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.

A TLR7-nanoparticle adjuvant promotes a broad immune response against heterologous strains of influenza and SARS-CoV-2.

The ideal vaccine against viruses such as influenza and SARS-CoV-2 must provide a robust, durable and broad immune protection against multiple viral variants. However, antibody responses to current vaccines often lack robust cross-reactivity. Here we describe a polymeric Toll-like receptor 7 agonist nanoparticle (TLR7-NP) adjuvant, which enhances lymph node targeting, and leads to persistent activation of immune cells and broad immune responses. When mixed with alum-adsorbed antigens, this TLR7-NP adjuvant elicits cross-reactive antibodies for both dominant and subdominant epitopes and antigen-specific CD8+ T-cell responses in mice. This TLR7-NP-adjuvanted influenza subunit vaccine successfully protects mice against viral challenge of a different strain. This strategy also enhances the antibody response to a SARS-CoV-2 subunit vaccine against multiple viral variants that have emerged. Moreover, this TLR7-NP augments antigen-specific responses in human tonsil organoids. Overall, we describe a nanoparticle adjuvant to improve immune responses to viral antigens, with promising implications for developing broadly protective vaccines.

KIR+CD8+ T cells suppress pathogenic T cells and are active in autoimmune diseases and COVID-19.

In this work, we find that CD8+ T cells expressing inhibitory killer cell immunoglobulin-like receptors (KIRs) are the human equivalent of Ly49+CD8+ regulatory T cells in mice and are increased in the blood and inflamed tissues of patients with a variety of autoimmune diseases. Moreover, these CD8+ T cells efficiently eliminated pathogenic gliadin-specific CD4+ T cells from the leukocytes of celiac disease patients in vitro. We also find elevated levels of KIR+CD8+ T cells, but not CD4+ regulatory T cells, in COVID-19 patients, correlating with disease severity and vasculitis. Selective ablation of Ly49+CD8+ T cells in virus-infected mice led to autoimmunity after infection. Our results indicate that in both species, these regulatory CD8+ T cells act specifically to suppress pathogenic T cells in autoimmune and infectious diseases.

Phenotypes of disease severity in a cohort of hospitalized COVID-19 patients: Results from the IMPACC study.

BACKGROUND: Better understanding of the association between characteristics of patients hospitalized with coronavirus disease 2019 (COVID-19) and outcome is needed to further improve upon patient management. METHODS: Immunophenotyping Assessment in a COVID-19 Cohort (IMPACC) is a prospective, observational study of 1164 patients from 20 hospitals across the United States. Disease severity was assessed using a 7-point ordinal scale based on degree of respiratory illness. Patients were prospectively surveyed for 1 year after discharge for post-acute sequalae of COVID-19 (PASC) through quarterly surveys. Demographics, comorbidities, radiographic findings, clinical laboratory values, SARS-CoV-2 PCR and serology were captured over a 28-day period. Multivariable logistic regression was performed. FINDINGS: The median age was 59 years (interquartile range [IQR] 20); 711 (61%) were men; overall mortality was 14%, and 228 (20%) required invasive mechanical ventilation. Unsupervised clustering of ordinal score over time revealed distinct disease course trajectories. Risk factors associated with prolonged hospitalization or death by day 28 included age ≥ 65 years (odds ratio [OR], 2.01; 95% CI 1.28-3.17), Hispanic ethnicity (OR, 1.71; 95% CI 1.13-2.57), elevated baseline creatinine (OR 2.80; 95% CI 1.63- 4.80) or troponin (OR 1.89; 95% 1.03-3.47), baseline lymphopenia (OR 2.19; 95% CI 1.61-2.97), presence of infiltrate by chest imaging (OR 3.16; 95% CI 1.96-5.10), and high SARS-CoV2 viral load (OR 1.53; 95% CI 1.17-2.00). Fatal cases had the lowest ratio of SARS-CoV-2 antibody to viral load levels compared to other trajectories over time (p=0.001). 589 survivors (51%) completed at least one survey at follow-up with 305 (52%) having at least one symptom consistent with PASC, most commonly dyspnea (56% among symptomatic patients). Female sex was the only associated risk factor for PASC. INTERPRETATION: Integration of PCR cycle threshold, and antibody values with demographics, comorbidities, and laboratory/radiographic findings identified risk factors for 28-day outcome severity, though only female sex was associated with PASC. Longitudinal clinical phenotyping offers important insights, and provides a framework for immunophenotyping for acute and long COVID-19. FUNDING: NIH.

Multiomic analysis reveals cell-type-specific molecular determinants of COVID-19 severity.

The determinants of severe COVID-19 in healthy adults are poorly understood, which limits the opportunity for early intervention. We present a multiomic analysis using machine learning to characterize the genomic basis of COVID-19 severity. We use single-cell multiome profiling of human lungs to link genetic signals to cell-type-specific functions. We discover >1,000 risk genes across 19 cell types, which account for 77% of the SNP-based heritability for severe disease. Genetic risk is particularly focused within natural killer (NK) cells and T cells, placing the dysfunction of these cells upstream of severe disease. Mendelian randomization and single-cell profiling of human NK cells support the role of NK cells and further localize genetic risk to CD56bright NK cells, which are key cytokine producers during the innate immune response. Rare variant analysis confirms the enrichment of severe-disease-associated genetic variation within NK-cell risk genes. Our study provides insights into the pathogenesis of severe COVID-19 with potential therapeutic targets.

