COVID-19: Using high-throughput flow cytometry to dissect clinical heterogeneity.

Here we consider how high-content flow cytometric methodology at appropriate scale and throughput rapidly provided meaningful biological data in our recent studies of COVID-19, which we discuss in the context of other similar investigations. In our work, high-throughput flow cytometry was instrumental to identify a consensus immune signature in COVID-19 patients, and to investigate the impact of SARS-CoV-2 exposure on patients with either solid or hematological cancers. We provide here some examples of our 'holistic' approach, in which flow cytometry data generated by lymphocyte and myelomonocyte panels were integrated with other analytical metrics, including SARS-CoV-2-specific serum antibody titers, plasma cytokine/chemokine levels, and in-depth clinical annotation. We report how selective differences between T cell subsets were revealed by a newly described flow cytometric TDS assay to distinguish actively cycling T cells in the peripheral blood. By such approaches, our and others' high-content flow cytometry studies collectively identified overt abnormalities and subtle but critical changes that discriminate the immuno-signature of COVID-19 patients from those of healthy donors and patients with non-COVID respiratory infections. Thereby, these studies offered several meaningful biomarkers of COVID-19 severity that have the potential to improve the management of patients and of hospital resources. In sum, flow cytometry provides an important means for rapidly obtaining data that can guide clinical decision-making without requiring highly expensive, sophisticated equipment, and/or "-omics" capabilities. We consider how this approach might be further developed.

Boosting of Waned Humoral and Cellular Responses to SARS-CoV-2 Variants of Concern Among Patients with Cancer.

This study offers longitudinal insight into the impact of three SARS-CoV-2 vaccinations on humoral and cellular immunity in patients with solid cancers, patients with hematologic malignancies, and persons without cancer. For all cohorts, virus-neutralizing immunity was significantly depleted over a period of up to 9 months following the second vaccine dose, the one striking exception being IL2 production by SARS-CoV-2 antigen-specific T cells. Immunity was restored by the third vaccine dose, except in a substantial number of patients with hematologic malignancy, for whom both cancer type and treatment schedule were associated with nonresponse. Thus, whereas most patients with myelodysplastic syndrome were conspicuously good responders, some patients with other hematologic malignancies receiving cancer therapies within 2 weeks of vaccination showed no seroconversion despite three vaccine doses. Moreover, SARS-CoV-2 exposure during the course of the study neither prevented immunity waning, even in healthy controls, nor guaranteed vaccine responsiveness. These data offer real-world human immunologic insights that can inform health policy for patients with cancer.

Antibodies as drugs-a Keystone Symposia report.

Therapeutic antibodies have broad indications across diverse disease states, such as oncology, autoimmune diseases, and infectious diseases. New research continues to identify antibodies with therapeutic potential as well as methods to improve upon endogenous antibodies and to design antibodies de novo. On April 27-30, 2022, experts in antibody research across academia and industry met for the Keystone symposium "Antibodies as Drugs" to present the state-of-the-art in antibody therapeutics, repertoires and deep learning, bispecific antibodies, and engineering.

Durable CD8 T Cell Memory against SARS-CoV-2 by Prime/Boost and Multi-Dose Vaccination: Considerations on Inter-Dose Time Intervals.

Facing the COVID-19 pandemic, anti-SARS-CoV-2 vaccines were developed at unprecedented pace, productively exploiting contemporary fundamental research and prior art. Large-scale use of anti-SARS-CoV-2 vaccines has greatly limited severe morbidity and mortality. Protection has been correlated with high serum titres of neutralizing antibodies capable of blocking the interaction between the viral surface protein spike and the host SARS-CoV-2 receptor, ACE-2. Yet, vaccine-induced protection subsides over time, and breakthrough infections are commonly observed, mostly reflecting the decay of neutralizing antibodies and the emergence of variant viruses with mutant spike proteins. Memory CD8 T cells are a potent weapon against viruses, as they are against tumour cells. Anti-SARS-CoV-2 memory CD8 T cells are induced by either natural infection or vaccination and can be potentially exploited against spike-mutated viruses. We offer here an overview of current research about the induction of anti-SARS-CoV-2 memory CD8 T cells by vaccination, in the context of prior knowledge on vaccines and on fundamental mechanisms of immunological memory. We focus particularly on how vaccination by two doses (prime/boost) or more (boosters) promotes differentiation of memory CD8 T cells, and on how the time-length of inter-dose intervals may influence the magnitude and persistence of CD8 T cell memory.

