Clinical & Translational Immunology

BackgroundThe fetal immune system undergoes pivotal development during gestation, preparing for postnatal antigenic challenges. Bacterial extracellular vesicles (bEVs), bioactive particles shed by bacteria, are emerging as modulators of host immunity. However, their role in shaping fetal intestinal immune development remains largely unexplored. ObjectivesThis study aimed to investigate the effects of bEV exposure on lymphoid and myeloid populations in the fetal murine gut, focusing on their role in priming intestinal immunity, promoting differentiation, and modulating immune cell phenotypes in both normal and germ-free (GF) environments. Materials and MethodsWe used a murine model to evaluate the immune-modulating effects of bEVs during fetal development. bEVs were isolated from bacterial cultures and introduced into the amniotic sac of embryonic day 15.5 (E15.5) fetuses through intra-amniotic injection. Fetal and neonatal mice were either raised under conventional conditions (normal environment, NE) or in germ-free (GF) environments to assess microbiota-dependent effects. Immune profiling of fetal (E17) and postnatal (4 weeks) gut tissues was performed using high-dimensional mass cytometry (CyTOF) in both conventionally housed and germ-free (GF) mice. Clustering and differential expression analyses identified lymphoid and myeloid subpopulations, including progenitors, antigen-presenting cells, and intestinal stem cells (ISCs). secondary immune challenge (LPS or TSST-1) was conducted in postnatal bEV-primed mice to assess immune memory responses. ResultsbEV exposure significantly increased the prevalence of CD45- CD24+ CD44+ ISCs, promoting intestinal renewal and defense via differentiation into Paneth and tuft cells. These ISCs exhibited potential antigen-presenting capabilities through MHC expression. CD45+ lymphoid progenitors were upregulated, highlighting their role in early differentiation pathways. Myeloid progenitors, particularly monocyte-dendritic progenitor subsets, showed a bias toward antigen-presenting phenotypes.Germ-free models revealed heightened sensitivity to bEVs, with pronounced activation of progenitors and a reduction in exhaustion markers. Interestingly, macrophage and neutrophil populations displayed dose-dependent modulation, with low bEV concentrations promoting their expansion and higher doses leading to reduced incidence. Our findings suggest that bEVs act as immune priming agents in the fetal gut, promoting progenitor expansion and differentiation while preparing the intestine for postnatal challenges. Differences in responses between NE and GF models emphasize the importance of environmental influences, including microbiota, on bEV-mediated immune modulation. ConclusionbEVs play a pivotal role in shaping fetal intestinal immunity by priming lymphoid and myeloid progenitors and enhancing ISC function. These results open potential avenues for leveraging bEVs in immunomodulation and vaccine strategies. Future studies should explore the functional responses of bEV-primed cells and their translational relevance in humans.

A better understanding of sepsis-induced immunosuppression pathophysiology is desirable for the development of novel therapeutic strategies to prevent and reduce the rates of secondary infections and their associated mortality. Here we demonstrate that PD-L1+CD44+B220LowCD138+IgM+ regulatory plasma cells (PCs) are induced in a murine model of sepsis-induced immune alterations and in critically ill patients with bacterial sepsis and COVID-19. This was revealed both by detailed analysis of their phenotypical features and gene expression profile and by functional explorations comparing capacity of purified B cells and PCs to suppress T cell proliferation and IFN{gamma} secretion ex vivo. Sepsis-induced regulatory PCs exerted their suppressive function on T cells through IL-10 production and increased PD-L1 expression independently of regulatory T cells. Our findings thus reveal a novel pathophysiological mechanism of sepsis-induced immunosuppression that involves regulatory PCs. As such, these PCs constitute valid therapeutic targets to improve immune cell functions impaired by sepsis.

