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Abstract Background There is limited epidemiologic data on patients with acute myelogenous (myeloid) leukemia (AML) requiring life-sustaining therapies in the intensive care unit (ICU). Our objectives were to describe the clinical characteristics and outcomes in critically ill AML patients. Methods This was a retrospective case-control study. Cases were defined as adult patients with a primary diagnosis of AML admitted to ICU at the University of Alberta Hospital between January 1st 2002 and June 30th 2008. Each case was matched by age, sex, and illness severity (ICU only) to two control groups: hospitalized AML controls, and non-AML ICU controls. Data were extracted on demographics, course of hospitalization, and clinical outcomes. Results In total, 45 AML patients with available data were admitted to ICU. Mean (SD) age was 54.8 (13.1) years and 28.9% were female. Primary diagnoses were sepsis (32.6%) and respiratory failure (37.3%). Mean (SD) APACHE II score was 30.3 (10.3), SOFA score 12.6 (4.0) with 62.2% receiving mechanical ventilation, 55.6% vasoactive therapy, and 26.7% renal replacement therapy. Crude in-hospital, 90-day and 1-year mortality was 44.4%, 51.1% and 71.1%, respectively. AML cases had significantly higher adjusted-hazards of death (HR 2.23; 95% CI, 1.38-3.60, p = 0.001) compared to both non-AML ICU controls (HR 1.69; 95% CI, 1.11-2.58, p = 0.02) and hospitalized AML controls (OR 1.0, reference variable). Factors associated with ICU mortality by univariate analysis included older age, AML subtype, higher baseline SOFA score, no change or an increase in early SOFA score, shock, vasoactive therapy and mechanical ventilation. Active chemotherapy in ICU was associated with lower mortality. Conclusions AML patients may represent a minority of all critically ill admissions; however, are not uncommonly supported in ICU. These AML patients are characterized by high illness severity, multi-organ dysfunction, and high treatment intensity and have a higher risk of death when compared with matched hospitalized AML or non-AML ICU controls. The absence of early improvement in organ failure may be a useful predictor for mortality for AML patients admitted to ICU.

Chikungunya patients may develop chronic joint pain that can persist for months to years. This study aimed to determine the factors associated with Chikungunya chronicity. This case-control study involved data from patients with laboratory-confirmed Chikungunya reported from March 2016 to December 2020 in Vitória, Espírito Santo state, Brazil. The data were accessed from compulsory notification databases (SINAN and eSUS VS) and electronic medical reports (Rede Bem-Estar). For each patient who developed chronic symptoms, we included a control patient who did not develop chronic symptoms by random sampling. A total of 183 chronic and 183 non-chronic patients were included in the study. Most of them were female (73.2%), with a median age of 49.5 years (interquartile range = 37-61), and presented fever (89.6%), myalgia (89.6%), arthralgia (89.3%), and headache (82.0%). Chronic patients were older (median = 53; interquartile range = 41-61) than non-chronic cases (median = 46; interquartile age = 31-61) (OR = 0.979, 95% CI = 0.968-0.991) and more frequently presented nausea (58.5% vs. 40.4%; OR = 2.109, 95% CI = 1.374-3.238), and leukopenia (20.2% vs. 10.9%; OR = 2.060, 95% CI = 1.122-3.779). Therefore, these characteristics should be monitored for the better clinical management of cases prone to chronicity.

Vaccination has successfully controlled several infectious diseases although better vaccines remain desirable. Host response to vaccination studies have identified correlates of vaccine immunogenicity that could be useful to guide development and selection of future vaccines. However, it remains unclear whether these findings represent mere statistical correlations or reflect functional associations with vaccine immunogenicity. Functional associations, rather than statistical correlates, would offer mechanistic insights into vaccine-induced adaptive immunity. Through a human experimental study to test the immunomodulatory properties of metformin, an anti-diabetic drug, we chanced upon a functional determinant of neutralizing antibodies. Although vaccine viremia is a known correlate of antibody response, we found that in healthy volunteers with no detectable or low yellow fever 17D viremia, metformin-treated volunteers elicited higher neutralizing antibody titers than placebo-treated volunteers. Transcriptional and metabolomic analyses collectively showed that a brief course of metformin, started 3 days prior to YF17D vaccination and stopped at 3 days after vaccination, expanded oxidative phosphorylation and protein translation capacities. These increased capacities directly correlated with YF17D neutralizing antibody titers, with reduced reactive oxygen species response compared to placebo-treated volunteers. Our findings thus demonstrate a functional association between cellular respiration and vaccine-induced humoral immunity and suggest potential approaches to enhancing vaccine immunogenicity., (© 2024. The Author(s).)

