The I-SPY COVID Adaptive Platform Trial for COVID-19 Acute Respiratory Failure: Rationale, Design and Operations

IntroductionThe COVID-19 pandemic brought an urgent need to discover novel effective therapeutics for patients hospitalized with severe COVID-19. The ISPY COVID trial was designed and implemented in early 2020 to evaluate investigational agents rapidly and simultaneously on a phase 2 adaptive platform. This manuscript outlines the design, rationale, implementation, and challenges of the ISPY COVID trial during the first phase of trial activity from April 2020 until December 2021. Methods and analysisThe ISPY COVID Trial is a multi-center open label phase 2 platform trial in the United States designed to evaluate therapeutics that may have a large effect on improving outcomes from severe COVID-19. The ISPY COVID Trial network includes academic and community hospitals with significant geographic diversity across the country. Enrolled patients are randomized to receive one of up to four investigational agents or a control and are evaluated for a family of two primary outcomes--time to recovery and mortality. The statistical design uses a Bayesian model with "stopping" and "graduation" criteria designed to efficiently discard ineffective therapies and graduate promising agents for definitive efficacy trials. Each investigational agent arm enrolls to a maximum of 125 patients per arm and is compared to concurrent controls. As of December 2021, 11 investigational agent arms had been activated, and 8 arms were complete. Enrollment and adaptation of the trial design is ongoing. Ethics and disseminationISPY COVID operates under a central institutional review board via Wake Forest School of Medicine IRB00066805. Data generated from this trial will be reported in peer reviewed medical journals. Trial registration numberClinicaltrials.gov registration number NCT04488081 Strengths and limitations of this studyO_LIThe ISPY COVID Trial was developed in early 2020 to rapidly and simultaneously evaluate therapeutics for severe COVID-19 on an adaptive open label phase 2 platform C_LIO_LIThe ISPY COVID Adaptive Platform Trial Network is an academic-industry partnership that includes academic and community hospitals spanning a wide geographic area across the United States C_LIO_LIOf December 2021, 11 investigational agent arms have been activated on the ISPY COVID Trial Platform C_LIO_LIThe ISPY COVID Trial was designed to identify therapeutic agents with a large clinical effect for further testing in definitive efficacy trials--limitations to this approach include the risk of a type 2 error C_LI

Signaling through FcγRIIA and the C5a-C5aR pathway mediates platelet hyperactivation in COVID-19

Patients with COVID-19 present with a wide variety of clinical manifestations. Thromboembolic events constitute a significant cause of morbidity and mortality in patients infected with SARS-CoV-2. Severe COVID-19 has been associated with hyperinflammation and pre-existing cardiovascular disease. Platelets are important mediators and sensors of inflammation and are directly affected by cardiovascular stressors. In this report, we found that platelets from severely ill, hospitalized COVID-19 patients exhibit higher basal levels of activation measured by P-selectin surface expression, and have a poor functional reserve upon in vitro stimulation. Correlating clinical features to the ability of plasma from COVID-19 patients to stimulate control platelets identified ferritin as a pivotal clinical marker associated with platelet hyperactivation. The COVID-19 plasma-mediated effect on control platelets was highest for patients that subsequently developed inpatient thrombotic events. Proteomic analysis of plasma from COVID-19 patients identified key mediators of inflammation and cardiovascular disease that positively correlated with in vitro platelet activation. Mechanistically, blocking the signaling of the Fc{gamma}RIIa-Syk and C5a-C5aR pathways on platelets, using antibody-mediated neutralization, IgG depletion or the Syk inhibitor fostamatinib, reversed this hyperactivity driven by COVID-19 plasma and prevented platelet aggregation in endothelial microfluidic chamber conditions, thus identifying these potentially actionable pathways as central for platelet activation and/or vascular complications in COVID-19 patients. In conclusion, we reveal a key role of platelet-mediated immunothrombosis in COVID-19 and identify distinct, clinically relevant, targetable signaling pathways that mediate this effect. These studies have implications for the role of platelet hyperactivation in complications associated with SARS-CoV-2 infection. Cover illustration O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY One-sentence summaryThe Fc{gamma}RIIA and C5a-C5aR pathways mediate platelet hyperactivation in COVID-19

Signatures of COVID-19 severity and immune response in the respiratory tract microbiome