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BackgroundThe antibody response to SARS-CoV-2 does not follow the immunoglobulin isotype pattern expected in a primary response and is inconsistent with the current interpretation of COVID-19 immunopathology as the result of a primary infection. To better understand the immune response to SARS-CoV-2, it is essential to determine whether it is primary or secondary (or recall). The analysis of highly granular immunological variable trajectories of a homogeneous cohort of patients receiving standardised medical care should discern between primary and secondary responses. MethodsThis is a prospective cohort study of 191 SARS-CoV-2 infection cases and 44 healthy controls from the second wave of COVID-19 in the Barcelona area. The study stratified patients by severity and analysed the trajectories of SARS-CoV-2 antibodies and multiple immune variables for features associated with primary and recall immune responses. FindingsIsotype-specific antibody trajectories to SARS-CoV-2 proteins revealed a pattern of recall response in 94{middle dot}2% of cases. In these cases, the detailed trajectories of plasmablasts, B cells, cTfh high-resolution subsets, and cytokines were consistent with a secondary response. The transcriptomic data indicated that this cohort is strictly comparable to contemporary cohorts. ConclusionsIn most cases, the immune response to SARS-CoV-2 is a recall response. This opens the possibility that most COVID-19 cases are subjected to immune imprinting by endemic coronavirus, which, in turn, can contribute to severity by interfering with the immune response to SARS-CoV-2 and by antibody-dependent enhancement. Considering the immune responses to SARS-CoV-2 secondary provides a better perspective to interpret COVID-19 pathology. FundingGrants COV20/00416, Cov20/00654, and COV20/00388 from Instituto de Salud Carlos III (ISCIII), Madrid, Spain, co-financed by the European Regional Development Fund (ERDF).

Emerging evidence suggests that SARS-CoV-2 infections are characterized by systemic immune responses that appear to be dysregulated with more severe CoViD-19 disease. Lymphopenia and delayed antibody responses are commonly identified in CoViD-19 subjects, and recent reports have demonstrated abrogation of germinal centers in severe CoViD-19. This work assessed a potential mechanistic basis for impaired humoral responses, focusing on the T follicular helper (Tfh) and B cell interface that is critical for germinal center reactions. Here we demonstrated that Tfh activity is impaired in hospitalized relative to ambulatory CoViD-19 subjects, potentially due to decreased expression of the costimulatory molecule ICOS-L on B cells. Functional impairment manifested as a diminished ability to stimulated Tfh derived IFN{gamma} and IL-21, the latter of which is critical for B cell proliferation and differentiation. Activation of Tfh cells by agonism of the ICOS receptor ex vivo by an agonistic antibody stimulated the generation of IFN{gamma}/IL-21 double positive cells from hospitalized CoViD-19 subjects. This report establishes an immunological defect that differentiates ambulatory from hospitalized CoViD and suggests that agents that could restore impaired mechanisms at the Tfh-B cell interface may be of therapeutic value.

A multivariant total subclass analysis has been performed for a control cohort (n=15) and a long COVID patient cohort (n=15) measuring the IgG1, IgG2, IgG3, IgG4 and IgE response to the following 14 variants of SARS-CoV-2: Wuhan, Alpha, Delta, BA.1, BA.2, BA.5, EG.5.1, XBB.1.5, BA.2.75, CH.1.1, BA.2.12.1, BQ.1.1, JN.1, and KP.3. Significant differences (p < 0.05 and p < 0.005) between concentrations of IgG subclasses by variant were found in 24% of variants and in mean-normalised distributions. The medians of the mean-normalised distributions were significantly lower for IgG1 (p < 0.05) in long COVID patients compared with controls, and significantly higher (p < 0.005) for levels of IgG3, IgG4 and IgE for long COVID patients. A preliminary diagnostics classification analysis performed by variation of the mean-normalised upper and lower percentiles symmetrically for IgG3 showed a long COVID diagnostic sensitivity of 80%, and specificity of 80% for the 60th percentile threshold of the control cohort. Three types of long COVID can be identified: patients with at least one variant below the threshold (hypo-immune), patients with at least one variant above the threshold (hyperimmune) and patients with IgG3 levels within the reference range. The multivariant subclass spectrum indicates IgG4 and IgE elevations due to potential chronic antigen exposure from persistent virus or autoimmunity and may indicate potential therapeutic interventions.

