Robust T cell responses to Pfizer/BioNTech vaccine compared to infection and evidence of attenuated peripheral CD8+ T cell responses due to COVID-19

T cells are a critical component of the response to SARS-CoV-2, but their kinetics after infection and vaccination are insufficiently understood. Using "spheromer” peptide-MHC multimer reagents, we analyzed healthy subjects receiving two doses of the Pfizer/BioNTech BNT162b2 vaccine. Vaccination resulted in robust Spike-specific T cell responses for the dominant CD4+ (HLA-DRB1∗15:01/S191) and CD8+ (HLA-A∗02/S691) T cell epitopes. Antigen-specific CD4+ and CD8+ T cell responses were asynchronous, with the peak CD4+ T cell responses occurring one week post the second vaccination (boost), whereas CD8+ T cells peaked two weeks later. These peripheral T cell responses were elevated compared to COVID-19 patients. We also found that prior SARS-CoV-2 infection resulted in decreased CD8+ T cell activation and expansion, suggesting that prior infection can influence the T cell response to vaccination. Graphical Our understanding of T cell responses in COVID-19 and vaccination is incomplete. Gao et al. examine SARS-CoV-2-specific T cell responses to infection and vaccination, revealing disparate kinetics between CD4+ and CD8+ T cells. Furthermore, compared to vaccination alone, circulating CD8+ T cells are attenuated during infection and in subsequent vaccination.

Spheromers reveal robust T cell responses to the Pfizer/BioNTech vaccine and attenuated peripheral CD8+ T cell responses post SARS-CoV-2 infection.

T cells are a critical component of the response to SARS-CoV-2, but their kinetics after infection and vaccination are insufficiently understood. Using "spheromer" peptide-MHC multimer reagents, we analyzed healthy subjects receiving two doses of the Pfizer/BioNTech BNT162b2 vaccine. Vaccination resulted in robust spike-specific T cell responses for the dominant CD4+ (HLA-DRB1∗15:01/S191) and CD8+ (HLA-A∗02/S691) T cell epitopes. Antigen-specific CD4+ and CD8+ T cell responses were asynchronous, with the peak CD4+ T cell responses occurring 1 week post the second vaccination (boost), whereas CD8+ T cells peaked 2 weeks later. These peripheral T cell responses were elevated compared with COVID-19 patients. We also found that previous SARS-CoV-2 infection resulted in decreased CD8+ T cell activation and expansion, suggesting that previous infection can influence the T cell response to vaccination.

TNF-α+ CD4+ T cells dominate the SARS-CoV-2 specific T cell response in COVID-19 outpatients and are associated with durable antibodies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific CD4+ T cells are likely important in immunity against coronavirus 2019 (COVID-19), but our understanding of CD4+ longitudinal dynamics following infection and of specific features that correlate with the maintenance of neutralizing antibodies remains limited. Here, we characterize SARS-CoV-2-specific CD4+ T cells in a longitudinal cohort of 109 COVID-19 outpatients enrolled during acute infection. The quality of the SARS-CoV-2-specific CD4+ response shifts from cells producing interferon gamma (IFNγ) to tumor necrosis factor alpha (TNF-α) from 5 days to 4 months post-enrollment, with IFNγ-IL-21-TNF-α+ CD4+ T cells the predominant population detected at later time points. Greater percentages of IFNγ-IL-21-TNF-α+ CD4+ T cells on day 28 correlate with SARS-CoV-2-neutralizing antibodies measured 7 months post-infection (⍴ = 0.4, p = 0.01). mRNA vaccination following SARS-CoV-2 infection boosts both IFNγ- and TNF-α-producing, spike-protein-specific CD4+ T cells. These data suggest that SARS-CoV-2-specific, TNF-α-producing CD4+ T cells may play an important role in antibody maintenance following COVID-19.

Autoantibodies are highly prevalent in non-SARS-CoV-2 respiratory infections and critical illness.

The widespread presence of autoantibodies in acute infection with SARS-CoV-2 is increasingly recognized, but the prevalence of autoantibodies in non-SARS-CoV-2 infections and critical illness has not yet been reported. We profiled IgG autoantibodies in 267 patients from 5 independent cohorts with non-SARS-CoV-2 viral, bacterial, and noninfectious critical illness. Serum samples were screened using Luminex arrays that included 58 cytokines and 55 autoantigens, many of which are associated with connective tissue diseases (CTDs). Samples positive for anti-cytokine antibodies were tested for receptor blocking activity using cell-based functional assays. Anti-cytokine antibodies were identified in > 50% of patients across all 5 acutely ill cohorts. In critically ill patients, anti-cytokine antibodies were far more common in infected versus uninfected patients. In cell-based functional assays, 11 of 39 samples positive for select anti-cytokine antibodies displayed receptor blocking activity against surface receptors for Type I IFN, GM-CSF, and IL-6. Autoantibodies against CTD-associated autoantigens were also commonly observed, including newly detected antibodies that emerged in longitudinal samples. These findings demonstrate that anti-cytokine and autoantibodies are common across different viral and nonviral infections and range in severity of illness.

