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BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), significantly threatens public health. Accumulating evidence suggests increased neutrophil activation and endothelial glycocalyx (EG) damage to be independently associated with severe COVID-19 outcomes. However, pathways that are involved in EG breakdown in COVID-19 remains unclear. Here, we hypothesised that increased blood neutrophil myeloperoxidase (MPO) level is associated with soluble EG breakdown, and inhibiting MPO activities can reduce EG damage. MethodsFrom a subset of acute and convalescent COVID-19 plasma, 10 severe and 15 non-severe COVID-19 cases, and 9 pre-COVID-19 controls (single timepoint), we determined MPO, MPO activities and soluble EG proteins (syndecan-1 and glypican-1) levels by enzyme-linked immunosorbent assay. In vitro primary human aortic endothelial cells were cultured in the presence of untreated or treated plasma with specific MPO inhibitors (MPO-IN-28, AZD-5904) to determine soluble EG shedding. We then investigated the association between MPO and soluble EG breakdown, and whether inhibiting MPO activities decrease EG disruption.ResultsIn COVID-19 plasma, MPO levels, MPO activity and soluble EG proteins levels were significantly raised compared to controls, and proteins levels increased in proportion to disease severity. Despite clinical recovery, proteins concentrations remained significantly elevated compared to controls. Interestingly, there is a trend of increasing MPO activity in convalescent plasma in both severe and non-severe groups. Importantly, MPO levels and MPO activity correlated significantly with soluble EG levels in plasma, and inhibiting MPO activity led to reduced syndecan-1 shedding, in vitro. ConclusionsOur work highlights the link between neutrophil MPO involvement and EG shedding in COVID-19, and inhibiting MPO activity may protect against EG disruption. Further research is needed to evaluate the utility of MPO inhibitors as potential therapeutics against severe COVID-19 outcomes.

Background Waning antibody levels post-vaccination and the emergence of variants of concern (VOCs) capable of evading protective immunity have raised the need for booster vaccinations. However, which combination of coronavirus disease 2019 (COVID-19) vaccines offers the strongest immune response against the Omicron variant is unknown. Methods This randomized, participant-blinded, controlled trial assessed the reactogenicity and immunogenicity of different COVID-19 vaccine booster combinations. A total of 100 BNT162b2-vaccinated individuals were enrolled and randomized 1:1 to either homologous (BNT162b2 + BNT162b2 + BNT162b2; “BBB”) or heterologous messenger RNA (mRNA) (BNT162b2 + BNT162b2 + mRNA-1273; “BBM”) booster vaccine. The primary end point was the level of neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) wild-type and VOCs at day 28. Results A total of 51 participants were allocated to BBB and 49 to BBM; 50 and 48, respectively, were analyzed for safety and immunogenicity outcomes. At day 28 post-boost, mean SARS-CoV-2 spike antibody titers were lower with BBB (22 382 IU/mL; 95% confidence interval [CI], 18 210 to 27 517) vs BBM (29 751 IU/mL; 95% CI, 25 281 to 35 011; P = .034) as was the median level of neutralizing antibodies: BBB 99.0% (interquartile range [IQR], 97.9% to 99.3%) vs BBM 99.3% (IQR, 98.8% to 99.5%; P = .021). On subgroup analysis, significant higher mean spike antibody titer, median surrogate neutralizing antibody level against all VOCs, and live Omicron neutralization titer were observed only in older adults receiving BBM. Both vaccines were well tolerated. Conclusions Heterologous mRNA-1273 booster vaccination compared with homologous BNT123b2 induced a stronger neutralizing response against the Omicron variant in older individuals. Clinical Trials Registration NCT05142319.