BackgroundMucosal antibodies can prevent virus entry and replication in mucosal epithelial cells and hence virus shedding. Preclinical and clinical studies have shown that a parenteral booster injection of a vaccine against a mucosal pathogen promotes stronger mucosal immune responses following prior infection compared to two injections of a parenteral vaccine. We investigated whether this was also the case for a COVID-19 mRNA vaccine. MethodsTwenty-three COVID-19 convalescent patients and 20 SARS-CoV-2-naive subjects were vaccinated with respectively one and two doses of the Pfizer-BioNTech COVID-19 RNA vaccine. Nasal Epithelial Lining Fluid (NELF) and plasma were collected before and after vaccination and assessed for Immunoglobulin (Ig)G and IgA to Spike and for their ability to inhibit the binding of Spike to its ACE-2 receptor. Blood was analyzed one week after vaccination for the number of Spike-specific Antibody Secreting Cells (ASCs) with a mucosal tropism. ResultsIn COVID-19 convalescent patients, a single dose of vaccine amplified pre-existing Spike-specific IgG and IgA antibody responses in both NELF and blood against both vaccine homologous and variant strains, including delta. These responses were associated with Spike-specific IgG and IgA ASCs with a mucosal tropism in blood. Nasal IgA and IgG antibody responses were lower in magnitude in SARS-CoV-2-naive subjects after two vaccine doses ConclusionThis study showed that a parenteral booster injection of a COVID-19 RNA vaccine promoted stronger mucosal immune responses in COVID-19 convalescent patients compared to SARS-CoV-2 naive subjects who had received a first vaccine dose.

Pediatric SARS-CoV-2 infection results in clinical presentations ranging from asymptomatic/mild infection to severe pulmonary COVID-19, to Multisystem Inflammatory Syndrome in Children (MIS-C), characterized by hyperinflammation and multi-organ involvement. While various aspects of antibody responses to pediatric SARS-CoV-2 infection manifestations have been reported, parallel studies of antibody responses to viral and self-antigens are understudied. We tested whether clinical presentations of increasing severity corresponded to different antiviral antibody and autoantibody signatures. Using custom arrays, we found that, relative to uninfected subjects, all SARS-CoV-2 infection manifestations were associated with increased autoantibody production, suggesting pediatric SARS-CoV-2 infection as a risk factor for autoimmune complications. Subtle differences were seen in autoantibody patterns among infection groups, with some autoantibodies more associated with mild manifestations and others with severe ones. When we compared MIS-C and severe COVID-19 subjects, we found differences in IgG (mostly IgG1) abundance but not in Fc-mediated effector functions. Thus, MIS-C may be associated with abnormal antibody function, suggesting that this syndrome, and perhaps other post-acute sequelae of SARS-CoV-2 infection, may be associated with antibody dysfunction. Our study shows that the antibody repertoire varies with clinical presentation of SARS-CoV-2 in children and its analysis may help understand long COVID pathogenesis. ImpactOur study shows that the antibody repertoire varies with the clinical presentation of SARS-CoV-2 in children, which has implications for understanding long COVID pathogenesis.

PurposePatients with common variable immunodeficiency (CVID) frequently exhibit impaired antibody responses to vaccination, yet the dynamics of humoral and cellular immunity following mRNA immunisation remain incompletely defined. This study aimed to characterise the temporal evolution of anti-SARS-CoV-2 antibody and T cell responses across successive vaccine doses in a well-characterised CVID cohort, and to identify key determinants of vaccine responsiveness in this population. MethodsWe performed a longitudinal and cross-sectional analysis of serum and peripheral blood mononuclear cell (PBMC) samples collected from 88 CVID patients after two, three, or four doses of mRNA vaccine (Moderna/mRNA-1273 or Pfizer-BioNTech/BNT162b2). Anti-receptor-binding domain (RBD) IgG titers were quantified in relation to vaccine dose, time since last vaccination, and clinical characteristics. Vaccine-specific CD4+ and CD8+ T cell responses were assessed ex vivo using an activation-induced marker (AIM) assay by flow cytometry. ResultsThe proportion of patients with detectable anti-RBD IgG increased from 35% after two doses to more than 80% after four doses. Boosting-dependent increases in IgG titers were observed exclusively in samples collected more than three months after the last dose, and antibody levels correlated positively with time since vaccination, consistent with delayed but progressive humoral kinetics that stabilised after the third dose. In contrast, spike-specific CD4+ and CD8+ T cell responses were rapidly induced and remained stable across all timepoints. ConclusionVaccine-induced immunity in CVID is characterised by delayed humoral responses alongside preserved cellular immunity. Early post-vaccination serology may systematically underestimate vaccine responsiveness, and booster vaccination supports stabilisation of antibody responses in this population.