RationaleViral infection of the respiratory tract can be associated with propagating effects on the airway microbiome, and microbiome dysbiosis may influence viral disease. ObjectiveTo define the respiratory tract microbiome in COVID-19 and relationship disease severity, systemic immunologic features, and outcomes. Methods and MeasurementsWe examined 507 oropharyngeal, nasopharyngeal and endotracheal samples from 83 hospitalized COVID-19 patients, along with non-COVID patients and healthy controls. Bacterial communities were interrogated using 16S rRNA gene sequencing, commensal DNA viruses Anelloviridae and Redondoviridae were quantified by qPCR, and immune features were characterized by lymphocyte/neutrophil (L/N) ratios and deep immune profiling of peripheral blood mononuclear cells (PBMC). Main ResultsCOVID-19 patients had upper respiratory microbiome dysbiosis, and greater change over time than critically ill patients without COVID-19. Diversity at the first time point correlated inversely with disease severity during hospitalization, and microbiome composition was associated with L/N ratios and PBMC profiles in blood. Intubated patients showed patient-specific and dynamic lung microbiome communities, with prominence of Staphylococcus. Anelloviridae and Redondoviridae showed more frequent colonization and higher titers in severe disease. Machine learning analysis demonstrated that integrated features of the microbiome at early sampling points had high power to discriminate ultimate level of COVID-19 severity. ConclusionsThe respiratory tract microbiome and commensal virome are disturbed in COVID-19, correlate with systemic immune parameters, and early microbiome features discriminate disease severity. Future studies should address clinical consequences of airway dysbiosis in COVID-19, possible use as biomarkers, and role of bacterial and viral taxa identified here in COVID-19 pathogenesis.

New-Onset IgG Autoantibodies in Hospitalized Patients with COVID-19

Coronavirus Disease 2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), is associated with a wide range of clinical manifestations, including autoimmune features and autoantibody production. We developed three different protein arrays to measure hallmark IgG autoantibodies associated with Connective Tissue Diseases (CTDs), Anti-Cytokine Antibodies (ACA), and anti-viral antibody responses in 147 hospitalized COVID-19 patients in three different centers. Autoantibodies were identified in approximately 50% of patients, but in <15% of healthy controls. When present, autoantibodies largely targeted autoantigens associated with rare disorders such as myositis, systemic sclerosis and CTD overlap syndromes. Anti-nuclear antibodies (ANA) were observed in [~]25% of patients. Patients with autoantibodies tended to demonstrate one or a few specificities whereas ACA were even more prevalent, and patients often had antibodies to multiple cytokines. Rare patients were identified with IgG antibodies against angiotensin converting enzyme-2 (ACE-2). A subset of autoantibodies and ACA developed de novo following SARS-CoV-2 infection while others were transient. Autoantibodies tracked with longitudinal development of IgG antibodies that recognized SARS-CoV-2 structural proteins such as S1, S2, M, N and a subset of non-structural proteins, but not proteins from influenza, seasonal coronaviruses or other pathogenic viruses. COVID-19 patients with one or more autoantibodies tended to have higher levels of antibodies against SARS-CoV-2 Nonstructural Protein 1 (NSP1) and Methyltransferase (ME). We conclude that SARS-CoV-2 causes development of new-onset IgG autoantibodies in a significant proportion of hospitalized COVID-19 patients and are positively correlated with immune responses to SARS-CoV-2 proteins.

Innate lymphoid cells and disease tolerance in SARS-CoV-2 infection

Risk of severe COVID-19 increases with age, is greater in males, and is associated with lymphopenia, but not with higher burden of SARS-CoV-2. It is unknown whether effects of age and sex on abundance of specific lymphoid subsets explain these correlations. This study found that the abundance of innate lymphoid cells (ILCs) decreases more than 7-fold over the human lifespan -- T cell subsets decrease less than 2-fold -- and is lower in males than in females. After accounting for effects of age and sex, ILCs, but not T cells, were lower in adults hospitalized with COVID-19, independent of lymphopenia. Among SARS-CoV-2-infected adults, the abundance of ILCs, but not of T cells, correlated inversely with odds and duration of hospitalization, and with severity of inflammation. ILCs were also uniquely decreased in pediatric COVID-19 and the numbers of these cells did not recover during follow-up. In contrast, children with MIS-C had depletion of both ILCs and T cells, and both cell types increased during follow-up. In both pediatric COVID-19 and MIS-C, ILC abundance correlated inversely with inflammation. Blood ILC mRNA and phenotype tracked closely with ILCs from lung. Importantly, blood ILCs produced amphiregulin, a protein implicated in disease tolerance and tissue homeostasis, and the percentage of amphiregulin-producing ILCs was higher in females than in males. These results suggest that, by promoting disease tolerance, homeostatic ILCs decrease morbidity and mortality associated with SARS-CoV-2 infection, and that lower ILC abundance accounts for increased COVID-19 severity with age and in males.

Seasonal human coronavirus antibodies are boosted upon SARS-CoV-2 infection but not associated with protection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread within the human population. Although SARS-CoV-2 is a novel coronavirus, most humans had been previously exposed to other antigenically distinct common seasonal human coronaviruses (hCoVs) before the COVID-19 pandemic. Here, we quantified levels of SARS-CoV-2-reactive antibodies and hCoV-reactive antibodies in serum samples collected from 204 humans before the COVID-19 pandemic. We then quantified pre-pandemic antibody levels in serum from a separate cohort of 252 individuals who became PCR-confirmed infected with SARS-CoV-2. Finally, we longitudinally measured hCoV and SARS-CoV-2 antibodies in the serum of hospitalized COVID-19 patients. Our studies indicate that most individuals possessed hCoV-reactive antibodies before the COVID-19 pandemic. We determined that [~]23% of these individuals possessed non-neutralizing antibodies that cross-reacted with SARS-CoV-2 spike and nucleocapsid proteins. These antibodies were not associated with protection against SARS-CoV-2 infections or hospitalizations, but paradoxically these hCoV cross-reactive antibodies were boosted upon SARS-CoV-2 infection.