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

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) that have become dominant as the pandemic progresses bear the ORF8 mutation together with multiple spike mutations. A 382-nucleotide deletion (Δ382) in the ORF7b and ORF8 regions has been associated with milder disease phenotype and less systemic inflammation in COVID-19 patients. However, its impact on host immunity against SARS-CoV-2 remains undefined. Here, RNA-sequencing was performed to elucidate whole blood transcriptomic profiles and identify contrasting immune signatures between patients infected with either wildtype or Δ382 SARS-CoV-2 variant. Interestingly, the immune landscape of Δ382 SARS-CoV-2 infected patients featured an increased adaptive immune response, evidenced by enrichment of genes related to T cell functionality, a more robust SARS-CoV-2-specific T cell immunity, as well as a more rapid antibody response. At the molecular level, eukaryotic initiation factor 2 signaling was found to be upregulated in patients bearing Δ382, and its associated genes were correlated with systemic levels of T cell-associated and pro-inflammatory cytokines. This study provides more in-depth insight into the host–pathogen interactions of ORF8 with great promise as a therapeutic target to combat SARS-CoV-2 infection. Supplementary Information The online version contains supplementary material available at 10.1007/s10875-021-01142-z.

IntroductionCOVID-19 has a wide disease spectrum ranging from asymptomatic to severe. While humoral immune responses are critical in preventing infection, the immune mechanisms leading to severe disease, and the identification of biomarkers of disease progression and/or resolution of the infection remains to be determined.MethodsPlasma samples were obtained from infections during the initial wave of ancestral wildtype SARS-CoV-2 and from vaccine breakthrough infections during the wave of Delta variant, up to six months post infection. The spike-specific antibody profiles were compared across different severity groups and timepoints.ResultsWe found an association between spike-specific IgM, IgA and IgG and disease severity in unvaccinated infected individuals. In addition to strong IgG1 and IgG3 response, patients with severe disease develop a robust IgG2 and IgG4 response. A comparison of the ratio of IgG1 and IgG3 to IgG2 and IgG4 showed that disease progression is associated with a smaller ratio in both the initial wave of WT and the vaccine breakthrough Delta infections. Time-course analysis revealed that smaller (IgG1 and IgG3)/(IgG2 and IgG4) ratio is associated with disease progression, while the reverse associates with clinical recovery.DiscussionWhile each IgG subclass is associated with disease severity, the balance within the four IgG subclasses may affect disease outcome. Acute disease progression or infection resolution is associated with a specific immunological phenotype that is conserved in both the initial wave of WT and the vaccine breakthrough Delta infections.

Summary Background 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. Methods 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. Findings 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. Interpretation 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. Funding National Medical Research Council, Biomedical Research Council, and A*STAR, Singapore.

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.

BackgroundOn 26 November 2021, the World Health Organization designated the B.1.1.529 lineage of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) as the fifth variant of concern, Omicron. Infections have quickly spread worldwide, but understanding of the viral dynamics and the cytokine and cellular immunological response during infection remain limited.MethodsDetailed patient-level data from 174 age-matched patients with sequence confirmed Omicron or Delta infection admitted to the National Centre for Infectious Diseases, Singapore were analyzed in an observational cohort study. Peripheral blood samples for measurement of SARS-CoV-2 immunological parameters were obtained from a subset. Respiratory samples were collected for viral cultures and correlated to corresponding PCR cycle threshold (Ct) values. ResultsOmicron and Delta variant infections in this hospitalized cohort were mild with only 3 (3%) and 14 (16%) developing pneumonia respectively. Omicron infections were more likely to present with sore throat (46.0 vs x23.0%, p=0.005). Neutrophil counts and C-reactive protein (CRP) were significantly lower among the Omicron cohort (Median neutrophil 2.95 [IQR 2.16 – 3.96] vs 4.60 [IQR 3.76 – 6.10] x 109/L , pConclusionsOmicron infections resulted in mild vaccine breakthrough illness in the majority of patients. Compared with Delta, Omicron infections were more frequently associated with upper respiratory tract infections, had lower viral loads, lower levels of pro-inflammatory cytokines and less dysregulated immune cell profiles.

