Exosomes Recovered From the Plasma of COVID-19 Patients Expose SARS-CoV-2 Spike-Derived Fragments and Contribute to the Adaptive Immune Response.

Coronavirus disease 2019 (COVID-19) is an infectious disease caused by beta-coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that has rapidly spread across the globe starting from February 2020. It is well established that during viral infection, extracellular vesicles become delivery/presenting vectors of viral material. However, studies regarding extracellular vesicle function in COVID-19 pathology are still scanty. Here, we performed a comparative study on exosomes recovered from the plasma of either MILD or SEVERE COVID-19 patients. We show that although both types of vesicles efficiently display SARS-CoV-2 spike-derived peptides and carry immunomodulatory molecules, only those of MILD patients are capable of efficiently regulating antigen-specific CD4+ T-cell responses. Accordingly, by mass spectrometry, we show that the proteome of exosomes of MILD patients correlates with a proper functioning of the immune system, while that of SEVERE patients is associated with increased and chronic inflammation. Overall, we show that exosomes recovered from the plasma of COVID-19 patients possess SARS-CoV-2-derived protein material, have an active role in enhancing the immune response, and possess a cargo that reflects the pathological state of patients in the acute phase of the disease.

Circulating Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Vaccine Antigen Detected in the Plasma of mRNA-1273 Vaccine Recipients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins were measured in longitudinal plasma samples collected from 13 participants who received two doses of mRNA-1273 vaccine. Eleven of 13 participants showed detectable levels of SARS-CoV-2 protein as early as day 1 after first vaccine injection. Clearance of detectable SARS-CoV-2 protein correlated with production of immunoglobulin G (IgG) and immunoglobulin A (IgA).

Comparative Investigation of Methods for Analysis of SARS-CoV-2-Spike-Specific Antisera.

In light of an increasing number of vaccinated and convalescent individuals, there is a major need for the development of robust methods for the quantification of neutralizing antibodies; although, a defined correlate of protection is still missing. Sera from hospitalized COVID-19 patients suffering or not suffering from acute respiratory distress syndrome (ARDS) were comparatively analyzed by plaque reduction neutralization test (PRNT) and pseudotype-based neutralization assays to quantify their neutralizing capacity. The two neutralization assays showed comparable data. In case of the non-ARDS sera, there was a distinct correlation between the data from the neutralization assays on the one hand, and enzyme-linked immune sorbent assay (ELISA), as well as biophysical analyses, on the other hand. As such, surface plasmon resonance (SPR)-based assays for quantification of binding antibodies or analysis of the stability of the antigen-antibody interaction and inhibition of syncytium formation, determined by cell fusion assays, were performed. In the case of ARDS sera, which are characterized by a significantly higher fraction of RBD-binding IgA antibodies, there is a clear correlation between the neutralization assays and the ELISA data. In contrast to this, a less clear correlation between the biophysical analyses on the one hand and ELISAs and neutralization assays on the other hand was observed, which might be explained by the heterogeneity of the antibodies. To conclude, for less complex immune sera-as in cases of non-ARDS sera-combinations of titer quantification by ELISA with inhibition of syncytium formation, SPR-based analysis of antibody binding, determination of the stability of the antigen-antibody complex, and competition of the RBD-ACE2 binding represent alternatives to the classic PRNT for analysis of the neutralizing potential of SARS-CoV-2-specific sera, without the requirement for a BSL3 facility.

Assessing the performance of a serological point-of-care test in measuring detectable antibodies against SARS-CoV-2.