BackgroundIn lung transplantation, de novo immunodominant donor-specific anti-HLA antibodies recognizing HLA-DQ antigens (dn-iDSA-DQ) are predominant and can induce chronic lung allograft dysfunction (CLAD). We previously developed a method to measure the active concentration of dn-iDSA-DQ. We aimed to determine whether this new quantitative biomarker is associated with transplantation outcomes. MethodsThis retrospective multicentre cohort study included 90 lung transplant recipients (LTRs) developing dn-iDSA-DQ, evidenced through single antigen flow beads (SAFB) follow-up. We measured the active concentration of dn-iDSA-DQ at the time of their first detection (T0) for all LTRs, and within the 2 years after DSA detection, whenever possible. SAFB dn-iDSA-DQ characteristics and clinical data were retrieved up to 5 years after DSA detection. ResultsWe tested 184 sera with SPR (n=90 at T0, n=94 within the 2 years after DSA detection), among which 63 (34.4%) had a quantifiable concentration of the dn-iDSA-DQ ([&ge;]0.3 nM). The median SAFB mean fluorescence intensity (MFI) of the dn-iDSA-DQ with a concentration [&ge;]0.3 nM was higher (p<0.0001), yet the correlation between SAFB MFI and active concentration was low (r=0.758, p<0.0001). In multivariate analysis, a concentration of the dn-iDSA-DQ [&ge;]0.3 nM at T0 was independently associated with a lower 2-year CLAD-free survival (HR 2.06, p=0.02). A concentration of the dn-iDSA-DQ [&ge;]0.3 nM within the 2 years from DSA detection was associated with a lower graft survival in univariate analysis. ConclusionsActive concentration of dn-iDSA-DQ appears as a valuable biomarker to identify pathogenic DSA at their first detection because of its association with CLAD.

BackgroundSerological and clinical features with similarities to systemic autoimmunity have been reported in severe COVID-19, but there is a lack of studies that include contemporaneous controls who do not have COVID-19. MethodsObservational cohort study of adult patients admitted to an intensive care unit with acute respiratory failure. Patients were divided into COVID+ and COVID- based on SARS-CoV-2 PCR from nasopharyngeal swabs and/or endotracheal aspirates. No COVID-19 specific interventions were given. The primary clinical outcome was death in the ICU within 3 months; secondary outcomes included in-hospital death and disease severity measures. Measurements including autoantibodies, were done longitudinally. ANOVA and Fishers exact test were used with =0.05, with a false discovery rate of q=0.05. Bayesian analysis was performed to provide credible estimates of the possible states of nature compatible with our results. Results22 COVID+ and 20 COVID- patients were recruited, 69% males, median age 60.5 years. Overall, 64% had anti-nuclear antibodies, 38% had antigen-specific autoantibodies, 31% had myositis related autoantibodies, and 38% had high levels of anti-cytokine autoantibodies. There were no statistically significant differences between COVID+ and COVID- for any of the clinical or autoantibody parameters. A specific pattern of anti-nuclear antibodies was associated with worse clinical severity for both cohorts. ConclusionsSevere COVID+ patients have similar humoral autoimmune features as comparably ill COVID- patients, suggesting that autoantibodies are a feature of critical illness regardless of COVID-19 status. The clinical significance of autoimmune serology and the correlation with severity in critical illness remains to be elucidated.

Zoonotic spillover of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to humans in December 2019 caused the coronavirus disease 2019 (COVID-19) pandemic. Serological monitoring is critical for detailed understanding of individual immune responses to infection and protection to guide clinical therapeutic and vaccine strategies. We developed a high throughput multiplexed SARS-CoV-2 antigen microarray incorporating spike (S) and nucleocapsid protein (NP) and fragments expressed in various hosts which allowed simultaneous assessment of serum IgG, IgA, and IgM responses. Antigen glycosylation influenced antibody binding, with S glycosylation generally increasing and NP glycosylation decreasing binding. Purified antibody isotypes demonstrated a binding pattern and intensity that differed from the same isotype in the presence of other isotypes in whole serum, probably due to competition. Using purified antibody isotypes from naive Irish COVID-19 patients, we correlated antibody isotype binding to different panels of antigens with disease severity, with significance for binding to the S region S1 expressed in insect cells (S1 Sf21) for all three antibody isotypes. Assessing longitudinal response for constant concentrations of antibody isotypes for a subset of patients demonstrated that while the relative proportion of antigen-specific IgGs decreased over time for severe disease, the relative proportion of antigen-specific IgA binding remained at the same magnitude at 5 and 9 months post-first symptom onset. Further, the relative proportion of IgM binding decreased for S antigens but remained the same for NP antigens. This may support antigen specific serum IgA and IgM playing a role in maintaining longer-term protection, of importance for developing and assessing vaccine strategies. Overall, these data demonstrate the multiplexed platform as a sensitive and useful platform for expanded humoral immunity studies, allowing detailed elucidation of antibody isotypes response against multiple antigens. This approach will be useful for monoclonal antibody therapeutic studies and screening of donor polyclonal antibodies for patient infusions.

