PD-1highCXCR5-CD4+ peripheral helper T cells promote CXCR3+ plasmablasts in human acute viral infection.

T cell-B cell interaction is the key immune response to protect the host from severe viral infection. However, how T cells support B cells to exert protective humoral immunity in humans is not well understood. Here, we use COVID-19 as a model of acute viral infections and analyze CD4+ T cell subsets associated with plasmablast expansion and clinical outcome. Peripheral helper T cells (Tph cells; denoted as PD-1highCXCR5-CD4+ T cells) are significantly increased, as are plasmablasts. Tph cells exhibit "B cell help" signatures and induce plasmablast differentiation in vitro. Interestingly, expanded plasmablasts show increased CXCR3 expression, which is positively correlated with higher frequency of activated Tph cells and better clinical outcome. Mechanistically, Tph cells help B cell differentiation and produce more interferon γ (IFNγ), which induces CXCR3 expression on plasmablasts. These results elucidate a role for Tph cells in regulating protective B cell response during acute viral infection.

Lack of association between pandemic chilblains and SARS-CoV-2 infection.

An increased incidence of chilblains has been observed during the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic and attributed to viral infection. Direct evidence of this relationship has been limited, however, as most cases do not have molecular evidence of prior SARS-CoV-2 infection with PCR or antibodies. We enrolled a cohort of 23 patients who were diagnosed and managed as having SARS-CoV-2-associated skin eruptions (including 21 pandemic chilblains [PC]) during the first wave of the pandemic in Connecticut. Antibody responses were determined through endpoint titration enzyme-linked immunosorbent assay and serum epitope repertoire analysis. T cell responses to SARS-CoV-2 were assessed by T cell receptor sequencing and in vitro SARS-CoV-2 antigen-specific peptide stimulation assays. Immunohistochemical and PCR studies of PC biopsies and tissue microarrays for evidence of SARS-CoV-2 were performed. Among patients diagnosed and managed as "covid toes" during the pandemic, we find a percentage of prior SARS-CoV-2 infection (9.5%) that approximates background seroprevalence (8.5%) at the time. Immunohistochemistry studies suggest that SARS-CoV-2 staining in PC biopsies may not be from SARS-CoV-2. Our results do not support SARS-CoV-2 as the causative agent of pandemic chilblains; however, our study does not exclude the possibility of SARS-CoV-2 seronegative abortive infections.

Multiscale PHATE identifies multimodal signatures of COVID-19.

As the biomedical community produces datasets that are increasingly complex and high dimensional, there is a need for more sophisticated computational tools to extract biological insights. We present Multiscale PHATE, a method that sweeps through all levels of data granularity to learn abstracted biological features directly predictive of disease outcome. Built on a coarse-graining process called diffusion condensation, Multiscale PHATE learns a data topology that can be analyzed at coarse resolutions for high-level summarizations of data and at fine resolutions for detailed representations of subsets. We apply Multiscale PHATE to a coronavirus disease 2019 (COVID-19) dataset with 54 million cells from 168 hospitalized patients and find that patients who die show CD16hiCD66blo neutrophil and IFN-γ+ granzyme B+ Th17 cell responses. We also show that population groupings from Multiscale PHATE directly fed into a classifier predict disease outcome more accurately than naive featurizations of the data. Multiscale PHATE is broadly generalizable to different data types, including flow cytometry, single-cell RNA sequencing (scRNA-seq), single-cell sequencing assay for transposase-accessible chromatin (scATAC-seq), and clinical variables.

Longitudinal Immune Profiling of a Severe Acute Respiratory Syndrome Coronavirus 2 Reinfection in a Solid Organ Transplant Recipient.

BACKGROUND: The underlying immunologic deficiencies enabling severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reinfection are currently unknown. We describe deep longitudinal immune profiling of a transplant recipient hospitalized twice for coronavirus disease 2019 (COVID-19). METHODS: A 66-year-old male renal transplant recipient was hospitalized with COVID-19 March 2020 then readmitted to the hospital with COVID-19 233 days after initial diagnosis. Virologic and immunologic investigations were performed on samples from the primary and secondary infections. RESULTS: Whole viral genome sequencing and phylogenetic analysis revealed that viruses causing both infections were caused by distinct genetic lineages without evidence of immune escape mutations. Longitudinal comparison of cellular and humoral responses during primary SARS-CoV-2 infection revealed that this patient responded to the primary infection with low neutralization titer anti-SARS-CoV-2 antibodies that were likely present at the time of reinfection. CONCLUSIONS: The development of neutralizing antibodies and humoral memory responses in this patient failed to confer protection against reinfection, suggesting that they were below a neutralizing titer threshold or that additional factors may be required for efficient prevention of SARS-CoV-2 reinfection. Development of poorly neutralizing antibodies may have been due to profound and relatively specific reduction in naive CD4 T-cell pools. Seropositivity alone may not be a perfect correlate of protection in immunocompromised patients.