Deep Immune Profiling of MIS-C demonstrates marked but transient immune activation compared to adult and pediatric COVID-19

Pediatric COVID-19 following SARS-CoV-2 infection is associated with fewer hospitalizations and often milder disease than in adults. A subset of children, however, present with Multisystem Inflammatory Syndrome in Children (MIS-C) that can lead to vascular complications and shock, but rarely death. The immune features of MIS-C compared to pediatric COVID-19 or adult disease remain poorly understood. We analyzed peripheral blood immune responses in hospitalized SARS-CoV-2 infected pediatric patients (pediatric COVID-19) and patients with MIS-C. MIS-C patients had patterns of T cell-biased lymphopenia and T cell activation similar to severely ill adults, and all patients with MIS-C had SARS-CoV-2 spike-specific antibodies at admission. A distinct feature of MIS-C patients was robust activation of vascular patrolling CX3CR1+ CD8 T cells that correlated with use of vasoactive medication. Finally, whereas pediatric COVID-19 patients with acute respiratory distress syndrome (ARDS) had sustained immune activation, MIS-C patients displayed clinical improvement over time, concomitant with decreasing immune activation. Thus, non-MIS-C versus MIS-C SARS-CoV-2 associated illnesses are characterized by divergent immune signatures that are temporally distinct and implicate CD8 T cells in clinical presentation and trajectory of MIS-C. One Sentence SummaryMIS-C is defined by generalized lymphocyte activation that corrects during hospitalization, including elevated plasmablast frequencies and marked activation of vascular patrolling CX3CR1+ CD8 T cells.

Erythrocytes Reveal Complement Activation in Patients with COVID-19

COVID-19, the disease caused by the SARS-CoV-2 virus, can progress to multi-organ failure characterized by respiratory insufficiency, arrhythmias, thromboembolic complications and shock 1-5. The mortality of patients hospitalized with COVID-19 is unacceptably high and new strategies are urgently needed to rapidly identify and treat patients at risk for organ failure. Clinical epidemiologic studies demonstrate that vulnerability to organ failure is greatest after viral clearance from the upper airway 6-8, which suggests that dysregulation of the host immune response is a critical mediator of clinical deterioration and death. Autopsy and pre-clinical evidence implicate aberrant complement activation in endothelial injury and organ failure 9,10. A potential therapeutic strategy warranting investigation is to inhibit complement, with case reports of successful treatment of COVID-19 with inhibitors of complement 10-13. However, this approach requires careful balance between the host protective and potential injurious effects of complement activation, and biomarkers to identify the optimal timing and candidates for therapy are lacking. Here we report the presence of complement activation products on circulating erythrocytes from hospitalized COVID-19 patients using flow cytometry. These findings suggest that novel erythrocyte-based diagnostics provide a method to identify patients with dysregulated complement activation.

Deep immune profiling of COVID-19 patients reveals patient heterogeneity and distinct immunotypes with implications for therapeutic interventions

COVID-19 has become a global pandemic. Immune dysregulation has been implicated, but immune responses remain poorly understood. We analyzed 71 COVID-19 patients compared to recovered and healthy subjects using high dimensional cytometry. Integrated analysis of [~]200 immune and >30 clinical features revealed activation of T cell and B cell subsets, but only in some patients. A subgroup of patients had T cell activation characteristic of acute viral infection and plasmablast responses could reach >30% of circulating B cells. However, another subgroup had lymphocyte activation comparable to uninfected subjects. Stable versus dynamic immunological signatures were identified and linked to trajectories of disease severity change. These analyses identified three "immunotypes" associated with poor clinical trajectories versus improving health. These immunotypes may have implications for therapeutics and vaccines.

Immunologic perturbations in severe COVID-19/SARS-CoV-2 infection

Although critical illness has been associated with SARS-CoV-2-induced hyperinflammation, the immune correlates of severe COVID-19 remain unclear. Here, we comprehensively analyzed peripheral blood immune perturbations in 42 SARS-CoV-2 infected and recovered individuals. We identified broad changes in neutrophils, NK cells, and monocytes during severe COVID-19, suggesting excessive mobilization of innate lineages. We found marked activation within T and B cells, highly oligoclonal B cell populations, profound plasmablast expansion, and SARS-CoV-2-specific antibodies in many, but not all, severe COVID-19 cases. Despite this heterogeneity, we found selective clustering of severe COVID-19 cases through unbiased analysis of the aggregated immunological phenotypes. Our findings demonstrate broad immune perturbations spanning both innate and adaptive leukocytes that distinguish dysregulated host responses in severe SARS-CoV-2 infection and warrant therapeutic investigation. One Sentence SummaryBroad immune perturbations in severe COVID-19