Global patterns of antigen receptor repertoire disruption across adaptive immune compartments in COVID-19.

Whereas pathogen-specific T and B cells are a primary focus of interest during infectious disease, we have used COVID-19 to ask whether their emergence comes at a cost of broader B cell and T cell repertoire disruption. We applied a genomic DNA-based approach to concurrently study the immunoglobulin-heavy (IGH) and T cell receptor (TCR) ß and δ chain loci of 95 individuals. Our approach detected anticipated repertoire focusing for the IGH repertoire, including expansions of clusters of related sequences temporally aligned with SARS-CoV-2-specific seroconversion, and enrichment of some shared SARS-CoV-2-associated sequences. No significant age-related or disease severity-related deficiencies were noted for the IGH repertoire. By contrast, whereas focusing occurred at the TCRß and TCRδ loci, including some TCRß sequence-sharing, disruptive repertoire narrowing was almost entirely limited to many patients aged older than 50 y. By temporarily reducing T cell diversity and by risking expansions of nonbeneficial T cells, these traits may constitute an age-related risk factor for COVID-19, including a vulnerability to new variants for which T cells may provide key protection.

Anti-type I interferon antibodies as a cause of severe COVID-19.

COVID-19 ranges from asymptomatic through to respiratory failure and death. Although specific pre-existing conditions such as age and male sex have been associated with poor outcomes, we remain largely ignorant of the mechanisms predisposing to severe disease. In this study, the authors discovered that approximately 10% of 987 patients with life-threatening COVID-19 harbored neutralizing antibodies to Type I interferons (IFNs)1. They demonstrated that these antibodies could neutralize high concentrations of the corresponding IFN and could rescue SARS-CoV-2 infection from inhibition by IFN in vitro. Importantly, anti-IFN antibodies were associated with low levels of serum IFN. These observations suggest that disease severity in these individuals results from a failure to control SARS-CoV-2 replication because of antibody-mediated IFN inhibition. The study suggests specific treatments and diagnostics for this class of severe COVID-19.

Safety and immunogenicity of one versus two doses of the COVID-19 vaccine BNT162b2 for patients with cancer: interim analysis of a prospective observational study.