Human Coronary Plaque T Cells Are Clonal and Cross-React to Virus and Self.

BACKGROUND: Coronary artery disease is an incurable, life-threatening disease that was once considered primarily a disorder of lipid deposition. Coronary artery disease is now also characterized by chronic inflammation' notable for the buildup of atherosclerotic plaques containing immune cells in various states of activation and differentiation. Understanding how these immune cells contribute to disease progression may lead to the development of novel therapeutic strategies. METHODS: We used single-cell technology and in vitro assays to interrogate the immune microenvironment of human coronary atherosclerotic plaque at different stages of maturity. RESULTS: In addition to macrophages, we found a high proportion of αß T cells in the coronary plaques. Most of these T cells lack high expression of CCR7 and L-selectin, indicating that they are primarily antigen-experienced memory cells. Notably, nearly one-third of these cells express the HLA-DRA surface marker, signifying activation through their TCRs (T-cell receptors). Consistent with this, TCR repertoire analysis confirmed the presence of activated αß T cells (CD4

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.

Immune imprinting, breadth of variant recognition, and germinal center response in human SARS-CoV-2 infection and vaccination.

During the SARS-CoV-2 pandemic, novel and traditional vaccine strategies have been deployed globally. We investigated whether antibodies stimulated by mRNA vaccination (BNT162b2), including third-dose boosting, differ from those generated by infection or adenoviral (ChAdOx1-S and Gam-COVID-Vac) or inactivated viral (BBIBP-CorV) vaccines. We analyzed human lymph nodes after infection or mRNA vaccination for correlates of serological differences. Antibody breadth against viral variants is lower after infection compared with all vaccines evaluated but improves over several months. Viral variant infection elicits variant-specific antibodies, but prior mRNA vaccination imprints serological responses toward Wuhan-Hu-1 rather than variant antigens. In contrast to disrupted germinal centers (GCs) in lymph nodes during infection, mRNA vaccination stimulates robust GCs containing vaccine mRNA and spike antigen up to 8 weeks postvaccination in some cases. SARS-CoV-2 antibody specificity, breadth, and maturation are affected by imprinting from exposure history and distinct histological and antigenic contexts in infection compared with vaccination.

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.

Durability of immune responses to the BNT162b2 mRNA vaccine.

Antibody responses to the Pfizer-BioNTech mRNA vaccine waned substantially 6 months after the second vaccination.

Integrated analysis of plasma and single immune cells uncovers metabolic changes in individuals with COVID-19.

A better understanding of the metabolic alterations in immune cells during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may elucidate the wide diversity of clinical symptoms experienced by individuals with coronavirus disease 2019 (COVID-19). Here, we report the metabolic changes associated with the peripheral immune response of 198 individuals with COVID-19 through an integrated analysis of plasma metabolite and protein levels as well as single-cell multiomics analyses from serial blood draws collected during the first week after clinical diagnosis. We document the emergence of rare but metabolically dominant T cell subpopulations and find that increasing disease severity correlates with a bifurcation of monocytes into two metabolically distinct subsets. This integrated analysis reveals a robust interplay between plasma metabolites and cell-type-specific metabolic reprogramming networks that is associated with disease severity and could predict survival.

Systems vaccinology of the BNT162b2 mRNA vaccine in humans.

The emergency use authorization of two mRNA vaccines in less than a year from the emergence of SARS-CoV-2 represents a landmark in vaccinology1,2. Yet, how mRNA vaccines stimulate the immune system to elicit protective immune responses is unknown. Here we used a systems vaccinology approach to comprehensively profile the innate and adaptive immune responses of 56 healthy volunteers who were vaccinated with the Pfizer-BioNTech mRNA vaccine (BNT162b2). Vaccination resulted in the robust production of neutralizing antibodies against the wild-type SARS-CoV-2 (derived from 2019-nCOV/USA_WA1/2020) and, to a lesser extent, the B.1.351 strain, as well as significant increases in antigen-specific polyfunctional CD4 and CD8 T cells after the second dose. Booster vaccination stimulated a notably enhanced innate immune response as compared to primary vaccination, evidenced by (1) a greater frequency of CD14+CD16+ inflammatory monocytes; (2) a higher concentration of plasma IFNγ; and (3) a transcriptional signature of innate antiviral immunity. Consistent with these observations, our single-cell transcriptomics analysis demonstrated an approximately 100-fold increase in the frequency of a myeloid cell cluster enriched in interferon-response transcription factors and reduced in AP-1 transcription factors, after secondary immunization. Finally, we identified distinct innate pathways associated with CD8 T cell and neutralizing antibody responses, and show that a monocyte-related signature correlates with the neutralizing antibody response against the B.1.351 variant. Collectively, these data provide insights into the immune responses induced by mRNA vaccination and demonstrate its capacity to prime the innate immune system to mount a more potent response after booster immunization.