This study investigated the performance of a rapid point-of-care antibody test, the BioMedomics COVID-19 IgM/IgG Rapid Test, in comparison with a high-quality, validated, laboratory-based platform, the Roche Elecsys Anti-SARS-CoV-2 assay. Serological testing was conducted on 709 individuals. Concordance metrics were estimated. Logistic regression was used to assess associations with seropositivity. SARS-CoV-2 seroprevalence was 63.5% (450/709; 95% CI 59.8%-67.0%) using the BioMedomics assay and 71.9% (510/709; 95% CI 68.5%-75.2%) using the Elecsys assay. There were 60 discordant results between the two assays, all of which were seropositive in the Elecsys assay, but seronegative in the BioMedomics assay. Overall, positive, and negative percent agreements between the two assays were 91.5% (95% CI 89.2%-93.5%), 88.2% (95% CI 85.1%-90.9%), and 100% (95% CI 98.2%-100%), respectively, with a Cohen's kappa of 0.81 (95% CI 0.78-0.84). Excluding specimens with lower (Elecsys) antibody titers, the agreement improved with overall, positive, and negative percent concordance of 94.4% (95% CI 92.3%-96.1%), 91.8% (95% CI 88.8%-94.3%), and 100% (95% CI 98.2%-100%), respectively, and a Cohen's kappa of 0.88 (95% CI 0.85-0.90). Logistic regression confirmed better agreement with higher antibody titers. The BioMedomics COVID-19 IgM/IgG Rapid Test demonstrated good performance in measuring detectable antibodies against SARS-CoV-2, supporting the utility of such rapid point-of-care serological testing to guide the public health responses and vaccine prioritization.

The SARS-CoV-2 Spike protein disrupts human cardiac pericytes function through CD147 receptor-mediated signalling: a potential non-infective mechanism of COVID-19 microvascular disease.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a broad range of clinical responses including prominent microvascular damage. The capacity of SARS-CoV-2 to infect vascular cells is still debated. Additionally, the SARS-CoV-2 Spike (S) protein may act as a ligand to induce non-infective cellular stress. We tested this hypothesis in pericytes (PCs), which are reportedly reduced in the heart of patients with severe coronavirus disease-2019 (COVID-19). Here we newly show that the in vitro exposure of primary human cardiac PCs to the SARS-CoV-2 wildtype strain or the α and δ variants caused rare infection events. Exposure to the recombinant S protein alone elicited signalling and functional alterations, including: (1) increased migration, (2) reduced ability to support endothelial cell (EC) network formation on Matrigel, (3) secretion of pro-inflammatory molecules typically involved in the cytokine storm, and (4) production of pro-apoptotic factors causing EC death. Next, adopting a blocking strategy against the S protein receptors angiotensin-converting enzyme 2 (ACE2) and CD147, we discovered that the S protein stimulates the phosphorylation/activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) through the CD147 receptor, but not ACE2, in PCs. The neutralisation of CD147, either using a blocking antibody or mRNA silencing, reduced ERK1/2 activation, and rescued PC function in the presence of the S protein. Immunoreactive S protein was detected in the peripheral blood of infected patients. In conclusion, our findings suggest that the S protein may prompt PC dysfunction, potentially contributing to microvascular injury. This mechanism may have clinical and therapeutic implications.

Microfluidic characterisation reveals broad range of SARS-CoV-2 antibody affinity in human plasma.

The clinical outcome of SARS-CoV-2 infections, which can range from asymptomatic to lethal, is crucially shaped by the concentration of antiviral antibodies and by their affinity to their targets. However, the affinity of polyclonal antibody responses in plasma is difficult to measure. Here we used microfluidic antibody affinity profiling (MAAP) to determine the aggregate affinities and concentrations of anti-SARS-CoV-2 antibodies in plasma samples of 42 seropositive individuals, 19 of which were healthy donors, 20 displayed mild symptoms, and 3 were critically ill. We found that dissociation constants, K d, of anti-receptor-binding domain antibodies spanned 2.5 orders of magnitude from sub-nanomolar to 43 nM. Using MAAP we found that antibodies of seropositive individuals induced the dissociation of pre-formed spike-ACE2 receptor complexes, which indicates that MAAP can be adapted as a complementary receptor competition assay. By comparison with cytopathic effect-based neutralisation assays, we show that MAAP can reliably predict the cellular neutralisation ability of sera, which may be an important consideration when selecting the most effective samples for therapeutic plasmapheresis and tracking the success of vaccinations.