BACKGROUND: The efficacy and safety profiles of vaccines against SARS-CoV-2 in patients with cancer is unknown. We aimed to assess the safety and immunogenicity of the BNT162b2 (Pfizer-BioNTech) vaccine in patients with cancer. METHODS: For this prospective observational study, we recruited patients with cancer and healthy controls (mostly health-care workers) from three London hospitals between Dec 8, 2020, and Feb 18, 2021. Participants who were vaccinated between Dec 8 and Dec 29, 2020, received two 30 µg doses of BNT162b2 administered intramuscularly 21 days apart; patients vaccinated after this date received only one 30 µg dose with a planned follow-up boost at 12 weeks. Blood samples were taken before vaccination and at 3 weeks and 5 weeks after the first vaccination. Where possible, serial nasopharyngeal real-time RT-PCR (rRT-PCR) swab tests were done every 10 days or in cases of symptomatic COVID-19. The coprimary endpoints were seroconversion to SARS-CoV-2 spike (S) protein in patients with cancer following the first vaccination with the BNT162b2 vaccine and the effect of vaccine boosting after 21 days on seroconversion. All participants with available data were included in the safety and immunogenicity analyses. Ongoing follow-up is underway for further blood sampling after the delayed (12-week) vaccine boost. This study is registered with the NHS Health Research Authority and Health and Care Research Wales (REC ID 20/HRA/2031). FINDINGS: 151 patients with cancer (95 patients with solid cancer and 56 patients with haematological cancer) and 54 healthy controls were enrolled. For this interim data analysis of the safety and immunogenicity of vaccinated patients with cancer, samples and data obtained up to March 19, 2021, were analysed. After exclusion of 17 patients who had been exposed to SARS-CoV-2 (detected by either antibody seroconversion or a positive rRT-PCR COVID-19 swab test) from the immunogenicity analysis, the proportion of positive anti-S IgG titres at approximately 21 days following a single vaccine inoculum across the three cohorts were 32 (94%; 95% CI 81-98) of 34 healthy controls; 21 (38%; 26-51) of 56 patients with solid cancer, and eight (18%; 10-32) of 44 patients with haematological cancer. 16 healthy controls, 25 patients with solid cancer, and six patients with haematological cancer received a second dose on day 21. Of the patients with available blood samples 2 weeks following a 21-day vaccine boost, and excluding 17 participants with evidence of previous natural SARS-CoV-2 exposure, 18 (95%; 95% CI 75-99) of 19 patients with solid cancer, 12 (100%; 76-100) of 12 healthy controls, and three (60%; 23-88) of five patients with haematological cancers were seropositive, compared with ten (30%; 17-47) of 33, 18 (86%; 65-95) of 21, and four (11%; 4-25) of 36, respectively, who did not receive a boost. The vaccine was well tolerated; no toxicities were reported in 75 (54%) of 140 patients with cancer following the first dose of BNT162b2, and in 22 (71%) of 31 patients with cancer following the second dose. Similarly, no toxicities were reported in 15 (38%) of 40 healthy controls after the first dose and in five (31%) of 16 after the second dose. Injection-site pain within 7 days following the first dose was the most commonly reported local reaction (23 [35%] of 65 patients with cancer; 12 [48%] of 25 healthy controls). No vaccine-related deaths were reported. INTERPRETATION: In patients with cancer, one dose of the BNT162b2 vaccine yields poor efficacy. Immunogenicity increased significantly in patients with solid cancer within 2 weeks of a vaccine boost at day 21 after the first dose. These data support prioritisation of patients with cancer for an early (day 21) second dose of the BNT162b2 vaccine. FUNDING: King's College London, Cancer Research UK, Wellcome Trust, Rosetrees Trust, and Francis Crick Institute.

Association of cardiometabolic microRNAs with COVID-19 severity and mortality.

AIMS: Coronavirus disease 2019 (COVID-19) can lead to multiorgan damage. MicroRNAs (miRNAs) in blood reflect cell activation and tissue injury. We aimed to determine the association of circulating miRNAs with COVID-19 severity and 28 day intensive care unit (ICU) mortality. METHODS AND RESULTS: We performed RNA-Seq in plasma of healthy controls (n = 11), non-severe (n = 18), and severe (n = 18) COVID-19 patients and selected 14 miRNAs according to cell- and tissue origin for measurement by reverse transcription quantitative polymerase chain reaction (RT-qPCR) in a separate cohort of mild (n = 6), moderate (n = 39), and severe (n = 16) patients. Candidates were then measured by RT-qPCR in longitudinal samples of ICU COVID-19 patients (n = 240 samples from n = 65 patients). A total of 60 miRNAs, including platelet-, endothelial-, hepatocyte-, and cardiomyocyte-derived miRNAs, were differentially expressed depending on severity, with increased miR-133a and reduced miR-122 also being associated with 28 day mortality. We leveraged mass spectrometry-based proteomics data for corresponding protein trajectories. Myocyte-derived (myomiR) miR-133a was inversely associated with neutrophil counts and positively with proteins related to neutrophil degranulation, such as myeloperoxidase. In contrast, levels of hepatocyte-derived miR-122 correlated to liver parameters and to liver-derived positive (inverse association) and negative acute phase proteins (positive association). Finally, we compared miRNAs to established markers of COVID-19 severity and outcome, i.e. SARS-CoV-2 RNAemia, age, BMI, D-dimer, and troponin. Whilst RNAemia, age and troponin were better predictors of mortality, miR-133a and miR-122 showed superior classification performance for severity. In binary and triplet combinations, miRNAs improved classification performance of established markers for severity and mortality. CONCLUSION: Circulating miRNAs of different tissue origin, including several known cardiometabolic biomarkers, rise with COVID-19 severity. MyomiR miR-133a and liver-derived miR-122 also relate to 28 day mortality. MiR-133a reflects inflammation-induced myocyte damage, whilst miR-122 reflects the hepatic acute phase response.