Heterogeneity in IgG-CD16 signaling in infectious disease outcomes.

In this review, we discuss how IgG antibodies can modulate inflammatory signaling during viral infections with a focus on CD16a-mediated functions. We describe the structural heterogeneity of IgG antibody ligands, including subclass and glycosylation that impact binding by and downstream activity of CD16a, as well as the heterogeneity of CD16a itself, including allele and expression density. While inflammation is a mechanism required for immune homeostasis and resolution of acute infections, we focus here on two infectious diseases that are driven by pathogenic inflammatory responses during infection. Specifically, we review and discuss the evolving body of literature showing that afucosylated IgG immune complex signaling through CD16a contributes to the overwhelming inflammatory response that is central to the pathogenesis of severe forms of dengue disease and coronavirus disease 2019 (COVID-19).

Early immune markers of clinical, virological, and immunological outcomes in patients with COVID-19: a multi-omics study.

Background: The great majority of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infections are mild and uncomplicated, but some individuals with initially mild COVID-19 progressively develop more severe symptoms. Furthermore, there is substantial heterogeneity in SARS-CoV-2-specific memory immune responses following infection. There remains a critical need to identify host immune biomarkers predictive of clinical and immunological outcomes in SARS-CoV-2-infected patients. Methods: Leveraging longitudinal samples and data from a clinical trial (N=108) in SARS-CoV-2-infected outpatients, we used host proteomics and transcriptomics to characterize the trajectory of the immune response in COVID-19 patients. We characterized the association between early immune markers and subsequent disease progression, control of viral shedding, and SARS-CoV-2-specific T cell and antibody responses measured up to 7 months after enrollment. We further compared associations between early immune markers and subsequent T cell and antibody responses following natural infection with those following mRNA vaccination. We developed machine-learning models to predict patient outcomes and validated the predictive model using data from 54 individuals enrolled in an independent clinical trial. Results: We identify early immune signatures, including plasma RIG-I levels, early IFN signaling, and related cytokines (CXCL10, MCP1, MCP-2, and MCP-3) associated with subsequent disease progression, control of viral shedding, and the SARS-CoV-2-specific T cell and antibody response measured up to 7 months after enrollment. We found that several biomarkers for immunological outcomes are shared between individuals receiving BNT162b2 (Pfizer-BioNTech) vaccine and COVID-19 patients. Finally, we demonstrate that machine-learning models using 2-7 plasma protein markers measured early within the course of infection are able to accurately predict disease progression, T cell memory, and the antibody response post-infection in a second, independent dataset. Conclusions: Early immune signatures following infection can accurately predict clinical and immunological outcomes in outpatients with COVID-19 using validated machine-learning models. Funding: Support for the study was provided from National Institute of Health/National Institute of Allergy and Infectious Diseases (NIH/NIAID) (U01 AI150741-01S1 and T32-AI052073), the Stanford's Innovative Medicines Accelerator, National Institutes of Health/National Institute on Drug Abuse (NIH/NIDA) DP1DA046089, and anonymous donors to Stanford University. Peginterferon lambda provided by Eiger BioPharmaceuticals.

Durable protection against the SARS-CoV-2 Omicron variant is induced by an adjuvanted subunit vaccine.

Despite the remarkable efficacy of COVID-19 vaccines, waning immunity and the emergence of SARS-CoV-2 variants such as Omicron represents a global health challenge. Here, we present data from a study in nonhuman primates demonstrating durable protection against the Omicron BA.1 variant induced by a subunit SARS-CoV-2 vaccine comprising the receptor binding domain of the ancestral strain (RBD-Wu) on the I53-50 nanoparticle adjuvanted with AS03, which was recently authorized for use in individuals 18 years or older. Vaccination induced neutralizing antibody (nAb) titers that were maintained at high concentrations for at least 1 year after two doses, with a pseudovirus nAb geometric mean titer (GMT) of 1978 and a live virus nAb GMT of 1331 against the ancestral strain but not against the Omicron BA.1 variant. However, a booster dose at 6 to 12 months with RBD-Wu or RBD-ß (RBD from the Beta variant) displayed on I53-50 elicited high neutralizing titers against the ancestral and Omicron variants. In addition, we observed persistent neutralization titers against a panel of sarbecoviruses, including SARS-CoV. Furthermore, there were substantial and persistent memory T and B cell responses reactive to Beta and Omicron variants. Vaccination resulted in protection against Omicron infection in the lung and suppression of viral burden in the nares at 6 weeks after the final booster immunization. Even at 6 months after vaccination, we observed protection in the lung and rapid control of virus in the nares. These results highlight the durable and cross-protective immunity elicited by the AS03-adjuvanted RBD-I53-50 nanoparticle vaccine.