The paucity of information on longevity of vaccine-induced immune responses and uncertainty of the correlates of protection hinder the development of evidence-based COVID-19 vaccination policies for new birth cohorts. Here, to address these knowledge gaps, we conducted a cohort study of healthy 5-12-year-olds vaccinated with BNT162b2. We serially measured binding and neutralizing antibody titers (nAbs), spike-specific memory B cell (MBC) and spike-reactive T cell responses over 1 year. We found that children mounted antibody, MBC and T cell responses after two doses of BNT162b2, with higher antibody and T cell responses than adults 6 months after vaccination. A booster (third) dose only improved antibody titers without impacting MBC and T cell responses. Among children with hybrid immunity, nAbs and T cell responses were highest in those infected after two vaccine doses. Binding IgG titers, MBC and T cell responses were predictive, with T cells being the most important predictor of protection against symptomatic infection before hybrid immunity; nAbs only correlated with protection after hybrid immunity. The stable MBC and T cell responses over time suggest sustained protection against symptomatic SARS-CoV-2 infection, even when nAbs wane. Booster vaccinations do not confer additional immunological protection to healthy children., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Severe COVID-19 is a major cause of morbidity and mortality worldwide, especially among those with co-morbidities, the elderly, and the immunocompromised. However, the molecular determinants critical for severe COVID-19 progression remain to be fully elucidated. Meta-analyses of transcriptomic RNAseq and single-cell sequencing datasets comparing severe and mild COVID-19 patients have demonstrated that the early expansion of myeloid-derived suppressor cells (MDSCs) could be a key feature of severe COVID-19 progression. Besides serving as potential early prognostic biomarkers for severe COVID-19 progression, several studies have also indicated the functional roles of MDSCs in severe COVID-19 pathogenesis and possibly even long COVID. Given the potential links between MDSCs and severe COVID-19, we examine the existing literature summarizing the characteristics of MDSCs, provide evidence of MDSCs in facilitating severe COVID-19 pathogenesis, and discuss the potential therapeutic avenues that can be explored to reduce the risk and burden of severe COVID-19. We also provide a web app where users can visualize the temporal changes in specific genes or MDSC-related gene sets during severe COVID-19 progression and disease resolution, based on our previous study.

Lung inflammation is a hallmark of Coronavirus disease 2019 (COVID-19) in patients who are severely ill, and the pathophysiology of disease is thought to be immune mediated. Mast cells (MCs) are polyfunctional immune cells present in the airways, where they respond to certain viruses and allergens and often promote inflammation. We observed widespread degranulation of MCs during acute and unresolved airway inflammation in SARS-CoV-2-infected mice and nonhuman primates. Using a mouse model of MC deficiency, MC-dependent interstitial pneumonitis, hemorrhaging, and edema in the lung were observed during SARS-CoV-2 infection. In humans, transcriptional changes in patients requiring oxygen supplementation also implicated cells with a MC phenotype in severe disease. MC activation in humans was confirmed through detection of MC-specific proteases, including chymase, the levels of which were significantly correlated with disease severity and with biomarkers of vascular dysregulation. These results support the involvement of MCs in lung tissue damage during SARS-CoV-2 infection in animal models and the association of MC activation with severe COVID-19 in humans, suggesting potential strategies for intervention.

Lipids can play diverse roles in metabolism, signaling, transport across membranes, regulating body temperature, and inflammation. Some viruses have evolved to exploit lipids in human cells to promote viral entry, fusion, replication, assembly, and energy production through fatty acid beta-oxidation. Hence, studying the virus-lipid interactions provides an opportunity to understand the biological processes involved in the viral life cycle, which can facilitate the development of antivirals. Due to the diversity and complexity of lipids, the assessment of lipid utilization in infected host cells can be challenging. However, the development of mass spectrometry, bioenergetics profiling, and bioinformatics has significantly advanced our knowledge on the study of lipidomics. Herein, we describe the detailed methods for lipid extraction, mass spectrometry, and assessment of fatty acid oxidation on cellular bioenergetics, as well as the bioinformatics approaches for detailed lipid analysis and utilization in host cells. These methods were employed for the investigation of lipid alterations in TMEM41B- and VMP1-deficient cells, where we previously found global dysregulations of the lipidome in these cells. Furthermore, we developed a web app to plot clustermaps or heatmaps for mass spectrometry data that is open source and can be hosted locally or at https://kuanrongchan-lipid-metabolite-analysis-app-k4im47.streamlit.app/. This protocol provides an efficient step-by-step methodology to assess lipid composition and usage in host cells., Competing Interests: Competing interestsThe authors describe no competing interests., (©Copyright : © 2023 The Authors; This is an open access article under the CC BY license.)

