Catching bird flu in a droplet.

It is assumed that a timely mass administration of antiviral drugs, backed by quarantines and social distancing, could contain a nascent influenza epidemic at its source, provided that the first clusters of cases were localized within a short time. However, effective routine surveillance may be impossible in countries lacking basic public health resources. For a global containment strategy to be successful, low-cost, easy-to-use handheld units that permit decentralized testing would be vital. Here we present a microfluidic platform that can detect the highly pathogenic avian influenza virus H5N1 in a throat swab sample by using magnetic forces to manipulate a free droplet containing superparamagnetic particles. In a sequential process, the viral RNA is isolated, purified, preconcentrated by 50,000% and subjected to ultrafast real-time RT-PCR. Compared to commercially available tests, the bioassay is equally sensitive and is 440% faster and 2,000-5,000% cheaper.

Crosstalk between CD64+MHCII+ macrophages and CD4+ T cells drives joint pathology during chikungunya.

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.

Comparative analysis of the genome sequences and replication profiles of chikungunya virus isolates within the East, Central and South African (ECSA) lineage.

BACKGROUND: A comparative analysis of the genomic and replication profiles of different geographical chikungunya virus (CHIKV) isolates of the East, Central and South African (ECSA) lineage was performed. FINDINGS: Analysis of the data revealed the different growth kinetics for the different isolates. Deep genome sequencing analysis further revealed specific amino acid mutations in the viral nsP1, nsP3, nsP4, E1 and E2 proteins in the different isolates. Despite the difference in viral genomic profiles, the virus-induced ultrastructural changes within infected cells remained highly conserved among the different chikungunya virus isolates. CONCLUSIONS: These findings provide insights into the genomic and replication profiles of the re-emerging chikungunya virus isolates of the ECSA lineage.

Acquired immunity against SARS-CoV-2 infection and vaccination.

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has caused more than 700 million confirmed infections and ~7 million fatalities worldwide since its emergence in December 2019. SARS-CoV-2 is part of a family of positive-sense, enveloped RNA viruses known as coronaviruses. Today, at least seven human coronaviruses have been identified and are known to cause respiratory tract illnesses with varying severity. The COVID-19 pandemic spurred the generation of a vast amount of scientific knowledge on coronaviruses in record time, leading to a broad understanding of host immunity against SARS-CoV-2, and the rapid development of life-saving vaccines (mainly mRNA and adenovirus- or inactivated virus-based vaccines). Real world data on licensed SARS-CoV-2 vaccines have shown that efficacy ranges from 50 to 95% depending on viral variants, pre-infections, and vaccine formulations, regimens, and combinations. While vaccination does markedly decrease the chances of infection and severe disease, breakthrough symptomatic and asymptomatic infections have occurred due to the emergence of immune escape virus variants. Therefore, despite these early successes, a better understanding of the mechanisms of protective immunity against infection is essential for the development of longer lasting and more efficient vaccines against SARS-CoV-2 and future coronaviruses.

Organ-specific immune response in lethal SARS-CoV-2 infection by deep spatial phenotyping.

Objectives: Immunopathology of ongoing COVID-19 global pandemic is not limited solely to pulmonary tissue, but is often associated with multi-organ complications, mechanisms of which are intensely being investigated. In this regard, the interplay between immune, stromal cells and cytokines in pulmonary and extrapulmonary infected tissues, especially in young adults (median age 46 years, range 30-53 years) without comorbidities, remains poorly characterised. Methods: We profiled lung, heart and intestinal autopsy samples from five SARS-CoV-2-infected cases for 18-20 targets to detect immune, cytokine and stromal cell status at subcellular resolution by a novel IHC-based deep-phenotyping technique, iSPOT (immunoSpatial histoPhenOmics using TSA-IHC), to assess spatial and functional patterns of immune response in situ, in lethal COVID-19 infection. Results: SARS-CoV-2-infected autopsy samples exhibit skewed counts of immune populations in all samples with organ-specific dysfunctions. Lung and ileal tissue reveal altered architecture with marked loss of tissue integrity, while lung and heart tissue show severe hyperinflammation marked by elevated TNF-α in heart tissue and additionally IL-6, IFN-γ and IL-10 cytokines in lung samples. Conclusion: With resurgence of infection in younger populations, single-cell cytokine localisation in immune and stromal structures provides important mechanistic insights into organ-specific immunopathology of naïve SARS-CoV-2 infection in the absence of other comorbidities.

Malaria abrogates O'nyong-nyong virus pathologies by restricting virus infection in nonimmune cells.

O'nyongnyong virus (ONNV) is a re-emerging alphavirus previously known to be transmitted by main malaria vectors, thus suggesting the possibility of coinfections with arboviruses in co-endemic areas. However, the pathological outcomes of such infections remain unknown. Using murine coinfection models, we demonstrated that a preexisting blood-stage Plasmodium infection suppresses ONNV-induced pathologies. We further showed that suppression of viremia and virus dissemination are dependent on Plasmodium-induced IFNγ and are associated with reduced infection of CD45- cells at the site of virus inoculation. We further proved that treatment with IFNγ or plasma samples from Plasmodium vivax-infected patients containing IFNγ are able to restrict ONNV infection in human fibroblast, synoviocyte, skeletal muscle, and endothelial cell lines. Mechanistically, the role of IFNγ in restricting ONNV infection was confirmed in in vitro infection assays through the generation of an IFNγ receptor 1 α chain (IFNγR1)-deficient cell line.

Decreased memory B cell frequencies in COVID-19 delta variant vaccine breakthrough infection.

