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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. [ABSTRACT FROM AUTHOR]

Background: Epidemics caused by the reemergence of Zika virus (ZIKV) warrant the need to develop new diagnostic measures to complement currently used detection methods. In this study, we explored the detection of ZIKV antigen in a defined leukocyte subset from patients' whole-blood specimens.Methods: Whole-blood samples were obtained at the acute and early convalescent phases from ZIKV-infected patients during the Singapore outbreak in August-September 2016. Presence of ZIKV antigen was determined by flow cytometry staining for intracellular ZIKV NS3, using a ZIKV-specific polyclonal antibody. The presence of ZIKV antigen was determined in CD45+CD14+ monocytes.Results: Data showed that ZIKV NS3 antigen could be detected in CD45+CD14+ monocytes. The levels of detection were further categorized into 3 groups: high (positivity among >40% of monocytes), moderate (positivity among 10%-40%), and low (positivity among <10%). While a majority of patients showed a decrease in the amount of ZIKV antigen detected at later time points, some patients displayed higher levels as the disease progressed.Conclusions: Our data highlights an alternative approach in using flow cytometry as a sensitive method for detecting ZIKV antigen in whole blood. Importantly, it further confirms the role of CD14+ monocytes as an important cellular target for ZIKV infection during the viremic phase. [ABSTRACT FROM AUTHOR]

Background: Biomarkers play a pivotal role in the diagnosis and management of patients with acute coronary syndrome. This study aimed to investigate the differences in level of several biomarkers, i.e. C-reactive protein, myeloperoxidase, soluble CD40 ligand and placental growth factor, between acute coronary syndrome and chronic stable angina patients. The relationship between these biomarkers in the coronary circulation and systemic circulation was also investigated. Methods: A total of 79 patients were recruited in this study. The coronary blood was sampled from occluded coronary artery, while the peripheral venous blood was withdrawn from antecubital fossa. The serum concentrations of C-reactive protein, soluble CD40 ligand and placental growth factor and plasma concentration of myeloperoxidase were measured using ELISA method. Results: The systemic level of the markers measured in the peripheral venous blood was significantly increased in acute coronary syndrome compared to chronic stable angina patients. The concentrations of the C-reactive protein, myeloperoxidase and soluble CD40 ligand taken from peripheral vein were closely similar to the concentration found in coronary blood of ACS patients. The level of placental growth factor was significantly higher in coronary circulation than its systemic level. Conclusion: The concentration of these C-reactive protein, myeloperoxidase, soluble CD40 ligand and placental growth factor were significantly increased in acute coronary syndrome patients. The concentration of the markers measured in the systemic circulation directly reflected those in the local coronary circulation. Thus, these markers have potential to become a useful tool in predicting plaque vulnerability in the future. [ABSTRACT FROM AUTHOR]

Objectives: The emergence of new SARS-CoV-2 variants has led to the development of Omicron-targeting bivalent mRNA vaccines. It is crucial to understand how bivalent vaccines may improve antibody responses against new variants. Methods: A total of 107 participants, who had three COVID-19 WT mRNA vaccine doses, were recruited, and given either a monovalent (WT) or a bivalent mRNA vaccination (Pfizer/BioNTech Bivalent (WT and BA.4/BA.5) or Moderna Bivalent (WT and BA.1). Blood samples were taken before booster and at 28 days post-booster. Results: We found significantly lower fold change in serum binding IgA responses against BA.1, BA.5 and EG.5.1 spike in the bivalent booster group, compared with the monovalent (WT) booster group, following vaccination. However, this was only observed in individuals with prior infection. The relative fold change in serum binding IgA response was more skewed towards WT over variant (BA.1, BA.5 or EG.5.1) spike in previously infected bivalent-booster-vaccinees, as compared with previously infected monovalent-(WT)-booster-vaccinees. Conclusion: The findings suggest imprinting of antibody responses that is shaped by the first vaccination (WT spike). Previous infection also affects the boosting effect of follow-up vaccination. Studies are needed to understand how to induce a robust and long-lasting IgA immunity for protection against COVID-19 infection.