Background: Post-mRNA vaccination-associated cardiac complication is a rare but life-threatening adverse event. Its risk has been well balanced by the benefit of vaccination-induced protection against severe COVID-19. As the rate of severe COVID-19 has consequently declined, future booster vaccination to sustain immunity, especially against infection with new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, may encounter benefit-risk ratios that are less favorable than at the start of the COVID-19 vaccination campaign. Understanding the pathogenesis of rare but severe vaccine-associated adverse events to minimize its risk is thus urgent., Methods: Here, we report a serendipitous finding of a case of cardiac complication following a third shot of COVID-19 mRNA vaccine. As this case was enrolled in a cohort study, pre-vaccination and pre-symptomatic blood samples were available for genomic and multiplex cytokine analyses., Findings: These analyses revealed the presence of subclinical chronic inflammation, with an elevated expression of RNASE2 at pre-booster baseline as a possible trigger of an acute-on-chronic inflammation that resulted in the cardiac complication. RNASE2 encodes for the ribonuclease RNase2, which cleaves RNA at the 3' side of uridine, which may thus remove the only Toll-like receptor (TLR)-avoidance safety feature of current mRNA vaccines., Conclusions: These pre-booster and pre-symptomatic gene and cytokine expression data provide unique insights into the possible pathogenesis of vaccine-associated cardiac complication and suggest the incorporation of additional nucleoside modification for an added safety margin., Funding: This work was funded by the NMRC Open Fund-Large Collaborative Grant on Integrated Innovations on Infectious Diseases (OFLCG19May-0034)., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Gene expression profiling has helped tremendously in the understanding of biological processes and diseases. However, interpreting processed data to gain insights into biological mechanisms remain challenging, especially to the non-bioinformaticians, as many of these data visualization and pathway analysis tools require extensive data formatting. To circumvent these challenges, we developed STAGEs (Static and Temporal Analysis of Gene Expression studies) that provides an interactive visualisation of omics analysis outputs. Users can directly upload data created from Excel spreadsheets and use STAGEs to render volcano plots, differentially expressed genes stacked bar charts, pathway enrichment analysis by Enrichr and Gene Set Enrichment Analysis (GSEA) against established pathway databases or customized gene sets, clustergrams and correlation matrices. Moreover, STAGEs takes care of Excel gene to date misconversions, ensuring that every gene is considered for pathway analysis. Output data tables and graphs can be exported, and users can easily customize individual graphs using widgets such as sliders, drop-down menus, text boxes and radio buttons. Collectively, STAGEs is an integrative platform for data analysis, data visualisation and pathway analysis, and is freely available at https://kuanrongchan-stages-stages-vpgh46.streamlitapp.com/ . In addition, developers can customise or modify the web tool locally based on our existing codes, which is publicly available at https://github.com/kuanrongchan/STAGES ., (© 2023. The Author(s).)

Vaccination induces an adaptive immune response that protects against infectious diseases. A defined magnitude of adaptive immune response that correlates with protection from the disease of interest, or correlates of protection (CoP), is useful for guiding vaccine development. Despite mounting evidence for the protective role of cellular immunity against viral diseases, studies on CoP have almost exclusively focused on humoral immune responses. Moreover, although studies have measured cellular immunity following vaccination, no study has defined if a "threshold" of T cells, both in frequency and functionality, is needed to reduce infection burden. We will thus conduct a double-blind, randomized clinical trial in 56 healthy adult volunteers, using the licensed live-attenuated yellow fever (YF17D) and chimeric Japanese encephalitis-YF17D (JE-YF17D) vaccines. These vaccines share the entire non-structural and capsid proteome where the majority of the T cell epitopes reside. The neutralizing antibody epitopes, in contrast, are found on the structural proteins which are not shared between the two vaccines and are thus distinct from one another. Study participants will receive JE-YF17D vaccination followed by YF17D challenge, or YF17D vaccination followed by JE-YF17D challenge. A separate cohort of 14 healthy adults will receive the inactivated Japanese Encephalitis virus (JEV) vaccine followed by YF17D challenge that controls for the effect of cross-reactive flaviviral antibodies. We hypothesize that a strong T cell response induced by YF17D vaccination will reduce JE-YF17D RNAemia upon challenge, as compared to JE-YF17D vaccination followed by YF17D challenge. The expected gradient of YF17D-specific T cell abundance and functionality would also allow us to gain insight into a T cell threshold for controlling acute viral infections. The knowledge gleaned from this study could guide the assessment of cellular immunity and vaccine development., Clinical Trial Registration: Clinicaltrials.gov, NCT05568953., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Kalimuddin, Chan, Sessions, Chan, Ong, Low, Bertoletti and Ooi.)