Despite the high vaccine efficacy of mRNA COVID-19 vaccines, there are individuals who developed excessive reactogenic and/or allergic responses after the first mRNA dose and were considered ineligible for further mRNA doses. CoronaVac, an inactivated SARS-CoV-2 vaccine, is recommended in Singapore as an alternative.Individuals, ineligible for further mRNA vaccines (BNT162b2 or mRNA-1273) because of excessive reactive responses to prime mRNA vaccination, were recruited and offered two doses of CoronaVac as booster vaccination 38-224 days post their mRNA vaccine dose. Individuals who did not develop any excessive reactive responses after the prime mRNA vaccination were also recruited and given another mRNA vaccine as booster vaccination. Blood samples were collected at days 0, 21 and 90 post first CoronaVac dose and mRNA dose, respectively, for analysis.We showed that two CoronaVac booster doses induced specific immunity in these mRNA vaccine-primed individuals. Although the spike-specific antibody response was lower, their memory B cell response against the receptor-binding domain (RBD) of the spike protein was similar, compared with individuals who received two BNT162b2 injections. The spike-specific memory T cell response also increased following CoronaVac booster doses. However, specific immunity against the Omicron variant was low, similar to individuals with two BNT162b2 doses.Our findings showed that while mRNA vaccine-primed individuals can opt for two subsequent doses of CoronaVac, an additional dose may be necessary to achieve protection, especially against newly emerging immune escape variants such as Omicron.

Efficient COVID-19 vaccines have been developed in record time. Here, we present findings from a comprehensive and integrated analysis of multiple compartments of the memory immune response in 312 individuals vaccinated with the BNT162b2 mRNA vaccine. Two vaccine doses induced high antibody and T cell responses in most individuals. However, antibody recognition of the Spike protein of delta variant was less efficient than that of the Wuhan strain. Age stratified analyses identified a group of low antibody responders where individuals ≥ 60 years were overrepresented. Waning of the antibody and cellular responses was observed in 30% of the vaccinees after six months. However, age did not influence the waning of these responses. Taken together, while individuals ≥ 60 years old took longer to acquire vaccine-induced immunity, they develop more sustained acquired immunity at six months post-vaccination. However, the higher proportion of older individuals in the group of antibody low responders and the lower antibody reactivity the Delta variant call for a booster immunization to increase immune responses and protection.

Severe SARS-CoV-2 infection can trigger uncontrolled innate and adaptive immune responses, which are commonly associated with lymphopenia and increased neutrophil counts. However, whether the immune abnormalities observed in mild to severely infected patients persist into convalescence remains unclear. Herein, comparisons were drawn between the immune responses of COVID-19 infected and convalescent adults. Strikingly, survivors of severe COVID-19 had decreased proportions of NKT and Vδ2 T cells, and increased proportions of low-density neutrophils, IgA+/CD86+/CD123+ non-classical monocytes and hyperactivated HLADR+CD38+ CD8+ T cells, and elevated levels of pro-inflammatory cytokines such as hepatocyte growth factor and vascular endothelial growth factor A, long after virus clearance. Our study suggests potential immune correlates of "long COVID-19", and defines key cells and cytokines that delineate true and quasi-convalescent states. Agency for Science, Technology and Research (A*STAR) National Medical Research Council (NMRC) National Research Foundation (NRF) Published version This work was supported by A*STAR Infectious Diseases Labs and Singapore Immunology Network (SIgN) core research grants, and the A*STAR COVID-19 Research funding (H/20/04/g1/006) provided to SIgN by the Biomedical Research Council (BMRC). Subject recruitment, sample collection and analyses were funded by the National Medical Research Council (NMRC) COVID-19 Research Fund (COVID19RF-001, COVID19-RF007, COVID190RF-060). The SIgN Immunomonitoring Platform is supported by a BMRC IAF 311006 grant and BMRC transition funds #H16/99/b0/011. The SIgN Flow Cytometry and the Multiple Analyte Platforms were supported by a grant from the National Research Foundation, Immunomonitoring Service Platform ISP) (#NRF2017_SISFP09) and the National Research Foundation Singapore (NRF).