Abstract Communications between immune cells are essential to ensure appropriate coordination of their activities. Here, we observed the infiltration of activated macrophages into the joint-footpads of chikungunya virus (CHIKV)-infected animals. Large numbers of CD64+MHCII+ and CD64+MHCII- macrophages were present in the joint-footpad, preceded by the recruitment of their CD11b+Ly6C+ inflammatory monocyte precursors. Recruitment and differentiation of these myeloid subsets were dependent on CD4+ T cells and GM-CSF. Transcriptomic and gene ontology analyses of CD64+MHCII+ and CD64+MHCII- macrophages revealed 89 differentially expressed genes, including genes involved in T cell proliferation and differentiation pathways. Depletion of phagocytes, including CD64+MHCII+ macrophages, from CHIKV-infected mice reduced disease pathology, demonstrating that these cells play a pro-inflammatory role in CHIKV infection. Together, these results highlight the synergistic dynamics of immune cell crosstalk in driving CHIKV immunopathogenesis. This study provides new insights in the disease mechanism and offers opportunities for development of novel anti-CHIKV therapeutics.

Abstract Due to the paucity of longitudinal molecular studies of COVID-19, particularly those covering the early stages of infection (Days 1-8 symptom onset), our understanding of host response over the disease course is limited. We perform longitudinal single cell RNA-seq on 286 blood samples from 108 age- and sex-matched COVID-19 patients, including 73 with early samples. We examine discrete cell subtypes and continuous cell states longitudinally, and we identify upregulation of type I IFN-stimulated genes (ISGs) as the predominant early signature of subsequent worsening of symptoms, which we validate in an independent cohort and corroborate by plasma markers. However, ISG expression is dynamic in progressors, spiking early and then rapidly receding to the level of severity-matched non-progressors. In contrast, cross-sectional analysis shows that ISG expression is deficient and IFN suppressors such as SOCS3 are upregulated in severe and critical COVID-19. We validate the latter in four independent cohorts, and SOCS3 inhibition reduces SARS-CoV-2 replication in vitro. In summary, we identify complexity in type I IFN response to COVID-19, as well as a potential avenue for host-directed therapy.

Abstract Identification of the risk factors and the high-risk groups which are most vulnerable is critical in COVID-19 disease management at a population level. Evaluating the efficacy of vaccination against infections is necessary to determine booster vaccination strategies for better protection in high-risk groups. In this study, we recruited 158 mRNA-vaccinated individuals during the Delta wave of SARS-CoV-2 infections in Singapore and examined the antibody profiles of infected individuals. We found that, despite high exposure due to communal living conditions in proximity, 4% of individuals (6/158) had PCR-confirmed infections and 96% (152/158) remained uninfected. Time-course analysis of the antibody profile at the start and the end of quarantine period showed Delta-specific boosting of anti-spike antibody response in 57% of the uninfected individuals (86/152). In the remaining 43% of the uninfected individuals (66/152) with no Delta-specific antibody boost, we found a higher Delta-specific antibody response at the start of quarantine period, which correlated with higher Delta pseudovirus neutralizing capacity. Our findings indicate that a higher basal variant-specific antibody response in the mRNA-vaccinated individuals contributes to better protection against infections by the new emerging SARS-CoV-2 variants.

Abstract The SARS‐CoV‐2 Delta (B.1.617.2) variant is capable of infecting vaccinated persons. An open question remains as to whether deficiencies in specific vaccine‐elicited immune responses result in susceptibility to vaccine breakthrough infection. We investigated 55 vaccine breakthrough infection cases (mostly Delta) in Singapore, comparing them against 86 vaccinated close contacts who did not contract infection. Vaccine breakthrough cases showed lower memory B cell frequencies against SARS‐CoV‐2 receptor‐binding domain (RBD). Compared to plasma antibodies, antibodies secreted by memory B cells retained a higher fraction of neutralizing properties against the Delta variant. Inflammatory cytokines including IL‐1β and TNF were lower in vaccine breakthrough infections than primary infection of similar disease severity, underscoring the usefulness of vaccination in preventing inflammation. This report highlights the importance of memory B cells against vaccine breakthrough and suggests that lower memory B cell levels may be a correlate of risk for Delta vaccine breakthrough infection.