Background: Mass vaccination has dramatically reduced the incidence of severe COVID-19, with most cases now presenting as self-limiting upper respiratory tract infections. However, those with co-morbidities, the elderly and immunocompromised, as well as the unvaccinated, remain disproportionately vulnerable to severe COVID-19 and its sequelae. Furthermore, as the effectiveness of vaccination wanes with time, immune escape SARS-CoV-2 variants could emerge to cause severe COVID-19. Reliable prognostic biomarkers for severe disease could be used as early indicator of re-emergence of severe COVID-19 as well as for triaging of patients for antiviral therapy., Methods: We performed a systematic review and re-analysis of 7 publicly available datasets, analysing a total of 140 severe and 181 mild COVID-19 patients, to determine the most consistent differentially regulated genes in peripheral blood of severe COVID-19 patients. In addition, we included an independent cohort where blood transcriptomics of COVID-19 patients were prospectively and longitudinally monitored previously, to track the time in which these gene expression changes occur before nadir of respiratory function. Single cell RNA-sequencing of peripheral blood mononuclear cells from publicly available datasets was then used to determine the immune cell subsets involved., Findings: The most consistent differentially regulated genes in peripheral blood of severe COVID-19 patients were MCEMP1, HLA-DRA and ETS1 across the 7 transcriptomics datasets. Moreover, we found significantly heightened MCEMP1 and reduced HLA-DRA expression as early as four days before the nadir of respiratory function, and the differential expression of MCEMP1 and HLA-DRA occurred predominantly in CD14+ cells. The online platform which we developed is publicly available at https://kuanrongchan-covid19-severity-app-t7l38g.streamlitapp.com/, for users to query gene expression differences between severe and mild COVID-19 patients in these datasets., Interpretation: Elevated MCEMP1 and reduced HLA-DRA gene expression in CD14+ cells during the early phase of disease are prognostic of severe COVID-19., Funding: K.R.C is funded by the National Medical Research Council (NMRC) of Singapore under the Open Fund Individual Research Grant (MOH-000610). E.E.O. is funded by the NMRC Senior Clinician-Scientist Award (MOH-000135-00). J.G.H.L. is funded by the NMRC under the Clinician-Scientist Award (NMRC/CSAINV/013/2016-01). S.K. is funded by the NMRC under the Transition Award. This study was sponsored in part by a generous gift from The Hour Glass., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

The four dengue viruses (DENVs) have evolved multiple mechanisms to ensure its survival. Among these mechanisms is the ability to regulate its replication rate, which may contribute to avoiding premature immune activation that limit infection dissemination: DENVs associated with dengue epidemics have shown slower replication rate than pre-epidemic strains. Correspondingly, wild-type DENVs replicate more slowly than their clinically attenuated derivatives. To understand how DENVs 'make haste slowly', we generated and screened for DENV2 mutants with accelerated replication that also induced high type-I interferon (IFN) expression in infected cells. We chanced upon a single NS2B-I114T amino acid substitution, in an otherwise highly conserved amino acid residue. Accelerated DENV2 replication damaged host DNA as mutant infection was dependent on host DNA damage repair factors, namely RAD21, EID3 and NEK5. DNA damage induced cGAS/STING signalling and activated early type-I IFN response that inhibited infection dissemination. Unexpectedly, STING activation also supported mutant DENV replication in infected cells through STING-induced autophagy. Our findings thus show that DENV NS2B has multi-faceted role in controlling DENV replication rate and immune evasion and suggest that the dual role of STING in supporting virus replication within infected cells but inhibiting infection dissemination could be particularly advantageous for live attenuated vaccine development.

Remarkable potency has been demonstrated for mRNA vaccines in reducing the global burden of the ongoing COVID-19 pandemic. An alternative form of the mRNA vaccine is the self-amplifying mRNA (sa-mRNA) vaccine, which encodes an alphavirus replicase that self-amplifies the full-length mRNA and SARS-CoV-2 spike (S) transgene. However, early-phase clinical trials of sa-mRNA COVID-19 vaccine candidates have questioned the potential of this platform to develop potent vaccines. We examined the immune gene response to a candidate sa-mRNA vaccine against COVID-19, ARCT-021, and compared our findings to the host response to other forms of vaccines. In blood samples from healthy volunteers that participated in a phase I/II clinical trial, greater induction of transcripts involved in Toll-like receptor (TLR) signalling, antigen presentation and complement activation at 1 day post-vaccination was associated with higher anti-S antibody titers. Conversely, transcripts involved in T-cell maturation at day 7 post-vaccination informed the magnitude of eventual S-specific T-cell responses. The transcriptomic signature for ARCT-021 vaccination strongly correlated with live viral vector vaccines, adjuvanted vaccines and BNT162b2 1 day post-vaccination. Moreover, the ARCT-021 signature correlated with day 7 YF17D live-attenuated vaccine transcriptomic responses. Altogether, our findings show that sa-mRNA vaccination induces innate immune responses that are associated with the development of adaptive immunity from other forms of vaccines, supporting further development of this vaccine platform for clinical application., (© 2022. The Author(s).)