Detection of cross-reactive immunoglobulin A against the severe acute respiratory syndrome-coronavirus-2 spike 1 subunit in saliva.

Abundant secretory immunoglobulin A (SIgA) in the mucus, breast milk, and saliva provides immunity against infection of mucosal surfaces. Pre-pandemic breast milk samples containing SIgA have been reported to cross-react with SARS-CoV-2; however, it remains unknown whether SIgA showing the cross-reaction with SARS-CoV-2 exists in saliva. We aimed to clarify whether SIgA in saliva cross-reacts with SARS-CoV-2 spike 1 subunit in individuals who have not been infected with this virus. The study involved 137 (men, n = 101; women, n = 36; mean age, 38.7; age range, 24-65 years) dentists and doctors from Kanagawa Dental University Hospital. Saliva and blood samples were analyzed by polymerase chain reaction (PCR) and immunochromatography for IgG and IgM, respectively. We then identified patients with saliva samples that were confirmed to be PCR-negative and IgM-negative for SARS-CoV-2. The cross-reactivity of IgA-positive saliva samples with SARS-CoV-2 was determined by enzyme-linked immunosorbent assay using a biotin-labeled spike recombinant protein (S1-mFc) covering the receptor-binding domain of SARS-CoV-2. The proportion of SARS-CoV-2 cross-reactive IgA-positive individuals was 46.7%, which correlated negatively with age (r = -0.218, p = 0.01). The proportion of IgA-positive individuals aged ≥50 years was significantly lower than that of patients aged ≤49 years (p = 0.008). SIgA was purified from the saliva of patients, which could partially suppress the binding of SARS-CoV-2 spike protein to the angiotensin converting enzyme-2 receptor. This study demonstrates the presence of SARS-CoV-2 cross-reactive SIgA in the saliva of individuals who had never been infected with the virus, suggesting that SIgA may help prevent SARS-CoV-2 infection.

High-throughput fabrication of high aspect ratio Ag/Al nanopillars for optical detection of biomarkers.

Nanomaterial-based optical techniques for biomarker detection have garnered tremendous attention from the nanofabrication community due to their high precision and enhanced limit of detection (LoD) features. These nanomaterials are highly responsive to local refractive index (RI) fluctuations, and their RI unit sensitivity can be tuned by varying the chemical composition, geometry, and dimensions of the utilized nanostructures. To improve the sensitivity and LoD values of these nanomaterials, it is common to increase both dimensions and aspect ratios of the fabricated nanostructures. However, limited by the complexity, prolonged duration, and elevated costs of the available nanofabrication techniques, mass production of these nanostructures remains challenging. To address not only high accuracy, but also speed and production effectiveness in these nanostructures' fabrication, our work reports, for the first time, a fast, high-throughput, and cost-effective nanofabrication protocol for routine manufacturing of polymer-based nanostructures with high sensitivity and calculated LoD in the pM range by utilizing anodized aluminum oxide (AAO) membranes as templates. Specifically, our developed platform consists of arrays of nearly uniform polystyrene nanopillars with an average diameter of ∼185 nm and aspect ratio of ∼11. We demonstrate that these nanostructures can be produced at a high speed and a notably low price, and that they can be efficiently applied for biosensing purposes after being coated with aluminum-doped silver (Ag/Al) thin films. Our platform successfully detected very low concentrations of human C-reactive protein (hCRP) and SARS-CoV-2 spike protein biomarkers in human plasma samples with LoDs of 11 and 5 pM, respectively. These results open new opportunities for day-to-day fabrication of high aspect ratio arrays of nanopillars that can be used as a base for nanoplasmonic sensors with competitive LoD values. This, in turn, contributes to the development of point-of-care devices and further improvement of the existing nanofabrication techniques, thereby enriching the fields of pharmacology, clinical analysis, and diagnostics.

Development of ACE2 autoantibodies after SARS-CoV-2 infection.