Single-cell multi-omics reveals dyssynchrony of the innate and adaptive immune system in progressive COVID-19.

Dysregulated immune responses against the SARS-CoV-2 virus are instrumental in severe COVID-19. However, the immune signatures associated with immunopathology are poorly understood. Here we use multi-omics single-cell analysis to probe the dynamic immune responses in hospitalized patients with stable or progressive course of COVID-19, explore V(D)J repertoires, and assess the cellular effects of tocilizumab. Coordinated profiling of gene expression and cell lineage protein markers shows that S100Ahi/HLA-DRlo classical monocytes and activated LAG-3hi T cells are hallmarks of progressive disease and highlights the abnormal MHC-II/LAG-3 interaction on myeloid and T cells, respectively. We also find skewed T cell receptor repertories in expanded effector CD8+ clones, unmutated IGHG+ B cell clones, and mutated B cell clones with stable somatic hypermutation frequency over time. In conclusion, our in-depth immune profiling reveals dyssynchrony of the innate and adaptive immune interaction in progressive COVID-19.

COVID-19 in an immunocompromised host: persistent shedding of viable SARS-CoV-2 and emergence of multiple mutations: a case report.

This article reports a case of a 21-year-old woman with refractory B-cell acute lymphocytic leukaemia who presented with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). She remained positive for SARS-CoV-2 by viral culture for 78 days and by polymerase chain reaction (PCR) for 97 days. Sequencing of repeat samples over time demonstrated an increasing and dynamic repertoire of mutations.

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

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 7 months after primary infection. To elucidate the immunological mechanisms responsible for 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 that was further compromised by intervening treatment for acute rejection of the renal allograft prior to reinfection. Importantly, we identified the development of neutralizing antibodies and 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.

Sex differences in immune responses that underlie COVID-19 disease outcomes.

There is increasing evidence that coronavirus disease 2019 (COVID-19) produces more severe symptoms and higher mortality among men than among women1-5. However, whether immune responses against severe acute respiratory syndrome coronavirus (SARS-CoV-2) differ between sexes, and whether such differences correlate with the sex difference in the disease course of COVID-19, is currently unknown. Here we examined sex differences in viral loads, SARS-CoV-2-specific antibody titres, plasma cytokines and blood-cell phenotyping in patients with moderate COVID-19 who had not received immunomodulatory medications. Male patients had higher plasma levels of innate immune cytokines such as IL-8 and IL-18 along with more robust induction of non-classical monocytes. By contrast, female patients had more robust T cell activation than male patients during SARS-CoV-2 infection. Notably, we found that a poor T cell response negatively correlated with patients' age and was associated with worse disease outcome in male patients, but not in female patients. By contrast, higher levels of innate immune cytokines were associated with worse disease progression in female patients, but not in male patients. These findings provide a possible explanation for the observed sex biases in COVID-19, and provide an important basis for the development of a sex-based approach to the treatment and care of male and female patients with COVID-19.

Kynurenic acid may underlie sex-specific immune responses to COVID-19.

Coronavirus disease 2019 (COVID-19) has poorer clinical outcomes in males than in females, and immune responses underlie these sex-related differences. Because immune responses are, in part, regulated by metabolites, we examined the serum metabolomes of COVID-19 patients. In male patients, kynurenic acid (KA) and a high KA-to-kynurenine (K) ratio (KA:K) positively correlated with age and with inflammatory cytokines and chemokines and negatively correlated with T cell responses. Males that clinically deteriorated had a higher KA:K than those that stabilized. KA inhibits glutamate release, and glutamate abundance was lower in patients that clinically deteriorated and correlated with immune responses. Analysis of data from the Genotype-Tissue Expression (GTEx) project revealed that the expression of the gene encoding the enzyme that produces KA, kynurenine aminotransferase, correlated with cytokine abundance and activation of immune responses in older males. This study reveals that KA has a sex-specific link to immune responses and clinical outcomes in COVID-19, suggesting a positive feedback between metabolites and immune responses in males.

Generating hard-to-obtain information from easy-to-obtain information: Applications in drug discovery and clinical inference.

Often when biological entities are measured in multiple ways, there are distinct categories of information: some information is easy-to-obtain information (EI) and can be gathered on virtually every subject of interest, while other information is hard-to-obtain information (HI) and can only be gathered on some. We propose building a model to make probabilistic predictions of HI using EI. Our feature mapping GAN (FMGAN), based on the conditional GAN framework, uses an embedding network to process conditions as part of the conditional GAN training to create manifold structure when it is not readily present in the conditions. We experiment on generating RNA sequencing of cell lines perturbed with a drug conditioned on the drug's chemical structure and generating FACS data from clinical monitoring variables on a cohort of COVID-19 patients, effectively describing their immune response in great detail.