The Flavivirus genus is made up of viruses that are either mosquito-borne or tick-borne and other viruses transmitted by unknown vectors. Flaviviruses present a significant threat to global health and infect up to 400 million of people annually. As the climate continues to change throughout the world, these viruses have become prominent infections, with increasing number of infections being detected beyond tropical borders. These include dengue virus (DENV), West Nile virus (WNV), Japanese encephalitis virus (JEV), and Zika virus (ZIKV). Several highly conserved epitopes of flaviviruses had been identified and reported to interact with antibodies, which lead to cross-reactivity results. The major interest of this review paper is mainly focused on the serological cross-reactivity between DENV serotypes, ZIKV, WNV, and JEV. Direct and molecular techniques are required in the diagnosis of Flavivirus -associated human disease. In this review, the serological assays such as neutralization tests, enzyme-linked immunosorbent assay, hemagglutination-inhibition test, Western blot test, and immunofluorescence test will be discussed. Serological assays that have been developed are able to detect different immunoglobulin isotypes (IgM, IgG, and IgA); however, it is challenging when interpreting the serological results due to the broad antigenic cross-reactivity of antibodies to these viruses. However, the neutralization tests are still considered as the gold standard to differentiate these flaviviruses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Chan, Ismail, Thergarajan, Raju, Yam, Rishya and Sekaran.)

Transmembrane Protein 41B (TMEM41B) and Vacuole Membrane Protein 1 (VMP1) are two ER-associated lipid scramblases that play a role in autophagosome formation and cellular lipid metabolism. TMEM41B is also a recently validated host factor required by flaviviruses and coronaviruses. However, the exact underlying mechanism of TMEM41B in promoting viral infections remains an open question. Here, we validated that both TMEM41B and VMP1 are essential host dependency factors for all four serotypes of dengue virus (DENV) and human coronavirus OC43 (HCoV-OC43), but not chikungunya virus (CHIKV). While HCoV-OC43 failed to replicate entirely in both TMEM41B- and VMP1-deficient cells, we detected diminished levels of DENV infections in these cell lines, which were accompanied by upregulation of the innate immune dsRNA sensors, RIG-I and MDA5. Nonetheless, this upregulation did not correspondingly induce the downstream effector TBK1 activation and Interferon-beta expression. Despite low levels of DENV replication, classical DENV replication organelles were undetectable in the infected TMEM41B-deficient cells, suggesting that the upregulation of the dsRNA sensors is likely a consequence of aberrant viral replication rather than a causal factor for reduced DENV infection. Intriguingly, we uncovered that the inhibitory effect of TMEM41B deficiency on DENV replication, but not HCoV-OC43, can be partially reversed using exogenous fatty acid supplements. In contrast, VMP1 deficiency cannot be rescued using the metabolite treatment. In line with the observed phenotypes, we found that both TMEM41B- and VMP1-deficient cells harbor higher levels of compromised mitochondria, especially in VMP1 deficiency which results in severe dysregulations of mitochondrial beta-oxidation. Using a metabolomic profiling approach, we revealed distinctive global dysregulations of the cellular metabolome, particularly lipidome, in TMEM41B- and VMP1-deficient cells. Our findings highlight a central role for TMEM41B and VMP1 in modulating multiple cellular pathways, including lipid mobilization, mitochondrial beta-oxidation, and global metabolic regulations, to facilitate the replication of flaviviruses and coronaviruses., Competing Interests: The authors have declared that no competing interests exist.

Opening and processing gene expression data files in Excel runs into the inadvertent risk of converting gene names to dates. As pathway analysis tools rely on gene symbols to query against pathway databases, the genes that are converted to dates will not be recognized, potentially causing voids in pathway analysis. Molecular pathways related to cell division, exocytosis, cilium assembly, protein ubiquitination and nitric oxide biosynthesis were found to be most affected by Excel auto-conversion. A plausible solution is hence to update these genes and dates to the newly approved gene names as recommended by the HUGO Gene Nomenclature Committee (HGNC), which are resilient to Excel auto-conversion. Herein, we developed a web tool with Streamlit that can convert old gene names and dates back into the new gene names recommended by HGNC. The web app is named Gene Updater, which is open source and can be either hosted locally or at https://share.streamlit.io/kuanrongchan/date-to-gene-converter/main/date_gene_tool.py . Additionally, as Mar-01 and Mar-02 can each be potentially mapped to 2 different gene names, users can assign the date terms to the appropriate gene names within the Gene Updater web tool. This user-friendly web tool ensures that the accuracy and integrity of gene expression data is preserved by minimizing errors in labelling gene names due to Excel auto-conversions., (© 2022. The Author(s).)