Abstract The immune responses and mechanisms limiting symptom progression in asymptomatic cases of SARS‐CoV‐2 infection remain unclear. We comprehensively characterized transcriptomic profiles, cytokine responses, neutralization capacity of antibodies, and cellular immune phenotypes of asymptomatic patients with acute SARS‐CoV‐2 infection to identify potential protective mechanisms. Compared to symptomatic patients, asymptomatic patients had higher counts of mature neutrophils and lower proportion of CD169+ expressing monocytes in the peripheral blood. Systemic levels of pro‐inflammatory cytokines were also lower in asymptomatic patients, accompanied by milder pro‐inflammatory gene signatures. Mechanistically, a more robust systemic Th2 cell signature with a higher level of virus‐specific Th17 cells and a weaker yet sufficient neutralizing antibody profile against SARS‐CoV‐2 was observed in asymptomatic patients. In addition, asymptomatic COVID‐19 patients had higher systemic levels of growth factors that are associated with cellular repair. Together, the data suggest that asymptomatic patients mount less pro‐inflammatory and more protective immune responses against SARS‐CoV‐2 indicative of disease tolerance. Insights from this study highlight key immune pathways that could serve as therapeutic targets to prevent disease progression in COVID‐19.

COVID-19 severity is associated with cytokine levels and lymphopenia, but the role of immune cell subsets is not well understood. Here the authors immunophenotype whole blood samples from 54 COVID-19 patients and find that the immature neutrophil-to-VD2 T-cell ratio is associated with severe COVID-19.

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.

Abstract Objectives The emergence of a SARS‐CoV‐2 variant with a point mutation in the spike (S) protein, D614G, has taken precedence over the original Wuhan isolate by May 2020. With an increased infection and transmission rate, it is imperative to determine whether antibodies induced against the D614 isolate may cross‐neutralise against the G614 variant. Methods Antibody profiling against the SARS‐CoV‐2 S protein of the D614 variant by flow cytometry and assessment of neutralising antibody titres using pseudotyped lentiviruses expressing the SARS‐CoV‐2 S protein of either the D614 or G614 variant tagged with a luciferase reporter were performed on plasma samples from COVID‐19 patients with known D614G status (n = 44 infected with D614, n = 6 infected with G614, n = 7 containing all other clades: O, S, L, V, G, GH or GR). Results Profiling of the anti‐SARS‐CoV‐2 humoral immunity reveals similar neutralisation profiles against both S protein variants, albeit waning neutralising antibody capacity at the later phase of infection. Of clinical importance, patients infected with either the D614 or G614 clade elicited a similar degree of neutralisation against both pseudoviruses, suggesting that the D614G mutation does not impact the neutralisation capacity of the elicited antibodies. Conclusions Cross‐reactivity occurs at the functional level of the humoral response on both the S protein variants, which suggests that existing serological assays will be able to detect both D614 and G614 clades of SARS‐CoV‐2. More importantly, there should be negligible impact towards the efficacy of antibody‐based therapies and vaccines that are currently being developed.