Anti-nucleocapsid antibody levels and pulmonary comorbid conditions are linked to post-COVID-19 syndrome.

BACKGROUNDProlonged symptoms after SARS-CoV-2 infection are well documented. However, which factors influence development of long-term symptoms, how symptoms vary across ethnic groups, and whether long-term symptoms correlate with biomarkers are points that remain elusive.METHODSAdult SARS-CoV-2 reverse transcription PCR-positive (RT-PCR-positive) patients were recruited at Stanford from March 2020 to February 2021. Study participants were seen for in-person visits at diagnosis and every 1-3 months for up to 1 year after diagnosis; they completed symptom surveys and underwent blood draws and nasal swab collections at each visit.RESULTSOur cohort (n = 617) ranged from asymptomatic to critical COVID-19 infections. In total, 40% of participants reported at least 1 symptom associated with COVID-19 six months after diagnosis. Median time from diagnosis to first resolution of all symptoms was 44 days; median time from diagnosis to sustained symptom resolution with no recurring symptoms for 1 month or longer was 214 days. Anti-nucleocapsid IgG level in the first week after positive RT-PCR test and history of lung disease were associated with time to sustained symptom resolution. COVID-19 disease severity, ethnicity, age, sex, and remdesivir use did not affect time to sustained symptom resolution.CONCLUSIONWe found that all disease severities had a similar risk of developing post-COVID-19 syndrome in an ethnically diverse population. Comorbid lung disease and lower levels of initial IgG response to SARS-CoV-2 nucleocapsid antigen were associated with longer symptom duration.TRIAL REGISTRATIONClinicalTrials.gov, NCT04373148.FUNDINGNIH UL1TR003142 CTSA grant, NIH U54CA260517 grant, NIEHS R21 ES03304901, Sean N Parker Center for Allergy and Asthma Research at Stanford University, Chan Zuckerberg Biohub, Chan Zuckerberg Initiative, Sunshine Foundation, Crown Foundation, and Parker Foundation.

Differential Peripheral Blood Glycoprotein Profiles in Symptomatic and Asymptomatic COVID-19.

Glycosylation is the most common form of post-translational modification of proteins, critically affecting their structure and function. Using liquid chromatography and mass spectrometry for high-resolution site-specific quantification of glycopeptides coupled with high-throughput artificial intelligence-powered data processing, we analyzed differential protein glycoisoform distributions of 597 abundant serum glycopeptides and nonglycosylated peptides in 50 individuals who had been seriously ill with COVID-19 and in 22 individuals who had recovered after an asymptomatic course of COVID-19. As additional comparison reference phenotypes, we included 12 individuals with a history of infection with a common cold coronavirus, 16 patients with bacterial sepsis, and 15 healthy subjects without history of coronavirus exposure. We found statistically significant differences, at FDR < 0.05, for normalized abundances of 374 of the 597 peptides and glycopeptides interrogated between symptomatic and asymptomatic COVID-19 patients. Similar statistically significant differences were seen when comparing symptomatic COVID-19 patients to healthy controls (350 differentially abundant peptides and glycopeptides) and common cold coronavirus seropositive subjects (353 differentially abundant peptides and glycopeptides). Among healthy controls and sepsis patients, 326 peptides and glycopeptides were found to be differentially abundant, of which 277 overlapped with biomarkers that showed differential expression between symptomatic COVID-19 cases and healthy controls. Among symptomatic COVID-19 cases and sepsis patients, 101 glycopeptide and peptide biomarkers were found to be statistically significantly abundant. Using both supervised and unsupervised machine learning techniques, we found specific glycoprotein profiles to be strongly predictive of symptomatic COVID-19 infection. LASSO-regularized multivariable logistic regression and K-means clustering yielded accuracies of 100% in an independent test set and of 96% overall, respectively. Our findings are consistent with the interpretation that a majority of glycoprotein modifications observed which are shared among symptomatic COVID-19 and sepsis patients likely represent a generic consequence of a severe systemic immune and inflammatory state. However, there are glycoisoform changes that are specific and particular to severe COVID-19 infection. These may be representative of either COVID-19-specific consequences or susceptibility to or predisposition for a severe course of the disease. Our findings support the potential value of glycoproteomic biomarkers in the biomedical understanding and, potentially, the clinical management of serious acute infectious conditions.