CD8+ T cells specific for conserved coronavirus epitopes correlate with milder disease in COVID-19 patients.

A central feature of the SARS-CoV-2 pandemic is that some individuals become severely ill or die, whereas others have only a mild disease course or are asymptomatic. Here we report development of an improved multimeric αß T cell staining reagent platform, with each maxi-ferritin "spheromer" displaying 12 peptide-MHC complexes. Spheromers stain specific T cells more efficiently than peptide-MHC tetramers and capture a broader portion of the sequence repertoire for a given peptide-MHC. Analyzing the response in unexposed individuals, we find that T cells recognizing peptides conserved amongst coronaviruses are more abundant and tend to have a "memory" phenotype, compared to those unique to SARS-CoV-2. Significantly, CD8+ T cells with these conserved specificities are much more abundant in COVID-19 patients with mild disease versus those with a more severe illness, suggesting a protective role.

Single-cell epigenomic landscape of peripheral immune cells reveals establishment of trained immunity in individuals convalescing from COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection often causes severe complications and even death. However, asymptomatic infection has also been reported, highlighting the difference in immune responses among individuals. Here we performed single-cell chromatin accessibility and T cell-receptor analyses of peripheral blood mononuclear cells collected from individuals convalescing from COVID-19 and healthy donors. Chromatin remodelling was observed in both innate and adaptive immune cells in the individuals convalescing from COVID-19. Compared with healthy donors, recovered individuals contained abundant TBET-enriched CD16+ and IRF1-enriched CD14+ monocytes with sequential trained and activated epigenomic states. The B-cell lineage in recovered individuals exhibited an accelerated developmental programme from immature B cells to antibody-producing plasma cells. Finally, an integrated analysis of single-cell T cell-receptor clonality with the chromatin accessibility landscape revealed the expansion of putative SARS-CoV-2-specific CD8+ T cells with epigenomic profiles that promote the differentiation of effector or memory cells. Overall, our data suggest that immune cells of individuals convalescing from COVID-19 exhibit global remodelling of the chromatin accessibility landscape, indicative of the establishment of immunological memory.

Systems biological assessment of human immunity to BNT162b2 mRNA vaccination.

The emergency use authorization of two COVID-19 mRNA vaccines in less than a year since the emergence of SARS-CoV-2, represents a landmark in vaccinology1,2. Yet, how mRNA vaccines stimulate the immune system to elicit protective immune responses is unknown. Here we used a systems biological approach to comprehensively profile the innate and adaptive immune responses in 56 healthy volunteers vaccinated with the Pfizer-BioNTech mRNA vaccine. Vaccination resulted in robust production of neutralizing antibodies (nAbs) against the parent strain and the variant of concern, B.1.351, but no induction of autoantibodies, and significant increases in antigen-specific polyfunctional CD4 and CD8 T cells after the second dose. The innate response induced within the first 2 days of booster vaccination was profoundly increased, relative to the response at corresponding times after priming. Thus, there was a striking increase in the: (i) frequency of CD14+CD16+ inflammatory monocytes; (ii) concentration of IFN- y in the plasma, which correlated with enhanced pSTAT3 and pSTAT1 levels in monocytes and T cells; and (iii) transcriptional signatures of innate responses characteristic of antiviral vaccine responses against pandemic influenza, HIV and Ebola, within 2 days following booster vaccination compared to primary vaccination. Consistent with these observations, single-cell transcriptomics analysis of 242,479 leukocytes demonstrated a ~100-fold increase in the frequency of a myeloid cluster, enriched in a signature of interferon-response transcription factors (TFs) and reduced in AP-1 TFs, one day after secondary immunization, at day 21. Finally, we delineated distinct molecular pathways of innate activation that correlate with CD8 T cell and nAb responses and identified an early monocyte-related signature that was associated with the breadth of the nAb response against the B1.351 variant strain. Collectively, these data provide insights into the immune responses induced by mRNA vaccines and demonstrate their capacity to stimulate an enhanced innate response following booster immunization.

Learning from HIV-1 to predict the immunogenicity of T cell epitopes in SARS-CoV-2.

We describe a physics-based learning model for predicting the immunogenicity of cytotoxic T lymphocyte (CTL) epitopes derived from diverse pathogens including SARS-CoV-2. The model was trained and optimized on the relative immunodominance of CTL epitopes in human immunodeficiency virus infection. Its accuracy was tested against experimental data from patients with COVID-19. Our model predicts that only some SARS-CoV-2 epitopes predicted to bind to HLA molecules are immunogenic. The immunogenic CTL epitopes across all SARS-CoV-2 proteins are predicted to provide broad population coverage, but those from the SARS-CoV-2 spike protein alone are unlikely to do so. Our model also predicts that several immunogenic SARS-CoV-2 CTL epitopes are identical to seasonal coronaviruses circulating in the population and such cross-reactive CD8+ T cells can indeed be detected in prepandemic blood donors, suggesting that some level of CTL immunity against COVID-19 may be present in some individuals before SARS-CoV-2 infection.