BACKGROUND: Activation of the immune system is implicated in the Post-Acute Sequelae after SARS-CoV-2 infection (PASC) but the mechanisms remain unknown. Angiotensin-converting enzyme 2 (ACE2) cleaves angiotensin II (Ang II) resulting in decreased activation of the AT1 receptor and decreased immune system activation. We hypothesized that autoantibodies against ACE2 may develop after SARS-CoV-2 infection, as anti-idiotypic antibodies to anti-spike protein antibodies. METHODS AND FINDINGS: We tested plasma or serum for ACE2 antibodies in 67 patients with known SARS-CoV-2 infection and 13 with no history of infection. None of the 13 patients without history of SARS-CoV-2 infection and 1 of the 20 outpatients that had a positive PCR test for SARS-CoV-2 had levels of ACE2 antibodies above the cutoff threshold. In contrast, 26/32 (81%) in the convalescent group and 14/15 (93%) of patients acutely hospitalized had detectable ACE2 antibodies. Plasma from patients with antibodies against ACE2 had less soluble ACE2 activity in plasma but similar amounts of ACE2 protein compared to patients without ACE2 antibodies. We measured the capacity of the samples to inhibit ACE2 enzyme activity. Addition of plasma from patients with ACE2 antibodies led to decreased activity of an exogenous preparation of ACE2 compared to patients that did not have antibodies. CONCLUSIONS: Many patients with a history of SARS-CoV-2 infection have antibodies specific for ACE2. Patients with ACE2 antibodies have lower activity of soluble ACE2 in plasma. Plasma from these patients also inhibits exogenous ACE2 activity. These findings are consistent with the hypothesis that ACE2 antibodies develop after SARS-CoV-2 infection and decrease ACE2 activity. This could lead to an increase in the abundance of Ang II, which causes a proinflammatory state that triggers symptoms of PASC.

Rapid measurement of SARS-CoV-2 spike T cells in whole blood from vaccinated and naturally infected individuals.

Defining the correlates of protection necessary to manage the COVID-19 pandemic requires the analysis of both antibody and T cell parameters, but the complexity of traditional tests limits virus-specific T cell measurements. We tested the sensitivity and performance of a simple and rapid SARS-CoV-2 spike protein-specific T cell test based on the stimulation of whole blood with peptides covering the SARS-CoV-2 spike protein, followed by cytokine (IFN-γ, IL-2) measurement in different cohorts including BNT162b2-vaccinated individuals (n = 112), convalescent asymptomatic and symptomatic COVID-19 patients (n = 130), and SARS-CoV-1-convalescent individuals (n = 12). The sensitivity of this rapid test is comparable to that of traditional methods of T cell analysis (ELISPOT, activation-induced marker). Using this test, we observed a similar mean magnitude of T cell responses between the vaccinees and SARS-CoV-2 convalescents 3 months after vaccination or virus priming. However, a wide heterogeneity of the magnitude of spike-specific T cell responses characterized the individual responses, irrespective of the time of analysis. The magnitude of these spike-specific T cell responses cannot be predicted from the neutralizing antibody levels. Hence, both humoral and cellular spike-specific immunity should be tested after vaccination to define the correlates of protection necessary to evaluate current vaccine strategies.

Longitudinal urinary biomarkers of immunological activation in covid-19 patients without clinically apparent kidney disease versus acute and chronic failure.