Antibodies elicited by SARS-CoV-2 infection or mRNA vaccines have reduced neutralizing activity against Beta and Omicron pseudoviruses.

Multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that have mutations associated with increased transmission and antibody escape have arisen over the course of the current pandemic. Although the current vaccines have largely been effective against past variants, the number of mutations found on the Omicron (B.1.1.529) spike protein appear to diminish the protection conferred by preexisting immunity. Using vesicular stomatitis virus (VSV) pseudoparticles expressing the spike protein of several SARS-CoV-2 variants, we evaluated the magnitude and breadth of the neutralizing antibody response over time in individuals after infection and in mRNA-vaccinated individuals. We observed that boosting increases the magnitude of the antibody response to wild-type (D614), Beta, Delta, and Omicron variants; however, the Omicron variant was the most resistant to neutralization. We further observed that vaccinated healthy adults had robust and broad antibody responses, whereas responses may have been reduced in vaccinated pregnant women, underscoring the importance of learning how to maximize mRNA vaccine responses in pregnant populations. Findings from this study show substantial heterogeneity in the magnitude and breadth of responses after infection and mRNA vaccination and may support the addition of more conserved viral antigens to existing SARS-CoV-2 vaccines.

Current and novel biomarkers of thrombotic risk in COVID-19: a Consensus Statement from the International COVID-19 Thrombosis Biomarkers Colloquium.

Coronavirus disease 2019 (COVID-19) predisposes patients to thrombotic and thromboembolic events, owing to excessive inflammation, endothelial cell activation and injury, platelet activation and hypercoagulability. Patients with COVID-19 have a prothrombotic or thrombophilic state, with elevations in the levels of several biomarkers of thrombosis, which are associated with disease severity and prognosis. Although some biomarkers of COVID-19-associated coagulopathy, including high levels of fibrinogen and D-dimer, were recognized early during the pandemic, many new biomarkers of thrombotic risk in COVID-19 have emerged. In this Consensus Statement, we delineate the thrombotic signature of COVID-19 and present the latest biomarkers and platforms to assess the risk of thrombosis in these patients, including markers of platelet activation, platelet aggregation, endothelial cell activation or injury, coagulation and fibrinolysis as well as biomarkers of the newly recognized post-vaccine thrombosis with thrombocytopenia syndrome. We then make consensus recommendations for the clinical use of these biomarkers to inform prognosis, assess disease acuity, and predict thrombotic risk and in-hospital mortality. A thorough understanding of these biomarkers might aid risk stratification and prognostication, guide interventions and provide a platform for future research.

Early non-neutralizing, afucosylated antibody responses are associated with COVID-19 severity.

A damaging inflammatory response is implicated in the pathogenesis of severe coronavirus disease 2019 (COVID-19), but mechanisms contributing to this response are unclear. In two prospective cohorts, early non-neutralizing, afucosylated immunoglobulin G (IgG) antibodies specific to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were associated with progression from mild to more severe COVID-19. To study the biology of afucosylated IgG immune complexes, we developed an in vivo model that revealed that human IgG-Fc-gamma receptor (FcγR) interactions could regulate inflammation in the lung. Afucosylated IgG immune complexes isolated from patients with COVID-19 induced inflammatory cytokine production and robust infiltration of the lung by immune cells. In contrast to the antibody structures that were associated with disease progression, antibodies that were elicited by messenger RNA SARS-CoV-2 vaccines were highly fucosylated and enriched in sialylation, both modifications that reduce the inflammatory potential of IgG. Vaccine-elicited IgG did not promote an inflammatory lung response. These results show that human IgG-FcγR interactions regulate inflammation in the lung and define distinct lung activities mediated by the IgG that are associated with protection against, or progression to, severe COVID-19.

Human B cell clonal expansion and convergent antibody responses to SARS-CoV-2

During virus infection B cells are critical for the production of antibodies and protective immunity. Here we show that the human B cell compartment in patients with diagnostically confirmed SARS-CoV-2 and clinical COVID-19 is rapidly altered with the early recruitment of B cells expressing a limited subset of IGHV genes, progressing to a highly polyclonal response of B cells with broader IGHV gene usage and extensive class switching to IgG and IgA subclasses with limited somatic hypermutation in the initial weeks of infection. We identify extensive convergence of antibody sequences across SARS-CoV-2 patients, highlighting stereotyped naive responses to this virus. Notably, sequence-based detection in COVID-19 patients of convergent B cell clonotypes previously reported in SARS-CoV infection predicts the presence of SARS-CoV/SARS-CoV-2 cross-reactive antibody titers specific for the receptor-binding domain. These findings offer molecular insights into shared features of human B cell responses to SARS-CoV-2 and other zoonotic spillover coronaviruses.