SARS-CoV-2 RNAemia and proteomic trajectories inform prognostication in COVID-19 patients admitted to intensive care.

Prognostic characteristics inform risk stratification in intensive care unit (ICU) patients with coronavirus disease 2019 (COVID-19). We obtained blood samples (n = 474) from hospitalized COVID-19 patients (n = 123), non-COVID-19 ICU sepsis patients (n = 25) and healthy controls (n = 30). Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA was detected in plasma or serum (RNAemia) of COVID-19 ICU patients when neutralizing antibody response was low. RNAemia is associated with higher 28-day ICU mortality (hazard ratio [HR], 1.84 [95% CI, 1.22-2.77] adjusted for age and sex). RNAemia is comparable in performance to the best protein predictors. Mannose binding lectin 2 and pentraxin-3 (PTX3), two activators of the complement pathway of the innate immune system, are positively associated with mortality. Machine learning identified 'Age, RNAemia' and 'Age, PTX3' as the best binary signatures associated with 28-day ICU mortality. In longitudinal comparisons, COVID-19 ICU patients have a distinct proteomic trajectory associated with mortality, with recovery of many liver-derived proteins indicating survival. Finally, proteins of the complement system and galectin-3-binding protein (LGALS3BP) are identified as interaction partners of SARS-CoV-2 spike glycoprotein. LGALS3BP overexpression inhibits spike-pseudoparticle uptake and spike-induced cell-cell fusion in vitro.

To Ki or Not to Ki: Re-Evaluating the Use and Potentials of Ki-67 for T Cell Analysis.

This study discusses substantive advances in T cell proliferation analysis, with the aim to provoke a re-evaluation of the generally-held view that Ki-67 is a reliable proliferation marker per se, and to offer a more sensitive and effective method for T cell cycle analysis, with informative examples in mouse and human settings. We summarize recent experimental work from our labs showing that, by Ki-67/DNA dual staining and refined flow cytometric methods, we were able to identify T cells in the S-G2/M phases of the cell-cycle in the peripheral blood (collectively termed "T Double S" for T cells in S-phase in Sanguine: in short "TDS" cells). Without our refinement, such cells may be excluded from conventional lymphocyte analyses. Specifically, we analyzed clonal expansion of antigen-specific CD8 T cells in vaccinated mice, and demonstrated the potential of TDS cells to reflect immune dynamics in human blood samples from healthy donors, and patients with type 1 diabetes, infectious mononucleosis, and COVID-19. The Ki-67/DNA dual staining, or TDS assay, provides a reliable approach by which human peripheral blood can be used to reflect the dynamics of human lymphocytes, rather than providing mere steady-state phenotypic snapshots. The method does not require highly sophisticated "-omics" capabilities, so it should be widely-applicable to health care in diverse settings. Furthermore, our results argue that the TDS assay can provide a window on immune dynamics in extra-lymphoid tissues, a long-sought potential of peripheral blood monitoring, for example in relation to organ-specific autoimmune diseases and infections, and cancer immunotherapy.

Neutralization potency of monoclonal antibodies recognizing dominant and subdominant epitopes on SARS-CoV-2 Spike is impacted by the B.1.1.7 variant.

Interaction of the SARS-CoV-2 Spike receptor binding domain (RBD) with the receptor ACE2 on host cells is essential for viral entry. RBD is the dominant target for neutralizing antibodies, and several neutralizing epitopes on RBD have been molecularly characterized. Analysis of circulating SARS-CoV-2 variants has revealed mutations arising in the RBD, N-terminal domain (NTD) and S2 subunits of Spike. To understand how these mutations affect Spike antigenicity, we isolated and characterized >100 monoclonal antibodies targeting epitopes on RBD, NTD, and S2 from SARS-CoV-2-infected individuals. Approximately 45% showed neutralizing activity, of which ∼20% were NTD specific. NTD-specific antibodies formed two distinct groups: the first was highly potent against infectious virus, whereas the second was less potent and displayed glycan-dependant neutralization activity. Mutations present in B.1.1.7 Spike frequently conferred neutralization resistance to NTD-specific antibodies. This work demonstrates that neutralizing antibodies targeting subdominant epitopes should be considered when investigating antigenic drift in emerging variants.