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 responses1-6. Although pathological innate immune activation is well-documented in severe disease1, the effect of autoantibodies on disease progression is less well-defined. Here we use a high-throughput autoantibody discovery technique known as rapid extracellular antigen profiling7 to screen a cohort of 194 individuals infected with SARS-CoV-2, comprising 172 patients with COVID-19 and 22 healthcare workers with mild disease or asymptomatic infection, for autoantibodies against 2,770 extracellular and secreted proteins (members of the exoproteome). We found that patients with COVID-19 exhibit marked increases in autoantibody reactivities as compared to uninfected individuals, and show 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 signalling and by altering peripheral immune cell composition, and found that mouse surrogates of these autoantibodies increase disease severity in a mouse model of SARS-CoV-2 infection. Our analysis of autoantibodies against tissue-associated antigens revealed associations with specific clinical characteristics. Our findings suggest a pathological role for exoproteome-directed autoantibodies in COVID-19, with diverse effects on immune functionality and associations with clinical outcomes.

Delayed production of neutralizing antibodies correlates with fatal COVID-19.

Recent studies have provided insights into innate and adaptive immune dynamics in coronavirus disease 2019 (COVID-19). However, the exact features of antibody responses that govern COVID-19 disease outcomes remain unclear. In this study, we analyzed humoral immune responses in 229 patients with asymptomatic, mild, moderate and severe COVID-19 over time to probe the nature of antibody responses in disease severity and mortality. We observed a correlation between anti-spike (S) immunoglobulin G (IgG) levels, length of hospitalization and clinical parameters associated with worse clinical progression. Although 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 discharged patients. However, they mounted a robust, yet delayed, response, measured by anti-S, anti-receptor-binding domain IgG and neutralizing antibody (NAb) levels compared to survivors. Delayed seroconversion kinetics correlated with impaired viral control in deceased patients. Finally, although sera from 85% of patients displayed some neutralization capacity during their disease course, NAb generation before 14 d 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.

Longitudinal analyses reveal immunological misfiring in severe COVID-19.

Recent studies have provided insights into the pathogenesis of coronavirus disease 2019 (COVID-19)1-4. However, the longitudinal immunological correlates of disease outcome remain unclear. Here we serially analysed immune responses in 113 patients with moderate or severe COVID-19. Immune profiling revealed an overall increase in innate cell lineages, with a concomitant reduction in T cell number. An early elevation in cytokine levels was associated with worse disease outcomes. Following an early increase in cytokines, patients with moderate COVID-19 displayed a progressive reduction in type 1 (antiviral) and type 3 (antifungal) responses. By contrast, patients with severe COVID-19 maintained these elevated responses throughout the course of the disease. Moreover, severe COVID-19 was accompanied by an increase in multiple type 2 (anti-helminths) effectors, including interleukin-5 (IL-5), IL-13, immunoglobulin E and eosinophils. Unsupervised clustering analysis identified four immune signatures, representing growth factors (A), type-2/3 cytokines (B), mixed type-1/2/3 cytokines (C), and chemokines (D) that correlated with three distinct disease trajectories. The immune profiles of patients who recovered from moderate COVID-19 were enriched in tissue reparative growth factor signature A, whereas the profiles of those with who developed severe disease had elevated levels of all four signatures. Thus, we have identified a maladapted immune response profile associated with severe COVID-19 and poor clinical outcome, as well as early immune signatures that correlate with divergent disease trajectories.

Analytical sensitivity and efficiency comparisons of SARS-CoV-2 RT-qPCR primer-probe sets.

The recent spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exemplifies the critical need for accurate and rapid diagnostic assays to prompt clinical and public health interventions. Currently, several quantitative reverse transcription-PCR (RT-qPCR) assays are being used by clinical, research and public health laboratories. However, it is currently unclear whether results from different tests are comparable. Our goal was to make independent evaluations of primer-probe sets used in four common SARS-CoV-2 diagnostic assays. From our comparisons of RT-qPCR analytical efficiency and sensitivity, we show that all primer-probe sets can be used to detect SARS-CoV-2 at 500 viral RNA copies per reaction. The exception for this is the RdRp-SARSr (Charité) confirmatory primer-probe set which has low sensitivity, probably due to a mismatch to circulating SARS-CoV-2 in the reverse primer. We did not find evidence for background amplification with pre-COVID-19 samples or recent SARS-CoV-2 evolution decreasing sensitivity. Our recommendation for SARS-CoV-2 diagnostic testing is to select an assay with high sensitivity and that is regionally used, to ease comparability between outcomes.