Given the ongoing SARS-CoV-2 pandemic, identification of immunogenic targets against the coronavirus spike glycoprotein will provide crucial advances towards the development of sensitive diagnostic ...tools and potential vaccine candidate targets. In this study, using pools of overlapping linear B-cell peptides, we report two IgG immunodominant regions on SARS-CoV-2 spike glycoprotein that are recognised by sera from COVID-19 convalescent patients. Notably, one is specific to SARS-CoV-2, which is located in close proximity to the receptor binding domain. The other region, which is localised at the fusion peptide, could potentially function as a pan-SARS target. Functionally, antibody depletion assays demonstrate that antibodies targeting these immunodominant regions significantly alter virus neutralisation capacities. Taken together, identification and validation of these neutralising B-cell epitopes will provide insights towards the design of diagnostics and vaccine candidates against this high priority coronavirus.
Numerous reports of vascular events after an initial recovery from COVID-19 form our impetus to investigate the impact of COVID-19 on vascular health of recovered patients. We found elevated levels ...of circulating endothelial cells (CECs), a biomarker of vascular injury, in COVID-19 convalescents compared to healthy controls. In particular, those with pre-existing conditions (e.g., hypertension, diabetes) had more pronounced endothelial activation hallmarks than non-COVID-19 patients with matched cardiovascular risk. Several proinflammatory and activated T lymphocyte-associated cytokines sustained from acute infection to recovery phase, which correlated positively with CEC measures, implicating cytokine-driven endothelial dysfunction. Notably, we found higher frequency of effector T cells in our COVID-19 convalescents compared to healthy controls. The activation markers detected on CECs mapped to counter receptors found primarily on cytotoxic CD8
T cells, raising the possibility of cytotoxic effector cells targeting activated endothelial cells. Clinical trials in preventive therapy for post-COVID-19 vascular complications may be needed.
SARS-CoV-2 is the novel coronavirus responsible for the current COVID-19 pandemic. Severe complications are observed only in a small proportion of infected patients but the cellular mechanisms ...underlying this progression are still unknown. Comprehensive flow cytometry of whole blood samples from 54 COVID-19 patients reveals a dramatic increase in the number of immature neutrophils. This increase strongly correlates with disease severity and is associated with elevated IL-6 and IP-10 levels, two key players in the cytokine storm. The most pronounced decrease in cell counts is observed for CD8 T-cells and VD2 γδ T-cells, which both exhibit increased differentiation and activation. ROC analysis reveals that the count ratio of immature neutrophils to VD2 (or CD8) T-cells predicts pneumonia onset (0.9071) as well as hypoxia onset (0.8908) with high sensitivity and specificity. It would thus be a useful prognostic marker for preventive patient management and improved healthcare resource management.
Understanding the impact of age on vaccinations is essential for the design and delivery of vaccines against SARS-CoV-2. Here, we present findings from a comprehensive analysis of multiple ...compartments of the memory immune response in 312 individuals vaccinated with the BNT162b2 SARS-CoV-2 mRNA vaccine. Two vaccine doses induce high antibody and T cell responses in most individuals. However, antibody recognition of the Spike protein of the Delta and Omicron variants is less efficient than that of the ancestral Wuhan strain. Age-stratified analyses identify a group of low antibody responders where individuals ≥60 years are overrepresented. Waning of the antibody and cellular responses is observed in 30% of the vaccinees after 6 months. However, age does not influence the waning of these responses. Taken together, while individuals ≥60 years old take longer to acquire vaccine-induced immunity, they develop more sustained acquired immunity at 6 months post-vaccination. A third dose strongly boosts the low antibody responses in the older individuals against the ancestral Wuhan strain, Delta and Omicron variants.
As part of the body's immune response, antibodies (Abs) have the ability to neutralize pathogenic viruses to prevent infection. To screen for neutralizing Abs (nAbs) from the immune repertoire, ...multiple screening techniques have been developed. However, conventional methods have a trade-off between screening throughput and the ability to screen for nAbs
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their functional efficacy. Although droplet microfluidic platforms have the ability to bridge this disparity, the majority of such reported platforms still rely on Ab-binding assays as a proxy for function, which results in irrelevant hits. Herein, we report the multi-module Droplet-based Platform for Effective Antibody RetrievaL (DROP-PEARL) platform, which can achieve high-throughput enrichment of Ab-secreting cells (ASCs) based on the neutralizing activity of secreted nAbs against the a target virus. In this study, in-droplet Chikungunya virus (CHIKV) infection of host cells and neutralization was demonstrated
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sequential delivery of viruses and host cells
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picoinjection. In addition, we demonstrate the ability of the sorting system to accurately discriminate and isolate uninfected droplets from a mixed population of droplets at a rate of 150 000 cells per hour. As a proof of concept, a single-cell neutralization assay was performed on two populations of cells (nAb-producing and non-Ab producing cells), and up to 2.75-fold enrichment of ASCs was demonstrated. Finally, we demonstrated that DROP-PEARL is able to achieve similar enrichment for low frequency (∼2%) functional nAb-producing cells in a background of excess cells secreting irrelevant antibodies, highlighting its potential prospect as a first round enrichment platform for functional ASCs. We envision that the DROP-PEARL platform could potentially be used to accelerate the discovery of nAbs against other pathogenic viral targets, and we believe it will be a useful in the ongoing fight against biological threats.
Drop-PEARL enables rapid screening and isolation of virus-neutralizing antibody secreting cells for high-throughput therapeutic antibody discovery.