Ensuring high vaccination and even booster vaccination coverage is critical in preventing severe Coronavirus Disease 2019 (COVID-19). Among the various COVID-19 vaccines currently in use, the mRNA vaccines have shown remarkable effectiveness. However, systemic adverse events (AEs), such as postvaccination fatigue, are prevalent following mRNA vaccination, and the underpinnings of which are not understood. Herein, we found that higher baseline expression of genes related to T and NK cell exhaustion and suppression were positively correlated with the development of moderately severe fatigue after Pfizer-BioNTech BNT162b2 vaccination; increased expression of genes associated with T and NK cell exhaustion and suppression reacted to vaccination were associated with greater levels of innate immune activation at 1 day postvaccination. We further found, in a mouse model, that altering the route of vaccination from intramuscular (i.m.) to subcutaneous (s.c.) could lessen the pro-inflammatory response and correspondingly the extent of systemic AEs; the humoral immune response to BNT162b2 vaccination was not compromised. Instead, it is possible that the s.c. route could improve cytotoxic CD8 T-cell responses to BNT162b2 vaccination. Our findings thus provide a glimpse of the molecular basis of postvaccination fatigue from mRNA vaccination and suggest a readily translatable solution to minimize systemic AEs., Competing Interests: The authors have declared no competing interests exists.

Aberrant cellular bioenergetics has detrimental consequences in host cells. For instance, pathogenic Zika virus strains can suppress mitochondria respiration and glycolytic functions, disrupting cellular bioenergetics that leads to apoptosis. Herein, we describe methods for flavivirus propagation, titering and infection, cell preparation, and procedures for mitochondrial and glycolytic stress tests. The protocol enables assessment of cellular respiration and glycolytic flux in flavivirus-infected cells. For complete details on the use and execution of this protocol, please refer to Yau et al. (2021)., Competing Interests: E.E.O. has an issued patent titled “Rapid method of generating live attenuated vaccines” (Singapore patent publication number: 10201602980W). This patent covers the Zika virus strain DN-1 used in Figure 5. The authors declare no other competing interests., (© 2022 The Author(s).)

Zika virus (ZIKV) is an Aedes-mosquito-borne flavivirus that causes debilitating congenital and developmental disorders. Improved understanding of ZIKV pathogenesis could assist efforts to fill the therapeutic and vaccine gap. We use several ZIKV strains, including a pair differing by a single phenylalanine-to-leucine substitution (M-F37L) in the membrane (M) protein, coupled with unbiased genomics to demarcate the border between attenuated and pathogenic infection. We identify infection-induced metabolic dysregulation as a minimal set of host alterations that differentiates attenuated from pathogenic ZIKV strains. Glycolytic rewiring results in impaired oxidative phosphorylation and mitochondrial dysfunction that trigger inflammation and apoptosis in pathogenic but not attenuated ZIKV strains. Critically, pyruvate supplementation prevents cell death, in vitro, and rescues fetal development in ZIKV-infected dams. Our findings thus demonstrate dysregulated metabolism as an underpinning of ZIKV pathogenicity and raise the potential of pyruvate supplementation in expectant women as a prophylaxis against congenital Zika syndrome., Competing Interests: Declaration of interests S.S.K. and E.E.O. have an issued patent titled “Rapid method of generating live attenuated vaccines” (Singapore patent publication number: 10201602980W). This patent covers the Zika virus strain DN-1 used in this study. The authors declare no other competing interests for this study., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Porcine deltacoronavirus (PDCoV) is a highly transmissible intestinal pathogen that causes mild to severe clinical symptoms, such as anorexia, vomiting, and watery diarrhea in pigs. By comparing the genetic sequences of the spike glycoprotein between historical and current Taiwanese PDCoV strains, we identified a novel PDCoV variant that displaced the PDCoV responsible for the 2015 epidemic. This PDCoV variant belongs to a young population within the US lineage, and infected pigs carry high concentrations of the virus. It also has several critical point mutations and an amino acid insertion at position 52 that may enhance the affinity between the B-cell epitopes located in the N-terminal domain with its complementarity regions, consequently facilitating binding or penetration between the fusion peptide and cellular membrane. Furthermore, viral protein structure prediction demonstrated that these amino acid changes may change the ability of the virus to bind to the receptor, which may consequently alter virus infectivity. Our results hence suggest the emergence of new PDCoV strains in Taiwan with the potential for greater transmission and pathogenesis., (© The Author(s) 2021. Published by Oxford University Press.)