Kidney function is affected in COVID-19, while kidney itself modulates the immune response. Here, hypothesize if COVID-19 urine biomarkers level can assess immune activation vs. clinical trajectory. Considering the kidney's critical role in modulating the immune response, we sought to analyze activation markers in patients with pre-existing dysfunction. This was a cross-sectional study of 68 patients. Blood and urine were collected within 48 h of hospital admission (H1), followed by 96 h (H2), seven days (H3), and up to 25 days (H4) from admission. Serum level ferritin, procalcitonin, IL-6 assessed immune activation overall, while the response to viral burden was gauged with serum level of spike protein and αspike IgM and IgG. 39 markers correlated highly between urine and blood. Age and race, and to a lesser extend gender, differentiated several urine markers. The burden of pre-existing conditions correlated with urine DCN, CAIX and PTN, but inversely with IL-5 or MCP-4. Higher urinary IL-12 and lower CAIX, CCL23, IL-15, IL-18, MCP-1, MCP-3, MUC-16, PD-L1, TNFRS12A, and TNFRS21 signified non-survivors. APACHE correlated with urine TNFRS12, PGF, CAIX, DCN, CXCL6, and EGF. Admission urine LAG-3 and IL-2 predicted death. Pre-existing kidney disease had a unique pattern of urinary inflammatory markers. Acute kidney injury was associated, and to a certain degree, predicted by IFNg, TWEAK, MMP7, and MUC-16. Remdesavir had a more profound effect on the urine biomarkers than steroids. Urinary biomarkers correlated with clinical status, kidney function, markers of the immune system activation, and probability of demise in COVID-19.

Long-Term Humoral Immune Response in Persons with Asymptomatic or Mild SARS-CoV-2 Infection, Vietnam.

Antibody response against nucleocapsid and spike proteins of SARS-CoV-2 in 11 persons with mild or asymptomatic infection rapidly increased after infection. At weeks 18-30 after diagnosis, all remained seropositive but spike protein-targeting antibody titers declined. These data may be useful for vaccine development.

A plug-and-play platform of ratiometric bioluminescent sensors for homogeneous immunoassays.

Heterogeneous immunoassays such as ELISA have become indispensable in modern bioanalysis, yet translation into point-of-care assays is hindered by their dependence on external calibration and multiple washing and incubation steps. Here, we introduce RAPPID (Ratiometric Plug-and-Play Immunodiagnostics), a mix-and-measure homogeneous immunoassay platform that combines highly specific antibody-based detection with a ratiometric bioluminescent readout. The concept entails analyte-induced complementation of split NanoLuc luciferase fragments, photoconjugated to an antibody sandwich pair via protein G adapters. Introduction of a calibrator luciferase provides a robust ratiometric signal that allows direct in-sample calibration and quantitative measurements in complex media such as blood plasma. We developed RAPPID sensors that allow low-picomolar detection of several protein biomarkers, anti-drug antibodies, therapeutic antibodies, and both SARS-CoV-2 spike protein and anti-SARS-CoV-2 antibodies. With its easy-to-implement standardized workflow, RAPPID provides an attractive, fast, and low-cost alternative to traditional immunoassays, in an academic setting, in clinical laboratories, and for point-of-care applications.

Improved production of SARS-CoV-2 spike receptor-binding domain (RBD) for serology assays.

The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a commonly used antigen for serology assays critical to determining the extent of SARS-CoV-2 exposure in the population. Different versions of the RBD protein have been developed and utilized in assays, with higher sensitivity attributed to particular forms of the protein. To improve the yield of these high-sensitivity forms of RBD and support the increased demand for this antigen in serology assays, we investigated several protein expression variables including DNA elements such as promoters and signal peptides, cell culture expression parameters, and purification processes. Through this investigation, we developed a simplified and robust purification strategy that consistently resulted in high levels of the high-sensitivity form of RBD and demonstrated that a carboxyterminal tag is responsible for the increased sensitivity in the ELISA. These improved reagents and processes produce high-quality proteins which are functional in serology assays and can be used to investigate seropositivity to SARS-CoV-2 infection.

An antigen microarray protocol for COVID-19 serological analysis.

The emergence of the coronavirus disease 2019 pandemic increased the interest in analysis of immunoglobulin responses. ELISA and lateral flow assays are widely used but are restricted by a single response value to an antigen or antigen pool. Here, we describe antigen microarrays, an alternative allowing simultaneous assessment of multiple interactions between antigens and the immunoglobulin content of patient sera. The technique requires minimal reagents and sample input and can be adapted to a wide variety of potential antigenic targets of interest.