PurposeCOVID-19, caused by Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2), has a wide disease spectrum ranging from asymptomatic to severe. While it is widely accepted that specific humoral immune responses are critical in controlling the infection, the relationship between the humoral immune response and disease severity is currently unclear.MethodsUsing a flow cytometry-based assay to detect specific antibodies against full length S protein, we compared the antibody levels between patients from different severity groups. We also analysed the cytokine profiles of patients from different severity groups by multiplex microbead-based immunoassay.ResultsWe found an association between specific IgM, IgA and IgG against the spike protein and disease severity. By comparing the ratio of Th1 IgG1 and IgG3 to Th2 IgG2 and IgG4, we observed that all severity groups exhibited a ratio that was skewed towards a stronger Th1 response over Th2 response. In addition to the strong Th1 response, patients with severe disease also developed a Th2 response, as exemplified by the smaller ratio of IgG1 and IgG3 over IgG2 and IgG4 and the smaller Th1/Th2 cytokine ratios, compared to patients with mild disease severity. ConclusionThe results suggest that acute severity or disease resolution is associated with a specific immunological phenotype. A smaller skew towards a Th1 response over Th2 response, during infection, may contribute to disease progression, while a greater skew towards a Th1 response over Th2 response may contribute to a better disease outcome. This may suggest potential therapeutic approaches to COVID-19 disease management.

A significant proportion of COVID-19 patients will progress to critical illness requiring invasive mechanical ventilation. This accentuates the need for a therapy that can reduce the severity of COVID-19. Clinical trials have shown the effectiveness of remdesivir in shortening recovery time and decreasing progression to respiratory failure and mechanical ventilation. However, some studies have highlighted its lack of efficacy in patients on high-flow oxygen and mechanical ventilation. This study uncovers some underlying immune response differences between responders and non-responders to remdesivir treatment. Immunological analyses revealed an upregulation of tissue repair factors BDNF, PDGF-BB and PIGF-1, as well as an increase in ratio of Th2-associated cytokine IL-4 to Th1-associated cytokine IFN-γ. Serological profiling of IgG subclasses corroborated this observation, with significantly higher magnitude of increase in Th2-associated IgG2 and IgG4 responses. These findings help to identify the mechanisms of immune regulation accompanying successful remdesivir treatment in severe COVID-19 patients. Agency for Science, Technology and Research (A*STAR) National Medical Research Council (NMRC) National Research Foundation (NRF) Published version This work was supported by Singapore Immunology Network (SIgN) core research grant and the A*STAR COVID-19 Research funding (H/20/04/g1/006) provided to SIgN by the Biomedical Research Council (BMRC). Subject recruitment and sample collection were funded by the National Medical Research Council (NMRC) COVID-19 Research Fund (COVID19RF001). The SIgN Immunomonitoring Platform is supported by a BMRC IAF 311006 grant and BMRC transition funds #H16/99/ b0/011. The SIgN Flow Cytometry and the Multiple Analyte Platforms were supported by a grant from the National Research Foundation, Immunomonitoring Service Platform ISP) (#NRF2017_SISFP09) and the National Research Foundation Singapore (NRF).

Key immune signatures of SARS-CoV-2 infection may associate with either adverse immune reactions (severity) or simply an ongoing anti-viral response (temporality); how immune signatures contribute to severe manifestations and/or temporal progression of disease and whether longer disease duration correlates with severity remain unknown. Patient blood was comprehensively immunophenotyped via mass cytometry and multiplex cytokine arrays, leading to the identification of 327 basic subsets that were further stratified into more than 5000 immunotypes and correlated with 28 plasma cytokines. Low-density neutrophil abundance was closely correlated with hepatocyte growth factor levels, which in turn correlated with disease severity. Deep analysis also revealed additional players, namely conventional type 2 dendritic cells, natural killer T cells, plasmablasts and CD16+ monocytes, that can influence COVID-19 severity independent of temporal progression. Herein, we provide interactive network analysis and data visualization tools to facilitate data mining and hypothesis generation for elucidating COVID-19 pathogenesis.