The ongoing coronavirus disease 2019 (COVID-19) crisis caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered a large-scale pandemic that is afflicting millions of individuals in over 200 countries. The clinical spectrum caused by SARS-CoV-2 infections can range from asymptomatic infection to mild undifferentiated febrile illness to severe respiratory disease with multiple complications. Elderly patients (aged 60 and above) with comorbidities such as cardiovascular diseases and diabetes mellitus appear to be at highest risk of a severe disease outcome. To protect against pulmonary immunopathology caused by SARS-CoV-2 infection, the host primarily depends on two distinct defense strategies: resistance and disease tolerance. Resistance is the ability of the host to suppress and eliminate incoming viruses. By contrast, disease tolerance refers to host responses that promote host health regardless of their impact on viral replication. Disruption of either resistance or disease tolerance mechanisms or both could underpin predisposition to elevated risk of severe disease during viral infection. Aging can disrupt host resistance and disease tolerance by compromising immune functions, weakening of the unfolded protein response, progressive mitochondrial dysfunction, and altering metabolic processes. A comprehensive understanding of the molecular mechanisms underlying declining host defense in elderly individuals could thus pave the way to provide new opportunities and approaches for the treatment of severe COVID-19., (© 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel coronavirus in humans, has expanded globally over the past year. COVID-19 remains an important subject of intensive research owing to its huge impact on economic and public health globally. Based on historical archives, the first coronavirus-related disease recorded was possibly animal-related, a case of feline infectious peritonitis described as early as 1912. Despite over a century of documented coronaviruses in animals, the global animal industry still suffers from outbreaks. Knowledge and experience handling animal coronaviruses provide a valuable tool to complement our understanding of the ongoing COVID-19 pandemic. In this review, we present an overview of coronaviruses, clinical signs, COVID-19 in animals, genome organization and recombination, immunopathogenesis, transmission, viral shedding, diagnosis, treatment, and prevention. By drawing parallels between COVID-19 in animals and humans, we provide perspectives on the pathophysiological mechanisms by which coronaviruses cause diseases in both animals and humans, providing a critical basis for the development of effective vaccines and therapeutics against these deadly viruses.