Severe acquired respiratory syndrome coronavirus-2 (SARS-CoV-2) is the cause of coronavirus disease (COVID-19). In severe COVID-19 cases, higher antibody titers against seasonal coronaviruses have been observed than in mild cases. To investigate antibody cross-reactivity as potential explanation for severe disease, we determined the kinetics, breadth, magnitude and level of cross-reactivity of IgG against SARS-CoV-2 and seasonal CoV nucleocapsid and spike from 17 severe COVID-19 cases at the clonal level. Although patients mounted a mostly type-specific SARS-CoV-2 response, B-cell clones directed against seasonal CoV dominated and strongly increased over time. Seasonal CoV IgG responses that did not neutralize SARS-CoV-2 were boosted well beyond detectable cross-reactivity, particularly for HCoV-OC43 spike. These findings support a back-boost of poorly protective coronavirus-specific antibodies in severe COVID-19 patients that may negatively impact de novo SARS-CoV-2 immunity, reminiscent of original antigenic sin.

Engineered long lived plasma cells have the potential to be a new area of cell therapy. A key step in developing this cell therapy is testing in a model with an intact immune system similar to humans. To that end, we have developed methods to purify, expand, and differentiate non-human primate (NHP; rhesus macaque) B cells ex vivo. We consistently achieved 10-fold expansion of NHP B cells using a readily available commercial supplement. After only seven days in culture, large percentages of cells in NHP B cell cultures were differentiated. These cells expressed surface markers found in human antibody secreting cells (CD38 and CD138) and secreted immunoglobulin G. From single cell transcriptome analysis of NHP, we verified the presence of plasma cell markers commonly shared with humans, and have unearthed less recognized markers such as CD59 and CD79A. In addition, we identified unique NHP plasma cell markers that are absent in humans including the immune checkpoint molecule CD274 (PD-L1, Programmed Death-Ligand 1). Furthermore, we found that MHC class I molecules were upregulated in NHP plasma cells, in contrast to the pattern observed in humans. Lastly, we also identified the serotypes (AAVD-J) and established the conditions for efficient transduction of NHP B cells with AAV vectors, achieving an editing rate of approximately 60%. We envision that this work will accelerate proof-of-concept in vivo studies using engineered protein-secreting B cells in the NHP model.

Immunoglobulin (Ig) replacement therapies (IgRT) including intravenous (IVIg) and subcutaneous (SCIg), are pooled IgG preparations widely used to restore humoral immunity and to suppress pathological inflammation in autoimmune and inflammatory disorders. Despite broad clinical use, the mechanisms underlying their immunomodulatory effects remain incompletely defined. Here, we identify extracellular vesicle (EV)-associated cytokines as mediators of IVIg activity. Multiplex bead-based flow cytometry revealed that EVs isolated by size exclusion followed by ultracentrifugation from IVIg were CD63 positive but depleted of platelet-derived and HLA markers relative to EVs from unprocessed human plasma. Luminex profiling demonstrated substantial reduction of pro-inflammatory cytokines in IVIg EVs. Notably, although IVIg EVs contained abundant IFN{gamma}, they failed to activate IFNGR/JAK/STAT1 signaling. Instead, prolonged exposure to IVIg EVs suppressed subsequent IFN{gamma}-induced STAT1 activation. Engineered IFN{gamma}-coated EVs (IFN{gamma}-eEVs) recapitulated both activating and inhibitory effects indicating context-dependent signaling bias. Critically, cold ethanol precipitation, a key step in IVIg manufacturing, selectively abrogated the activating function of IFN{gamma}-eEVs while preserving their inhibitory capacity. These findings define a previously unrecognized mechanism where IVIg processing generates EVs that bias IFN{gamma} signaling toward suppression. EV-associated cytokines therefore represent a generalizable pathway through which IVIg exerts anti-inflammatory effects across immune-mediated diseases.