The rapid rise of coronavirus disease 2019 patients who suffer from vascular events after their initial recovery is expected to lead to a worldwide shift in disease burden. We aim to investigate the impact of COVID-19 on the pathophysiological state of blood vessels in convalescent patients. Here, convalescent COVID-19 patients with or without preexisting conditions (i.e. hypertension, diabetes, hyperlipidemia) were compared to non-COVID-19 patients with matched cardiovascular risk factors or healthy participants. Convalescent patients had elevated circulating endothelial cells (CECs), and those with underlying cardiovascular risk had more pronounced endothelial activation hallmarks (ICAM1, P-selectin or CX3CL1) expressed by CECs. Multiplex microbead-based immunoassays revealed some levels of cytokine production sustained from acute infection to recovery phase. Several proinflammatory and activated T lymphocyte-associated cytokines correlated positively with CEC measures, implicating cytokine-driven endothelial dysfunction. Finally, the activation markers detected on CECs mapped to the counter receptors (i.e. ITGAL, SELPLG, and CX3CR1) found primarily on CD8+ T cells and natural killer cells, suggesting that activated endothelial cells could be targeted by cytotoxic effector cells. Clinical trials in preventive therapy for post-COVID-19 vascular complications may be needed.Graphical abstract

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 revealed a dramatic increase in the number of immature neutrophils. This increase strongly correlated with disease severity and was associated with elevated IL-6 and IP-10 levels, two key players in the cytokine storm. The most pronounced decrease in cell counts was observed for CD8 T-cells and VD2 γδ T-cells, which both exhibited increased differentiation and activation. ROC analysis revealed that the count ratio of immature neutrophils to CD8 or VD2 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.

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., Characterisation of the human antibody response to SARS-CoV-2 can help the design of serological tests and vaccines. Here, the authors identify two linear epitopes in SARS-CoV-2 spike protein that elicit neutralising antibodies in several patients and could thus be useful for serology and vaccine development.

Background: Host determinants of severe coronavirus disease 2019 include advanced age, comorbidities and male sex. Virologic factors may also be important in determining clinical outcome and transmission rates, but limited patient-level data is available. Methods: We conducted an observational cohort study at seven public hospitals in Singapore. Clinical and laboratory data were collected and compared between individuals infected with different SARS-CoV-2 clades. Firth’s logistic regression was used to examine the association between SARS-CoV-2 clade and development of hypoxia, and quasi-Poisson regression to compare transmission rates. Plasma samples were tested for immune mediator levels and the kinetics of viral replication in cell culture were compared. Findings: 319 patients with PCR-confirmed SARS-CoV-2 infection had clinical and virologic data available for analysis. 29 (9%) were infected with clade S, 90 (28%) with clade L/V, 96 (30%) with clade G (containing D614G variant), and 104 (33%) with other clades ‘O’ were assigned to lineage B.6. After adjusting for age and other covariates, infections with clade S (adjusted odds ratio (aOR) 0·030 (95% confidence intervals (CI): 0·0002-0·29)) or clade O (B·6) (aOR 0·26 (95% CI 0·064-0·93)) were associated with lower odds of developing hypoxia requiring supplemental oxygen compared with clade L/V. Patients infected with clade L/V had more pronounced systemic inflammation with higher concentrations of pro-inflammatory cytokines, chemokines and growth factors. No significant difference in the severity of clade G infections was observed (aOR 0·95 (95% CI: 0·35-2·52). Though viral loads were significantly higher, there was no evidence of increased transmissibility of clade G, and replicative fitness in cell culture was similar for all clades. Interpretation: Infection with clades L/V was associated with increased severity and more systemic release of pro-inflammatory cytokines. Infection with clade G was not associated with changes in severity, and despite higher viral loads there was no evidence of increased transmissibility. Funding Statement: This study was funded by grants from the Singapore National Medical Research Council (COVID19RF- 001, COVID19RF2-0001, COVID19RF-007, and COVID19RF-60) and Biomedical Research Council (project number H20/04/g1/006). Declaration of Interests: No conflicts of interest declared. Ethics Approval Statement: The epidemiological investigation was conducted under the Infectious Diseases Act (Singapore). Study protocols were approved by ethics committees of the National Healthcare Group and SingHealth. Written informed consent was obtained from participants for clinical data and biological sample collection as part of the PROTECT study (2012/00917; 2018/3045). A waiver of informed consent for retrospective data collection only was granted for individuals admitted to the National Centre of Infectious Diseases (2020/01122). Healthy donor samples were collected under study numbers 2017/2806 and NUS IRB 04-140.