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-1ß 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.

Heterologous booster vaccination with CoronaVac following prime vaccination with mRNA vaccine.

Objective: 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. Methods: 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. Results: 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. Conclusion: 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.

Convalescent COVID-19 patients are susceptible to endothelial dysfunction due to persistent immune activation.

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.

Human neutralising antibodies elicited by SARS-CoV-2 non-D614G variants offer cross-protection against the SARS-CoV-2 D614G variant.

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.

Association of SARS-CoV-2 clades with clinical, inflammatory and virologic outcomes: An observational study.

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.

Systematic analysis of disease-specific immunological signatures in patients with febrile illness from Saudi Arabia.

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.

Safety and potential efficacy of cyclooxygenase-2 inhibitors in coronavirus disease 2019.

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.

Type I interferon shapes the quantity and quality of the anti-Zika virus antibody response.

OBJECTIVES: Zika virus (ZIKV) is a mosquito-borne flavivirus that re-emerged in 2015. The association between ZIKV and neurological complications initiated the development of relevant animal models to understand the mechanisms underlying ZIKV-induced pathologies. Transient inhibition of the type I interferon (IFN) pathway through the use of an IFNAR1-blocking antibody, MAR1-5A3, could efficiently permit active virus replication in immunocompetent animals. Type I IFN signalling is involved in the regulation of humoral responses, and thus, it is crucial to investigate the potential effects of type I IFN blockade towards B-cell responses. METHODS: In this study, comparative analysis was conducted using serum samples collected from ZIKV-infected wild-type (WT) animals either administered with or without MAR1-5A3. RESULTS: Serological assays revealed a more robust ZIKV-specific IgG response and subtype switching upon inhibition of type I IFN due to the abundance of antigen availability. This observation was corroborated by an increase in germinal centres, plasma cells and germinal centre B cells. Interestingly, although both groups of animals recognised different B-cell linear epitopes in the E and NS1 regions, there was no difference in neutralising capacity. Further characterisation of these epitopes in the E protein revealed a detrimental role of antibodies that were generated in the absence of type I IFN. CONCLUSION: This study highlights the role of type I IFN in shaping the anti-ZIKV antibody response to generate beneficial antibodies and will help guide development of better vaccine candidates triggering efficient neutralising antibodies and avoiding detrimental ones.

Erratum: Novel differential linear B-cell epitopes to identify Zika and dengue virus infections in patients.

[This corrects the article DOI: 10.1002/cti2.1066.].

Associations of viral ribonucleic acid (RNA) shedding patterns with clinical illness and immune responses in Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection.

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-1ß 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.

Whole blood immunophenotyping uncovers immature neutrophil-to-VD2 T-cell ratio as an early marker for severe COVID-19.

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.

Viperin controls chikungunya virus-specific pathogenic T cell IFNγ Th1 stimulation 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.

Novel differential linear B-cell epitopes to identify Zika and dengue virus infections in patients.

OBJECTIVES: Recent Zika virus (ZIKV) outbreaks challenged existing laboratory diagnostic standards, especially for serology-based methods. Because of the genetic and structural similarity of ZIKV with other flaviviruses, this results in cross-reactive antibodies, which confounds serological interpretations. METHODS: Plasma from Singapore ZIKV patients was screened longitudinally for antibody responses and neutralising capacities against ZIKV. Samples from healthy controls, ZIKV patients and DENV patients were further assessed using ZIKV and DENV peptides of precursor membrane (prM), envelope (E) or non-structural 1 (NS1) viral proteins in a peptide-based ELISA for epitope identification. Identified epitopes were re-validated and diagnostically evaluated using sera of patients with DENV, bacteria or unknown infections from Thailand. RESULTS: Long-lasting ZIKV-neutralising antibodies were elicited during ZIKV infection. Thirteen potential linear B-cell epitopes were identified, and of these, four common flavivirus, three ZIKV-specific and one DENV-specific differential epitopes had more than 50% sensitivity and specificity. Notably, ZIKV-specific peptide 26 on domain I/II of E protein (amino acid residues 271-288) presented 80% sensitivity and 85.7% specificity. Importantly, the differential epitopes also showed significance in differentiating non-flavivirus patient samples. CONCLUSION: Linear B-cell epitope candidates to differentiate between ZIKV and DENV infections were identified, providing the first step towards the design of a much-needed serology-based assay.

Mutating chikungunya virus non-structural protein produces potent live-attenuated vaccine candidate.

Currently, there are no commercially available live-attenuated vaccines against chikungunya virus (CHIKV). Here, CHIKVs with mutations in non-structural proteins (nsPs) were investigated for their suitability as attenuated CHIKV vaccines. R532H mutation in nsP1 caused reduced infectivity in mouse tail fibroblasts but an enhanced type-I IFN response compared to WT-CHIKV Adult mice infected with this nsP-mutant exhibited a mild joint phenotype with low-level viremia that rapidly cleared. Mechanistically, ingenuity pathway analyses revealed a tilt in the anti-inflammatory IL-10 versus pro-inflammatory IL-1ß and IL-18 balance during CHIKV nsP-mutant infection that modified acute antiviral and cell signaling canonical pathways. Challenging CHIKV nsP-mutant-infected mice with WT-CHIKV or the closely related O'nyong-nyong virus resulted in no detectable viremia, observable joint inflammation, or damage. Challenged mice showed high antibody titers with efficient neutralizing capacity, indicative of immunological memory. Manipulating molecular processes that govern CHIKV replication could lead to plausible vaccine candidates against alphavirus infection.