Author Correction: A dynamic COVID-19 immune signature includes associations with poor prognosis.

A Correction to this paper has been published: https://doi.org/10.1038/s41591-020-01186-5.

Acute Immune Signatures and Their Legacies in Severe Acute Respiratory Syndrome Coronavirus-2 Infected Cancer Patients.

Given the immune system's importance for cancer surveillance and treatment, we have investigated how it may be affected by SARS-CoV-2 infection of cancer patients. Across some heterogeneity in tumor type, stage, and treatment, virus-exposed solid cancer patients display a dominant impact of SARS-CoV-2, apparent from the resemblance of their immune signatures to those for COVID-19+ non-cancer patients. This is not the case for hematological malignancies, with virus-exposed patients collectively displaying heterogeneous humoral responses, an exhausted T cell phenotype and a high prevalence of prolonged virus shedding. Furthermore, while recovered solid cancer patients' immunophenotypes resemble those of non-virus-exposed cancer patients, recovered hematological cancer patients display distinct, lingering immunological legacies. Thus, while solid cancer patients, including those with advanced disease, seem no more at risk of SARS-CoV-2-associated immune dysregulation than the general population, hematological cancer patients show complex immunological consequences of SARS-CoV-2 exposure that might usefully inform their care.

Longitudinal proteomic profiling reveals increased early inflammation and sustained apoptosis proteins in severe COVID-19.

SARS-CoV-2 infection has a risk to develop into life-threatening COVID-19 disease. Whereas age, hypertension, and chronic inflammatory conditions are risk factors, underlying host factors and markers for disease severity, e.g. requiring intensive care unit (ICU) treatment, remain poorly defined. To this end, we longitudinally profiled blood inflammation markers, antibodies, and 101 plasma proteins of hospitalized COVID-19 patients who did or did not require ICU admission. While essentially all patients displayed SARS-CoV-2-specific antibodies and virus-neutralization capacity within 12-15 days, a rapid, mostly transient upregulation of selective inflammatory markers including IL-6, CXCL10, CXCL11, IFNγ, IL-10, and monocyte-attracting CCL2, CCL7 and CCL8, was particularly evident in ICU patients. In addition, there was consistent and sustained upregulation of apoptosis-associated proteins CASP8, TNFSF14, HGF, and TGFB1, with HGF discriminating between ICU and non-ICU cohorts. Thus, COVID-19 is associated with a selective inflammatory milieu within which the apoptotic pathway is a cardinal feature with potential to aid risk-based patient stratification.

A dynamic COVID-19 immune signature includes associations with poor prognosis.

Improved understanding and management of COVID-19, a potentially life-threatening disease, could greatly reduce the threat posed by its etiologic agent, SARS-CoV-2. Toward this end, we have identified a core peripheral blood immune signature across 63 hospital-treated patients with COVID-19 who were otherwise highly heterogeneous. The signature includes discrete changes in B and myelomonocytic cell composition, profoundly altered T cell phenotypes, selective cytokine/chemokine upregulation and SARS-CoV-2-specific antibodies. Some signature traits identify links with other settings of immunoprotection and immunopathology; others, including basophil and plasmacytoid dendritic cell depletion, correlate strongly with disease severity; while a third set of traits, including a triad of IP-10, interleukin-10 and interleukin-6, anticipate subsequent clinical progression. Hence, contingent upon independent validation in other COVID-19 cohorts, individual traits within this signature may collectively and individually guide treatment options; offer insights into COVID-19 pathogenesis; and aid early, risk-based patient stratification that is particularly beneficial in phasic diseases such as COVID-19.