Long‐term complications from coronavirus disease 2019 (COVID‐19) are concerning, as survivors can develop subclinical multiorgan dysfunction. It is unknown if such complications are due to prolonged ...inflammation, and severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) vaccination may reduce sequela. We conducted a prospective longitudinal study on hospitalized patients over 24 months. Clinical symptoms were collected by self‐reporting during follow‐up, along with blood samples for quantification of inflammatory markers and immune cell frequencies. All patients were given one dose of mRNA vaccine at 12−16 months. Their immune profiles at 12 and 24 months were compared. Approximately 37% and 39% of our patients reported post‐COVID‐19 symptoms at 12 and 24 months, respectively. The proportion of symptomatic patients with more than one symptom decreased from 69% at 12 months to 56% at 24 months. Longitudinal cytokine profiling revealed a cluster of individuals with persistently high inflammatory cytokine levels 12 months after infection. Patients with prolonged inflammation showed elevated terminally differentiated memory T cells in their blood; 54% had symptoms at 12 months. The majority of inflammatory markers and dysregulated immune cells in vaccinated patients recovered to a healthy baseline at 24 months, even though symptoms persisted. Post‐COVID‐19 symptoms can linger for 2 years after the initial infection and are associated with prolonged inflammation. Prolonged inflammation in hospitalized patients resolves after 2 years. We define a set of analytes associated with persistent inflammation and presence of symptoms, which could be useful biomarkers for identifying and monitoring high‐risk survivors.
Studies have found different waning rates of neutralising antibodies compared with binding antibodies against SARS-CoV-2. The impact of neutralising antibody waning rate at the individual patient ...level on the longevity of immunity remains unknown. We aimed to investigate the peak levels and dynamics of neutralising antibody waning and IgG avidity maturation over time, and correlate this with clinical parameters, cytokines, and T-cell responses.
We did a longitudinal study of patients who had recovered from COVID-19 up to day 180 post-symptom onset by monitoring changes in neutralising antibody levels using a previously validated surrogate virus neutralisation test. Changes in antibody avidities and other immune markers at different convalescent stages were determined and correlated with clinical features. Using a machine learning algorithm, temporal change in neutralising antibody levels was classified into five groups and used to predict the longevity of neutralising antibody-mediated immunity.
We approached 517 patients for participation in the study, of whom 288 consented for outpatient follow-up and collection of serial blood samples. 164 patients were followed up and had adequate blood samples collected for analysis, with a total of 546 serum samples collected, including 128 blood samples taken up to 180 days post-symptom onset. We identified five distinctive patterns of neutralising antibody dynamics as follows: negative, individuals who did not, at our intervals of sampling, develop neutralising antibodies at the 30% inhibition level (19 12% of 164 patients); rapid waning, individuals who had varying levels of neutralising antibodies from around 20 days after symptom onset, but seroreverted in less than 180 days (44 27% of 164 patients); slow waning, individuals who remained neutralising antibody-positive at 180 days post-symptom onset (46 28% of 164 patients); persistent, although with varying peak neutralising antibody levels, these individuals had minimal neutralising antibody decay (52 32% of 164 patients); and delayed response, a small group that showed an unexpected increase of neutralising antibodies during late convalescence (at 90 or 180 days after symptom onset; three 2% of 164 patients). Persistence of neutralising antibodies was associated with disease severity and sustained level of pro-inflammatory cytokines, chemokines, and growth factors. By contrast, T-cell responses were similar among the different neutralising antibody dynamics groups. On the basis of the different decay dynamics, we established a prediction algorithm that revealed a wide range of neutralising antibody longevity, varying from around 40 days to many decades.
Neutralising antibody response dynamics in patients who have recovered from COVID-19 vary greatly, and prediction of immune longevity can only be accurately determined at the individual level. Our findings emphasise the importance of public health and social measures in the ongoing pandemic outbreak response, and might have implications for longevity of immunity after vaccination.
National Medical Research Council, Biomedical Research Council, and A*STAR, Singapore.
Alphaviruses are transmitted to humans via bites of infected mosquitoes. Although alphaviruses have caused a wide range of outbreaks and crippling disease, the availability of licensed vaccines or ...antiviral therapies remains limited. Mosquito vectors such as
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are the main culprits in the transmission of alphaviruses. This review explores how mosquito saliva may promote alphavirus infection. Identifying the roles of mosquito-derived factors in alphavirus pathogenesis will generate novel tools to circumvent and control mosquito-borne alphavirus infections in humans.
Waning antibody levels against severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) and the emergence of variants of concern highlight the need for booster vaccinations. This is particularly ...important for the elderly population, who are at a higher risk of developing severe coronavirus disease 2019 (COVID‐19) disease. While studies have shown increased antibody responses following booster vaccination, understanding the changes in T and B cell compartments induced by a third vaccine dose remains limited. We analyzed the humoral and cellular responses in subjects who received either a homologous messenger RNA(mRNA) booster vaccine (BNT162b2 + BNT162b2 + BNT162b2; ‘‘BBB”) or a heterologous mRNA booster vaccine (BNT162b2 + BNT162b2 + mRNA‐1273; ‘‘BBM”) at Day 0 (prebooster), Day 7, and Day 28 (postbooster). Compared with BBB, elderly individuals (≥60 years old) who received the BBM vaccination regimen display higher levels of neutralizing antibodies against the Wuhan and Delta strains along with a higher boost in immunoglobulin G memory B cells, particularly against the Omicron variant. Circulating T helper type 1(Th1), Th2, Th17, and T follicular helper responses were also increased in elderly individuals given the BBM regimen. While mRNA vaccines increase antibody, T cell, and B cell responses against SARS‐CoV‐2 1 month after receiving the third dose booster, the efficacy of the booster vaccine strategies may vary depending on age group and regimen combination.
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