Autoreactive antibodies (AAB) are currently being investigated as causative or aggravating factors during post-COVID. In this study we analyze the effect of immunoadsorption therapy on symptom improvement and the relationship with immunological parameters in post-COVID patients exhibiting symptoms of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS). This observational study includes 12 post-COVID patients exhibiting a predominance of ME/CFS symptoms alongside increased concentrations of autonomic nervous system receptors (ANSR) autoantibodies and neurological impairments. We found that following immunoadsorption therapy, the ANSR autoantibodies were nearly eliminated from the patients blood. The removal of IgG antibodies was accompanied by a decrease of pro-inflammatory cytokines including IL4, IL2, IL1{beta}, TNF and IL17A serum levels, and a significant reduction of soluble spike protein. Notably, a strong positive correlation between pro-inflammatory cytokines and ASNR-AABs {beta}1, {beta}2, M3, and M4 was observed in spike protein-positive patients, whereas no such correlation was evident in spike protein-negative patients. 30 days post-immunoadsorption therapy, patients exhibited notable improvement in neuropsychological function and a substantial amelioration of hand grip strength was observed. However, neither self-reported symptoms nor scores on ME/CSF questionnaires showed a significant improvement and a rebound of the removed proteins occurring within a month.

BackgroundMultiple factors affecting COVID19 vaccine induced antibody responses in SARS-CoV2 uninfected immunosuppressed solid organ transplant recipients have been reported; however, there is still a lack of information on non-ACE2 competing cross-CoV2 neutralizing functional antibodies induced in these cohorts, and similarly the vaccine efficacy in prior CoV2-infected immunosuppressed individuals is not well understood. MethodsCOVID19 vaccine efficacy was compared in a panel of kidney and heart transplant recipients who were either CoV2 uninfected (n=63) or CoV2 infected (n=13) prior to receiving two or three doses of mRNA vaccines using pseudoviral neutralization assays against eight CoV2 strains (the CoV2_D614G ancestral strain, alpha, beta, gamma, delta, kappa, lambda, and omicron-BA1 variants), while plasma antibody titers were determined by ELISA using recombinant CoV2-RBD-wt proteins. ResultsMinimally protective neutralizing plasma antibody titers (IC50 [&ge;] 1:50) against the variants were recorded 7-14% and 25-35% after the second and third doses respectively, with Omicron being the most resistant. In contrast, all previously infected vaccinees possessed minimal protective plasma titers against D614G after either two or three vaccine doses, with 11/13 exhibiting strong protection (IC50[&ge;] 1:500) and 10/13 exceeding the minimal protective titer against Omicron. Absorption of the selected plasma with immobilized parental RBD removed [&ge;] 90% of its neutralizing activity, indicating that the dominant neutralization targets were in the RBD. ConclusionsThis study showed that CoV2 infection followed by vaccination, but not vaccination alone, induces the presence of potent highly cross-reactive CoV2 neutralizing plasma antibodies that extend to Omicron variants, even in immunosuppressed SOTRs.

SARS-CoV-2 infection in children typically results in asymptomatic or mild disease. There is a paucity of studies on antiviral immunity in African children. We investigated SARS-CoV-2-specific T cell responses in 71 unvaccinated asymptomatic South African children who were seropositive or seronegative for SARS-CoV-2. SARS-CoV-2-specific CD4+ T cell responses were detectable in 83% of seropositive and 60% of seronegative children. Although the magnitude of the CD4+ T cell response did not differ significantly between the two groups, their functional profiles were distinct, with SARS-CoV-2 seropositive children exhibiting a higher proportion of polyfunctional T cells compared to their seronegative counterparts. The frequency of SARS-CoV-2-specific CD4+ T cells in seronegative children was associated with the endemic human coronavirus (HCoV) HKU1 IgG response. Overall, the presence of SARS-CoV-2-responding T cells in seronegative children may result from cross-reactivity to endemic coronaviruses and could contribute to the relative protection from disease observed in SARS-CoV-2-infected children.