Background: Given the unceasing worldwide surge in COVID-19 cases, there is an imperative need to develop highly specific and sensitive serology assays to define exposure to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Methods: Pooled plasma samples from PCR positive COVID-19 patients were used to identify linear B-cell epitopes from a SARS-CoV-2 peptide library of spike (S), envelope (E), membrane (M), and nucleocapsid (N) structural proteins by peptide-based ELISA. Hit epitopes were further validated with 79 COVID-19 patients with different disease severity status, 12 seasonal human CoV, 20 recovered SARS patients and 22 healthy donors. Findings: Four immunodominant epitopes, S14P5, S20P2, S21P2 and N4P5, were identified on the S and N viral proteins. IgG responses to all identified epitopes displayed a strong detection profile, with N4P5 achieving the highest level of specificity (100%) and sensitivity (>96%) against SARS-CoV-2. Furthermore, the magnitude of IgG responses to S14P5, S21P2 and N4P5 were strongly associated with disease severity. Interpretation: IgG responses to the peptide epitopes can serve as useful indicators for the degree of immunopathology in COVID-19 patients, and function as higly specific and sensitive sero-immunosurveillance tools for recent or past SARS-CoV-2 infections. The flexibility of these epitopes to be used alone or in combination will allow for the development of improved point-of-care-tests (POCTs). Funding Statement: Biomedical Research Council (BMRC), the A*ccelerate GAP-funded project (ACCL/19-GAP064-R20H-H) from Agency of Science, Technology and Research (A*STAR), and National Medical Research Council (NMRC) COVID-19 Research fund (COVID19RF-001). ATR is supported by the Singapore International Graduate Award (SINGA), A*STAR. Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: The study design and protocols for COVID-19, recovered SARS and seasonal hCoV patient cohorts were evaluated by National Healthcare Group (NHG) Domain Specific Review Board (DSRB) and approved under study numbers 2012/00917, 2020/00091 and 2020/00076, respectively. Healthy donor samples were collected under study numbers 2017/2806 and NUS IRB 04-140. Residual serum sample from National Healthy Survey (NHS) collected in 2010 under study number 006/2010 was used as a positive control in peptide-based ELISA (13). Written informed consent was obtained from participants in accordance with the Declaration of Helsinki for Human Research.

