinct SARS-CoV-2 variants, reinfections were reported infrequently - presumably due to the generation of durable and protective immune responses. However, case reports also suggested that rare, repeated infections may occur as soon as 48 days following initial disease onset. The underlying immunologic deficiencies enabling SARS-CoV-2 reinfections are currently unknown. Here we describe a renal transplant recipient who developed recurrent, symptomatic SARS-CoV-2 infection - confirmed by whole virus genome sequencing - 7 months after primary infection. To elucidate the immunological mechanisms responsible for SARS-CoV-2 reinfection, we performed longitudinal profiling of cellular and humoral responses during both primary and recurrent SARS-CoV-2 infection. We found that the patient responded to the primary infection with transient, poor-quality adaptive immune responses. The patients immune system was further compromised by intervening treatment for acute rejection of the renal allograft prior to reinfection. Importantly, we also identified the development of neutralizing antibodies and the formation of humoral memory responses prior to SARS-CoV-2 reinfection. However, these neutralizing antibodies failed to confer protection against reinfection, suggesting that additional factors are required for efficient prevention of SARS-CoV-2 reinfection. Further, we found no evidence supporting viral evasion of primary adaptive immune responses, suggesting that susceptibility to reinfection may be determined by host factors rather than pathogen adaptation in this patient. In summary, our study suggests that a low neutralizing antibody presence alone is not sufficient to confer resistance against reinfection. Thus, patients with solid organ transplantation, or patients who are otherwise immunosuppressed, who recover from infection with SARS-CoV-2 may not develop sufficient protective immunity and are at risk of reinfection.

Saliva viral load is a dynamic unifying correlate of COVID-19 severity and mortality

While several clinical and immunological parameters correlate with disease severity and mortality in SARS-CoV-2 infection, work remains in identifying unifying correlates of coronavirus disease 2019 (COVID-19) that can be used to guide clinical practice. Here, we examine saliva and nasopharyngeal (NP) viral load over time and correlate them with patient demographics, and cellular and immune profiling. We found that saliva viral load was significantly higher in those with COVID-19 risk factors; that it correlated with increasing levels of disease severity and showed a superior ability over nasopharyngeal viral load as a predictor of mortality over time (AUC=0.90). A comprehensive analysis of immune factors and cell subsets revealed strong predictors of high and low saliva viral load, which were associated with increased disease severity or better overall outcomes, respectively. Saliva viral load was positively associated with many known COVID-19 inflammatory markers such as IL-6, IL-18, IL-10, and CXCL10, as well as type 1 immune response cytokines. Higher saliva viral loads strongly correlated with the progressive depletion of platelets, lymphocytes, and effector T cell subsets including circulating follicular CD4 T cells (cTfh). Anti-spike (S) and anti-receptor binding domain (RBD) IgG levels were negatively correlated with saliva viral load showing a strong temporal association that could help distinguish severity and mortality in COVID-19. Finally, patients with fatal COVID-19 exhibited higher viral loads, which correlated with the depletion of cTfh cells, and lower production of anti-RBD and anti-S IgG levels. Together these results demonstrated that viral load - as measured by saliva but not nasopharyngeal -- is a dynamic unifying correlate of disease presentation, severity, and mortality over time.

Kinetics of antibody responses dictate COVID-19 outcome

Recent studies have provided insights into innate and adaptive immune dynamics in coronavirus disease 2019 (COVID-19). Yet, the exact feature of antibody responses that governs COVID-19 disease outcomes remain unclear. Here, we analysed humoral immune responses in 209 asymptomatic, mild, moderate and severe COVID-19 patients over time to probe the nature of antibody responses in disease severity and mortality. We observed a correlation between anti-Spike (S) IgG levels, length of hospitalization and clinical parameters associated with worse clinical progression. While high anti-S IgG levels correlated with worse disease severity, such correlation was time-dependent. Deceased patients did not have higher overall humoral response than live discharged patients. However, they mounted a robust, yet delayed response, measured by anti-S, anti-RBD IgG, and neutralizing antibody (NAb) levels, compared to survivors. Delayed seroconversion kinetics correlated with impaired viral control in deceased patients. Finally, while sera from 89% of patients displayed some neutralization capacity during their disease course, NAb generation prior to 14 days of disease onset emerged as a key factor for recovery. These data indicate that COVID-19 mortality does not correlate with the cross-sectional antiviral antibody levels per se, but rather with the delayed kinetics of NAb production.