BackgroundLung transplantation (LUTX) is frequently complicated by Primary Graft Dysfunction (PGD), a heterogeneous form of acute lung injury associated with multi-organ failure and rejection. We hypothesized that early, bedside plasma biomarkers could capture this biological heterogeneity, identifying phenotypes with differential clinical outcomes. MethodsThis two-year prospective single-center observational study enrolled 78 bilateral LUTX recipients. With a real-time point-of-care immunoanalyzer we measured IL-1{beta}, IL-2, IL-6, IFN-{gamma}, TNF, CCL-2, IL-15, Ferritin, and D-dimer 36 hours post-reperfusion. Outcome-agnostic Latent Profile Analysis (LPA) was applied to identify bio-signatures. PGD (grade 3) incidence and clinical outcomes were compared across classes. ResultsLPA identified three distinct classes, interpreted as biological sub-phenotypes. The Adaptive phenotype (68%) had the lowest inflammatory activation, shortest vasoactive support and Invasive Mechanical Ventilation (IMV) (both 1 day) and ICU stay (3 days), and lowest 72-hours PGD (8%) and Acute Kidney Injury (AKI) (28%) rates. The Hyperinflammatory phenotype (18%) showed the highest IL-6 and Ferritin levels with resolving PGD (69% to 23%, from 6 to 72 hours), but prolonged IMV (6 days), vasoactive support (3 days), ICU stay (7 days), with a high AKI rate (69%). The Coagulopathic phenotype (15%), requiring more intraoperative blood products, exhibited the highest TNF and D-dimer with persistent PGD (36% at 24 and 72 hours), intermediate vasoactive support (2 days), and AKI severity. Six-month rejection-free survival was similar across phenotypes. ConclusionsThis preliminary hypothesis-generating study suggests that point-of-care biomarkers after LUTX may identify biological phenotypes with potential clinical relevance. Future studies are needed to confirm such phenotyping.

BackgroundCharacterizing the kinetics of the antibody response to SARS{square}CoV{square}2 is of critical importance to developing strategies that may mitigate the public health burden of COVID-19. We sought to determine how circulating antibody levels change over time following natural infection. Methods/MaterialsWe conducted a prospective, longitudinal analysis of COVID-19 convalescent plasma (CCP) donors at multiple time points over a 9-month period. At each study visit, subjects either donated plasma or only had study samples drawn. In all cases, anti-SARS-CoV-2 donor testing was performed using semi-quantitative chemiluminescent immunoassays (ChLIA) targeting subunit 1 (S1) of the SARS-CoV-2 spike (S) protein, and an in-house fluorescence reduction neutralization assay (FRNA). ResultsFrom April to November 2020 we enrolled 202 donors, mean age 47.3 {+/-}14.7 years, 55% female, 75% Caucasian. Most donors reported a mild clinical course (91%, n=171) without hospitalization. One hundred and five (105) (52%) donors presented for repeat visits with a median 42 (12-163) days between visits. The final visit occurred at a median 160 (53-273) days post-symptom resolution. Total anti-SARS-CoV-2 antibodies (Ab), SARS-CoV-2 specific IgG and neutralizing antibodies were detected in 97.5%, 91.1%, and 74% of donors respectively at initial presentation. Neutralizing Ab titers based on FRNA50 were positively associated with mean IgG levels (p = <0.0001). Mean IgG levels and neutralizing titers were positively associated with COVID-19 severity, increased donor age and BMI (p=0.0006 and p=0.0028, p=0.0083 and p=0.0363, (p=0.0008 and p=0.0018, respectively). Over the course of repeat visits, IgG decreased in 74.1% of donors; FRNA50 decreased in 44.4% and remained unchanged in 33.3% of repeat donors. A weak negative correlation was observed between total Ab levels and number of days post-symptom recovery (r = 0.09). ConclusionAnti-SARS-CoV-2 antibodies were identified in 97% of convalescent donors at initial presentation. In a cohort that largely did not require hospitalization. IgG and neutralizing antibodies were positively correlated with age, BMI and clinical severity, and persisted for up to 9 months post-recovery from natural infection. On repeat presentation, IgG anti-SARS-CoV-2 levels decreased in 56% of repeat donors. Overall, these data suggest that CP donors possess a wide range of IgG and neutralizing antibody levels that are proportionally distributed across demographics, with the exception of age, BMI and clinical severity.