New-onset IgG autoantibodies in hospitalized patients with COVID-19.

COVID-19 is associated with a wide range of clinical manifestations, including autoimmune features and autoantibody production. Here we develop three protein arrays to measure IgG autoantibodies associated with connective tissue diseases, anti-cytokine antibodies, and anti-viral antibody responses in serum from 147 hospitalized COVID-19 patients. Autoantibodies are identified in approximately 50% of patients but in less than 15% of healthy controls. When present, autoantibodies largely target autoantigens associated with rare disorders such as myositis, systemic sclerosis and overlap syndromes. A subset of autoantibodies targeting traditional autoantigens or cytokines develop de novo following SARS-CoV-2 infection. Autoantibodies track with longitudinal development of IgG antibodies recognizing SARS-CoV-2 structural proteins and a subset of non-structural proteins, but not proteins from influenza, seasonal coronaviruses or other pathogenic viruses. 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.

Illuminating the Fc dependence of SARS-CoV-2 neutralization.

Monoclonal antibodies show efficacy in treating COVID-19, but the functional requirements for protection are unclear. In this issue of Immunity, Ullah et al. (2021) develop a stable SARS-CoV-2 reporter virus and use bioluminescence imaging to longitudinally monitor infection and assess neutralizing monoclonal antibody interventions in mice. They find that antibody-mediated protection depends on the Fc domain and Fc-gamma receptor-expressing immune cells.

An aberrant inflammatory response in severe COVID-19.

Severe COVID-19 arises from the convergence of inadequate pre-existing immunity and a host response that damages, rather than repairs, tissues. We outline clinical presentations of COVID-19 that are likely driven by dysregulated host immunity, discuss potential mechanisms underlying pathological responses, and highlight important areas for basic research on this topic.

Engineering luminescent biosensors for point-of-care SARS-CoV-2 antibody detection.

Current serology tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies mainly take the form of enzyme-linked immunosorbent assays, chemiluminescent microparticle immunoassays or lateral flow assays, which are either laborious, expensive or lacking sufficient sensitivity and scalability. Here we present the development and validation of a rapid, low-cost, solution-based assay to detect antibodies in serum, plasma, whole blood and to a lesser extent saliva, using rationally designed split luciferase antibody biosensors. This new assay, which generates quantitative results in 30 min, substantially reduces the complexity and improves the scalability of coronavirus disease 2019 (COVID-19) antibody tests. This assay is well-suited for point-of-care, broad population testing, and applications in low-resource settings, for monitoring host humoral responses to vaccination or viral infection.

Proinflammatory IgG Fc structures in patients with severe COVID-19.

Severe acute respiratory syndrome coronavirus 2 infections can cause coronavirus disease 2019 (COVID-19), which manifests with a range of severities from mild illness to life-threatening pneumonia and multi-organ failure. Severe COVID-19 is characterized by an inflammatory signature, including high levels of inflammatory cytokines, alveolar inflammatory infiltrates and vascular microthrombi. Here we show that patients with severe COVID-19 produced a unique serologic signature, including an increased likelihood of IgG1 with afucosylated Fc glycans. This Fc modification on severe acute respiratory syndrome coronavirus 2 IgGs enhanced interactions with the activating Fcγ receptor FcγRIIIa; when incorporated into immune complexes, Fc afucosylation enhanced production of inflammatory cytokines by monocytes, including interleukin-6 and tumor necrosis factor. These results show that disease severity in COVID-19 correlates with the presence of proinflammatory IgG Fc structures, including afucosylated IgG1.

SARS-CoV-2 vaccines in advanced clinical trials: Where do we stand?

The ongoing SARS-CoV-2 pandemic has led to the focused application of resources and scientific expertise toward the goal of developing investigational vaccines to prevent COVID-19. The highly collaborative global efforts by private industry, governments and non-governmental organizations have resulted in a number of SARS-CoV-2 vaccine candidates moving to Phase III trials in a period of only months since the start of the pandemic. In this review, we provide an overview of the preclinical and clinical data on SARS-CoV-2 vaccines that are currently in Phase III clinical trials and in few cases authorized for emergency use. We further discuss relevant vaccine platforms and provide a discussion of SARS-CoV-2 antigens that may be targeted to increase the breadth and durability of vaccine responses.