Diverse Functional Autoantibodies in Patients with COVID-19

COVID-19 manifests with a wide spectrum of clinical phenotypes that are characterized by exaggerated and misdirected host immune responses1-8. While pathological innate immune activation is well documented in severe disease1, the impact of autoantibodies on disease progression is less defined. Here, we used a high-throughput autoantibody discovery technique called Rapid Extracellular Antigen Profiling (REAP) to screen a cohort of 194 SARS-CoV-2 infected COVID-19 patients and healthcare workers for autoantibodies against 2,770 extracellular and secreted proteins (the "exoproteome"). We found that COVID-19 patients exhibit dramatic increases in autoantibody reactivities compared to uninfected controls, with a high prevalence of autoantibodies against immunomodulatory proteins including cytokines, chemokines, complement components, and cell surface proteins. We established that these autoantibodies perturb immune function and impair virological control by inhibiting immunoreceptor signaling and by altering peripheral immune cell composition, and found that murine surrogates of these autoantibodies exacerbate disease severity in a mouse model of SARS-CoV-2 infection. Analysis of autoantibodies against tissue-associated antigens revealed associations with specific clinical characteristics and disease severity. In summary, these findings implicate a pathological role for exoproteome-directed autoantibodies in COVID-19 with diverse impacts on immune functionality and associations with clinical outcomes.

Multiscale PHATE Exploration of SARS-CoV-2 Data Reveals Multimodal Signatures of Disease

1The biomedical community is producing increasingly high dimensional datasets, integrated from hundreds of patient samples, which current computational techniques struggle to explore. To uncover biological meaning from these complex datasets, we present an approach called Multiscale PHATE, which learns abstracted biological features from data that can be directly predictive of disease. Built on a continuous coarse graining process called diffusion condensation, Multiscale PHATE creates a tree of data granularities that can be cut at coarse levels for high level summarizations of data, as well as at fine levels for detailed representations on subsets. We apply Multiscale PHATE to study the immune response to COVID-19 in 54 million cells from 168 hospitalized patients. Through our analysis of patient samples, we identify CD16hi CD66blo neutrophil and IFN{gamma}+GranzymeB+ Th17 cell responses enriched in patients who die. Further, we show that population groupings Multiscale PHATE discovers can be directly fed into a classifier to predict disease outcome. We also use Multiscale PHATE-derived features to construct two different manifolds of patients, one from abstracted flow cytometry features and another directly on patient clinical features, both associating immune subsets and clinical markers with outcome.

Immunologically distinct responses occur in the CNS of COVID-19 patients

One third of COVID-19 patients develop significant neurological symptoms, yet SARS-CoV-2 is rarely detected in central nervous system (CNS) tissue, suggesting a potential role for parainfectious processes, including neuroimmune responses. We therefore examined immune parameters in cerebrospinal fluid (CSF) and blood samples from a cohort of patients with COVID-19 and significant neurological complications. We found divergent immunological responses in the CNS compartment, including increased levels of IL-12 and IL-12-associated innate and adaptive immune cell activation. Moreover, we found increased proportions of B cells in the CSF relative to the periphery and evidence of clonal expansion of CSF B cells, suggesting a divergent intrathecal humoral response to SARS-CoV-2. Indeed, all COVID-19 cases examined had anti-SARS-CoV-2 IgG antibodies in the CSF whose target epitopes diverged from serum antibodies. We directly examined whether CSF resident antibodies target self-antigens and found a significant burden of CNS autoimmunity, with the CSF from most patients recognizing neural self-antigens. Finally, we produced a panel of monoclonal antibodies from patients CSF and show that these target both anti-viral and anti-neural antigens--including one mAb specific for the spike protein that also recognizes neural tissue. This exploratory immune survey reveals evidence of a compartmentalized and self-reactive immune response in the CNS meriting a more systematic evaluation of neurologically impaired COVID-19 patients. One Sentence SummaryA subset of COVID-19 patients with neurologic impairment show cerebrospinal fluid-specific immune alterations that point to both neuroinvasion and anti-neural autoimmunity as potential causes of impairment.

Kynurenic acid underlies sex-specific immune responses to COVID-19

Coronavirus disease-2019 (COVID-19) has poorer clinical outcomes in males compared to females, and immune responses underlie these sex-related differences in disease trajectory. As immune responses are in part regulated by metabolites, we examined whether the serum metabolome has sex-specificity for immune responses in COVID-19. In males with COVID-19, kynurenic acid (KA) and a high KA to kynurenine (K) ratio was positively correlated with age, inflammatory cytokines, and chemokines and was negatively correlated with T cell responses, revealing that KA production is linked to immune responses in males. Males that clinically deteriorated had a higher KA:K ratio than those that stabilized. In females with COVID-19, this ratio positively correlated with T cell responses and did not correlate with age or clinical severity. KA is known to inhibit glutamate release, and we observed that serum glutamate is lower in patients that deteriorate from COVID-19 compared to those that stabilize, and correlates with immune responses. Analysis of Genotype-Tissue Expression (GTEx) data revealed that expression of kynurenine aminotransferase, which regulates KA production, correlates most strongly with cytokine levels and aryl hydrocarbon receptor activation in older males. This study reveals that KA has a sex-specific link to immune responses and clinical outcomes, in COVID-19 infection.