The impact of ORF8 382-nucleotide deletion (Δ382) on the cellular host immune response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains undefined. Here, RNA-sequencing was performed to elucidate whole blood transcriptomic profiles and identify contrasting immune signatures between patients infected with wildtype or Δ382 variant of SARS-CoV-2. Interestingly, immune landscape of Δ382 SARS-CoV-2 infected patients featured an increased T cell response, evidenced by enrichment of genes related to T cell functionality that correlated with up-regulation of T cell-associated cytokines, under-expression of neutrophil activation-associated genes, lowered systemic inflammation and effective antibody response. At the molecular level, eukaryotic initiation factor 2 signaling was found to be up-regulated in patients bearing Δ382 and its associated genes were correlated with systemic levels of T cell-associated and pro-inflammatory cytokines. These key insights could serve as an important foundation for future human challenge vaccine studies and highlights the prophylactic potential of Δ382 as vaccine candidate. Funding: The study was supported by core and COVID-19 (project number H20/04/g1/006) research grants provided to Singapore Immunology Network by the Biomedical Research Council (BMRC) and A*ccelerate GAP-funded project (ACCL/20-GAP001C20H-E) from the Singapore Ministry of Health’s National Medical Research Council. This study was also funded by the National Medical Research Council (NMRC) COVID-19 Research fund (COVID19RF-001). SIgN Immunomonitoring Platform is supported by a BMRC IAF 311006 grant and BMRC transition funds #H16/99/b0/011. ATR is supported by the Singapore International Graduate Award (SINGA), A*STAR. Conflict of Interest: The authors declare no conflict of interest. Ethical Approval: The study design and protocols for the COVID-19 PROTECT study group were evaluated by National Healthcare Group (NHG) Domain Specific Review Board (DSRB) and approved under study number 2012/00917. Collection of healthy donor samples was approved by SingHealth Centralised Institutional Review Board (CIRB) under study number 2017/2806 and NUS IRB 04-140. Written informed consent was obtained from participants in accordance with the Declaration of Helsinki for Human Research.

Background: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide, causing severe infections and death with hitherto no proven t

This study shows that Viperin controls the microenvironment pro-inflammatory response and CD4 T cell–mediated pathogenesis during anti-chikungunya virus immune response in mice., Chikungunya virus (CHIKV) has been a worldwide threat since its reemergence in La Reunion Island in 2004. Expression of the interferon-stimulated protein Viperin correlates with viral load burden in patients, and studies in mice have demonstrated its role to limit disease severity against CHIKV infection. Using Viperin−/− mice, we aimed to understand the contribution of Viperin to the T-cell immune response against CHIKV. CD4 T-cell depletion in Viperin−/− mice showed that increased late acute joint inflammation (5–8 d postinfection) was exclusively mediated by T cells. Specifically, CHIKV-infected Viperin−/− mice showed an increased INFγ Th1 profile of CD4 T cells, enhanced INFγ stimulation by APCs, an increased INFγ secretion profile in the joint microenvironment, and increased numbers of inflammatory monocytes in virus-infected joints compared with WT mice. Bone marrow grafting experiments showed that Viperin expression in both hematopoietic and non-hematopoietic cells is instrumental in reducing disease severity associated with a CD4 T-cell response.

The selection of stable endogenous control genes is critical for normalization of quantitative realtime PCR (qPCR) data. In this study, we aimed to identify a suitable set of control genes to be used as endogenous references for gene expression evaluation in human peripheral blood samples among coronary artery disease patients. The expression levels of 12 endogenous control genes procured from TATAA Biocenter (Goteborg, Sweden) were measured in five acute coronary syndrome patients and five chronic stable angina patients. Gene expression stability was analyzed using two different software applications i.e geNorm and NormFinder. Results suggested that beta-glucuronidase is the most stable endogenous control, followed by hypoxanthine-guanine phosphoribosyltransferase. The NormFinder analysis further confirmed that betaglucuronidase and hypoxanthine-guanine phosphoribosyltransferase were on the first rank order with the most stable expression among endogenous control genes analyzed and 60S acidic ribosomal protein P0. Besides, the expression levels of 18S rRNA were revealed to be highly variable between coronary heart disease patients. We thus recommend the use of beta-glucuronidase and hypoxanthine-guanine phosphoribosyltransferase as reference genes for accurate normalization of relative quantities of gene expression levels in coronary artery disease patients using qPCR. Also the use of 18S rRNA as a control gene should be avoided.