Decreased inhibition of exosomal miRNAs on SARS-CoV-2 replication underlies poor outcomes in elderly people and diabetic patients.

Elderly people and patients with comorbidities are at higher risk of COVID-19 infection, resulting in severe complications and high mortality. However, the underlying mechanisms are unclear. In this study, we investigate whether miRNAs in serum exosomes can exert antiviral functions and affect the response to COVID-19 in the elderly and people with diabetes. First, we identified four miRNAs (miR-7-5p, miR-24-3p, miR-145-5p and miR-223-3p) through high-throughput sequencing and quantitative real-time PCR analysis, that are remarkably decreased in the elderly and diabetic groups. We further demonstrated that these miRNAs, either in the exosome or in the free form, can directly inhibit S protein expression and SARS-CoV-2 replication. Serum exosomes from young people can inhibit SARS-CoV-2 replication and S protein expression, while the inhibitory effect is markedly decreased in the elderly and diabetic patients. Moreover, three out of the four circulating miRNAs are significantly increased in the serum of healthy volunteers after 8-weeks' continuous physical exercise. Serum exosomes isolated from these volunteers also showed stronger inhibitory effects on S protein expression and SARS-CoV-2 replication. Our study demonstrates for the first time that circulating exosomal miRNAs can directly inhibit SARS-CoV-2 replication and may provide a possible explanation for the difference in response to COVID-19 between young people and the elderly or people with comorbidities.

Duration of post-COVID-19 symptoms is associated with sustained SARS-CoV-2-specific immune responses.

A subset of COVID-19 patients exhibit post-acute sequelae of COVID-19 (PASC), but little is known about the immune signatures associated with these syndromes. We investigated longitudinal peripheral blood samples in 50 individuals with previously confirmed SARS-CoV-2 infection, including 20 who experienced prolonged duration of COVID-19 symptoms (lasting more than 30 days; median = 74 days) compared with 30 who had symptom resolution within 20 days. Individuals with prolonged symptom duration maintained antigen-specific T cell response magnitudes to SARS-CoV-2 spike protein in CD4+ and circulating T follicular helper cell populations during late convalescence, while those without persistent symptoms demonstrated an expected decline. The prolonged group also displayed increased IgG avidity to SARS-CoV-2 spike protein. Significant correlations between symptom duration and both SARS-CoV-2-specific T cells and antibodies were observed. Activation and exhaustion markers were evaluated in multiple immune cell types, revealing few phenotypic differences between prolonged and recovered groups, suggesting that prolonged symptom duration is not due to persistent systemic inflammation. These findings demonstrate that SARS-CoV-2-specific immune responses are maintained in patients suffering from prolonged post-COVID-19 symptom duration in contrast to those with resolved symptoms and may suggest the persistence of viral antigens as an underlying etiology.

Seroconversion rates following COVID-19 vaccination among patients with cancer.

As COVID-19 adversely affects patients with cancer, prophylactic strategies are critically needed. Using a validated antibody assay against SARS-CoV-2 spike protein, we determined a high seroconversion rate (94%) in 200 patients with cancer in New York City that had received full dosing with one of the FDA-approved COVID-19 vaccines. On comparison with solid tumors (98%), a significantly lower rate of seroconversion was observed in patients with hematologic malignancies (85%), particularly recipients following highly immunosuppressive therapies such as anti-CD20 therapies (70%) and stem cell transplantation (73%). Patients receiving immune checkpoint inhibitor therapy (97%) or hormonal therapies (100%) demonstrated high seroconversion post vaccination. Patients with prior COVID-19 infection demonstrated higher anti-spike IgG titers post vaccination. Relatively lower IgG titers were observed following vaccination with the adenoviral than with mRNA-based vaccines. These data demonstrate generally high immunogenicity of COVID-19 vaccination in oncology patients and identify immunosuppressed cohorts that need novel vaccination or passive immunization strategies.