Abstract Objectives Little is known about the prevalence of febrile illness in the Arabian region as clinical, laboratory and immunological profiling remains largely uncharacterised. Methods A total of 2018 febrile patients from Jazan, Saudi Arabia, were recruited between 2014 and 2017. Patients were screened for dengue and chikungunya virus, Plasmodium, Brucella, Neisseria meningitidis, group A streptococcus and Leptospira. Clinical history and biochemical parameters from blood tests were collected. Patient sera of selected disease‐confirmed infections were quantified for immune mediators by multiplex microbead‐based immunoassays. Results Approximately 20% of febrile patients were tested positive for one of the pathogens, and they presented overlapping clinical and laboratory parameters. Nonetheless, eight disease‐specific immune mediators were identified as potential biomarkers for dengue (MIP‐1α, MCP‐1), malaria (TNF‐α), streptococcal and meningococcal (eotaxin, GRO‐α, RANTES, SDF‐1α and PIGF‐1) infections, with high specificity and sensitivity profiles. Notably, based on the conditional inference model, six of these mediators (MIP‐1α, TNF‐α, GRO‐α, RANTES, SDF‐1α and PIGF‐1) were revealed to be 68.4% accurate in diagnosing different febrile infections, including those of unknown diseases. Conclusions This study is the first extensive characterisation of the clinical analysis and immune biomarkers of several clinically important febrile infections in Saudi Arabia. Importantly, an immune signature with robust accuracy, specificity and sensitivity in differentiating several febrile infections was identified, providing useful insights into patient disease management in the Arabian Peninsula.

Abstract Objectives While the safety of non‐steroidal anti‐inflammatory drugs in COVID‐19 has been questioned, they may be beneficial given the hyper‐inflammatory immune response associated with severe disease. We aimed to assess the safety and potential efficacy of cyclooxygenase‐2 (COX‐2) selective inhibitors in high‐risk patients. Methods Retrospective study of patients with COVID‐19 pneumonia and aged ≥ 50 years who were admitted to hospital. Adverse outcomes analysed included supplemental oxygen use, intensive care unit admission, mechanical ventilation and mortality, with the primary endpoint a composite of any of these. Plasma levels of inflammatory cytokines and chemokines were measured in a subset. Results Twenty‐two of 168 (13.1%) in the cohort received COX‐2 inhibitors [median duration 3 days, interquartile range (IQR) 3–4.25]. Median age was 61 (IQR 55–67.75), 44.6% were female, and 72.6% had at least one comorbidity. A lower proportion of patients receiving COX‐2 inhibitors met the primary endpoint: 4 (18.2%) versus 57 (39.0%), P = 0.062. This difference was less pronounced after adjusting for baseline difference in age, gender and comorbidities in a multivariate logistic regression model [adjusted odds ratio (AOR) 0.45, 95% CI 0.14–1.46]. The level of interleukin‐6 declined after treatment in five of six (83.3%) treatment group patients [compared to 15 of 28 (53.6%) in the control group] with a greater reduction in absolute IL‐6 levels (P‐value = 0.025). Conclusion Treatment with COX‐2 inhibitors was not associated with an increase in adverse outcomes. Its potential for therapeutic use as an immune modulator warrants further evaluation in a large randomised controlled trial.

Abstract Objectives A wide range of duration of viral RNA shedding in patients infected with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS‐CoV‐2) has been observed. We aimed to investigate factors associated with prolonged and intermittent viral RNA shedding in a retrospective cohort of symptomatic COVID‐19 patients. Methods Demographic, clinical and laboratory data from hospitalised COVID‐19 patients from a single centre with two consecutive negative respiratory reverse transcription‐polymerase chain reaction (RT‐PCR) results were extracted from electronic medical records. Kaplan–Meier survival curve analysis was used to assess the effect of clinical characteristics on the duration and pattern of shedding. Plasma levels of immune mediators were measured using Luminex multiplex microbead‐based immunoassay. Results There were 201 symptomatic patients included. Median age was 49 years (interquartile range 16–61), and 52.2% were male. Median RNA shedding was 14 days (IQR 9–18). Intermittent shedding was observed in 77 (38.3%). We did not identify any factor associated with prolonged or intermittent viral RNA shedding. Duration of shedding was inversely correlated with plasma levels of T‐cell cytokines IL‐1β and IL‐17A at the initial phase of infection, and patients had lower levels of pro‐inflammatory cytokines during intermittent shedding. Conclusions Less active T‐cell responses at the initial phase of infection were associated with prolonged viral RNA shedding, suggesting that early immune responses are beneficial to control viral load and prevent viral RNA shedding. Intermittent shedding is common and may explain re‐detection of viral RNA in recovered patients.