Longitudinal immunological analyses reveal inflammatory misfiring in severe COVID-19 patients

Recent studies have provided insights into the pathogenesis of coronavirus disease 2019 (COVID-19)1-4. Yet, longitudinal immunological correlates of disease outcome remain unclear. Here, we serially analysed immune responses in 113 COVID-19 patients with moderate (non-ICU) and severe (ICU) disease. Immune profiling revealed an overall increase in innate cell lineages with a concomitant reduction in T cell number. We identify an association between early, elevated cytokines and worse disease outcomes. Following an early increase in cytokines, COVID-19 patients with moderate disease displayed a progressive reduction in type-1 (antiviral) and type-3 (antifungal) responses. In contrast, patients with severe disease maintained these elevated responses throughout the course of disease. Moreover, severe disease was accompanied by an increase in multiple type 2 (anti-helminths) effectors including, IL-5, IL-13, IgE and eosinophils. Unsupervised clustering analysis of plasma and peripheral blood leukocyte data identified 4 immune signatures, representing (A) growth factors, (B) type-2/3 cytokines, (C) mixed type-1/2/3 cytokines, and (D) chemokines that correlated with three distinct disease trajectories of patients. The immune profile of patients who recovered with moderate disease was enriched in tissue reparative growth factor signature (A), while the profile for those with worsened disease trajectory had elevated levels of all four signatures. Thus, we identified development of a maladapted immune response profile associated with severe COVID-19 outcome and early immune signatures that correlate with divergent disease trajectories.

Sex differences in immune responses to SARS-CoV-2 that underlie disease outcomes

A growing body of evidence indicates sex differences in the clinical outcomes of coronavirus disease 2019 (COVID-19)1-4. However, whether immune responses against SARS-CoV-2 differ between sexes, and whether such differences explain male susceptibility to COVID-19, is currently unknown. In this study, we examined sex differences in viral loads, SARS-CoV-2-specific antibody titers, plasma cytokines, as well as blood cell phenotyping in COVID-19 patients. By focusing our analysis on patients with mild to moderate disease who had not received immunomodulatory medications, our results revealed that male patients had higher plasma levels of innate immune cytokines and chemokines including IL-8, IL-18, and CCL5, along with more robust induction of non-classical monocytes. In contrast, female patients mounted significantly more robust T cell activation than male patients during SARS-CoV-2 infection, which was sustained in old age. Importantly, we found that a poor T cell response negatively correlated with patients age and was predictive of worse disease outcome in male patients, but not in female patients. Conversely, higher innate immune cytokines in female patients associated with worse disease progression, but not in male patients. These findings reveal a possible explanation underlying observed sex biases in COVID-19, and provide important basis for the development of sex-based approach to the treatment and care of men and women with COVID-19.

Neuroinvasive potential of SARS-CoV-2 revealed in a human brain organoid model

Although COVID-19 is considered to be primarily a respiratory disease, SARS-CoV-2 affects multiple organ systems including the central nervous system (CNS). Yet, there is no consensus whether the virus can infect the brain, or what the consequences of CNS infection are. Here, we used three independent approaches to probe the capacity of SARS-CoV-2 to infect the brain. First, using human brain organoids, we observed clear evidence of infection with accompanying metabolic changes in the infected and neighboring neurons. However, no evidence for the type I interferon responses was detected. We demonstrate that neuronal infection can be prevented either by blocking ACE2 with antibodies or by administering cerebrospinal fluid from a COVID-19 patient. Second, using mice overexpressing human ACE2, we demonstrate in vivo that SARS-CoV-2 neuroinvasion, but not respiratory infection, is associated with mortality. Finally, in brain autopsy from patients who died of COVID-19, we detect SARS-CoV-2 in the cortical neurons, and note pathologic features associated with infection with minimal immune cell infiltrates. These results provide evidence for the neuroinvasive capacity of SARS-CoV2, and an unexpected consequence of direct infection of neurons by SARS-CoV-2.

Detection of SARS-CoV-2 RNA by multiplex RT-qPCR

The current RT-qPCR assay recommended for SARS-CoV-2 testing in the United States requires analysis of three genomic targets per sample: two viral and one host. To simplify testing and reduce the volume of required reagents, we developed a multiplex RT-qPCR assay to detect SARS-CoV-2 in a single reaction. We used existing N1, N2, and RP primer and probe sets by the CDC, but substituted fluorophores to allow multiplexing of the assay. The cycle threshold (Ct) values of our multiplex RT-qPCR were comparable to those obtained by the singleplex assay adapted for research purposes. Low copies (>500 copies / reaction) of SARS-CoV-2 RNA were consistently detected by the multiplex RT-qPCR. Our novel multiplex RT-qPCR improves upon current singleplex diagnostics by saving reagents, costs, time and labor.