Background: The coronavirus disease-19 (COVID-19) pandemic has cost lives and economic hardships globally. Various studies have found a number of different factors, such as hyperinflammation and exhausted/suppressed T cell responses to the etiological SARS coronavirus-2 (SARS-CoV-2), being associated with severe COVID-19. However, sieving the causative from associative factors of respiratory dysfunction has remained rudimentary., Methods: We postulated that the host responses causative of respiratory dysfunction would track most closely with disease progression and resolution and thus be differentiated from other factors that are statistically associated with but not causative of severe COVID-19. To track the temporal dynamics of the host responses involved, we examined the changes in gene expression in whole blood of 6 severe and 4 non-severe COVID-19 patients across 15 different timepoints spanning the nadir of respiratory function., Findings: We found that neutrophil activation but not type I interferon signaling transcripts tracked most closely with disease progression and resolution. Moreover, transcripts encoding for protein phosphorylation, particularly the serine-threonine kinases, many of which have known T cell proliferation and activation functions, were increased after and may thus contribute to the upswing of respiratory function. Notably, these associative genes were targeted by dexamethasone, but not methylprednisolone, which is consistent with efficacy outcomes in clinical trials., Interpretation: Our findings suggest neutrophil activation as a critical factor of respiratory dysfunction in COVID-19. Drugs that target this pathway could be potentially repurposed for the treatment of severe COVID-19., Funding: This study was sponsored in part by a generous gift from The Hour Glass. EEO and JGL are funded by the National Medical Research Council of Singapore, through the Clinician Scientist Awards awarded by the National Research Foundation of Singapore., Competing Interests: Declaration of Competing Interests Dr Eng Eong Ooi reports grants from National Medical Research Council, Singapore, during the conduct of the study; personal fees from Tychan Private Limited, outside the submitted work; the other authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: The emergence of Zika virus (ZIKV) as an important cause of congenital and childhood developmental disorders presents another challenge to global health. Efforts to develop a Zika vaccine have begun although vaccine development against flaviviruses, of which ZIKV belongs to, has proven to be time-consuming and challenging. Defining the vaccine attributes that elicit adaptive immune response necessary for preventing ZIKV infection could provide an evidence-based guide to Zika vaccine development., Methods: We used a previously described attenuated ZIKV DN-2 strain in a type-I interferon receptor deficient mouse model and tested the hypothesis that duration of vaccine burden rather than peak level of infection, is a determinant of immunogenicity. We quantified both humoral and cellular responses against ZIKV using plaque reduction neutralisation test and flow cytometry with ELISPOT assays, respectively. Vaccinated mice were challenged with wild-type ZIKV (H/PF/2013 strain) to determine the level of protection against infection., Findings: We found that the overall vaccine burden is directly correlated with neutralising antibody titres. Reduced duration of vaccine burden lowered neutralising antibody titres that resulted in subclinical infection, despite unchanged peak vaccine viraemia levels. We also found that sterilising immunity is dependant on both neutralising antibody and CD8 + T cell responses; depletion of CD8 + T cells in vaccinated animals led to wild-type ZIKV infection, especially in the male reproductive tract., Interpretation: Our findings indicate that duration of attenuated virus vaccine burden is a determinant of humoral and cellular immunity and also suggest that vaccines that elicit both arms of the adaptive immune response are needed to fully prevent ZIKV transmission., Funding: This study was supported by the National Medical Research Council through the Clinician-Scientist Award (Senior Investigator) to E.E.O. Salary support for S.W. was from a Competitive Research Programme grant awarded by the National Research Foundation of Singapore., Competing Interests: Declaration of Competing Interest E.E.O and S.S.K. have an issued patent titled “Rapid method of generating live attenuated vaccines” (Singapore patent publication number: 10201602980W), which includes the Zika virus strain DN-2 used in this study. The authors declare no other competing interests., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Dengue virus (DENV) is a medically important flavivirus and the aetiological agent of Dengue, a normally self-resolving febrile illness that, in some individuals, can progress into Severe Dengue (SD), a life-threatening disorder that manifests as organ impairment, bleeding and shock. Many different risk factors have been associated with the development of SD, one of which is obesity. In many countries where DENV is endemic, obesity is becoming more prevalent, therefore SD is becoming an increased public health concern. However, there is a paucity of research on the mechanistic links between obesity and SD. This is a narrative review based on original research and reviews sourced from PubMed and Google Scholar. Four key areas could possibly explain how obesity can promote viral pathogenesis. Firstly, obesity downregulates AMP-Protein Kinase (AMPK), which leads to an accumulation of lipids in the endoplasmic reticulum (ER) that facilitates viral replication. Secondly, the long-term production of pro-inflammatory adipokines found in obese individuals can cause endothelial and platelet dysfunction and can facilitate SD. Thirdly, obesity could also cause endothelial dysfunction in addition to chronic inflammation, through the production of reactive oxygen species (ROS) and possible damage to the glycocalyx found in the endothelium. Finally, obesity has several effects on immunomodulation that reduces NK cell function, B and T cell response and increased pre-disposition to stronger pro-inflammatory cytokine responses after viral infection. Together, these effects can lead to greater viral proliferation and greater tissue damage both of which could contribute to SD. The four mechanisms outlined in this review can be taken as reference starting points for investigating the link between obesity and SD, and to discover potential therapeutic strategies that can potentially reduce disease severity., Competing Interests: Declaration of Competing Interest Authors declare no conflict of interest., (Copyright © 2020 The British Infection Association. Published by Elsevier Ltd. All rights reserved.)

The molecular basis of dengue virus (DENV) attenuation remains ambiguous and hampers a targeted approach to derive safe but nonetheless immunogenic live vaccine candidates. Here, we take advantage of DENV serotype 2 PDK53 vaccine strain, which recently and successfully completed a phase-3 clinical trial, to identify how this virus is attenuated compared to its wild-type parent, DENV2 16681. Site-directed mutagenesis on a 16681 infectious clone identifies a single G53D substitution in the non-structural 1 (NS1) protein that reduces 16681 infection and dissemination in both Aedes aegypti, as well as in mammalian cells to produce the characteristic phenotypes of PDK53. Mechanistically, NS1 G53D impairs the function of a known host factor, the endoplasmic reticulum (ER)-resident ribophorin 1 protein, to properly glycosylate NS1 and thus induce a host antiviral gene through ER stress responses. Our findings provide molecular insights on DENV attenuation on a clinically tested strain., Competing Interests: Declaration of Interests E.E.O. served as a dengue vaccine advisory board member for Takeda Vaccines, which uses DENV2 PDK53 strain as a component of their dengue vaccine candidate, TAK-003., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Live-attenuated vaccines (LAVs) have achieved remarkable successes in controlling virus spread, as well as for other applications such as cancer immunotherapy. However, with rapid increases in international travel, globalization, geographic spread of viral vectors, and widespread use of vaccines, there is an increasing need to consider how pre-exposure to viruses which share similar antigenic regions can impact vaccine efficacy. Pre-existing antibodies, derived from either from maternal-fetal transmission, or by previous infection or vaccination, have been demonstrated to interfere with vaccine immunogenicity of measles, adenovirus, and influenza LAVs. Immune interference of LAVs can be caused by the formation of virus-antibody complexes that neutralize virus infection in antigen-presenting cells, or by the cross-linking of the B-cell receptor with the inhibitory receptor, FcgRIIB. On the other hand, pre-existing antibodies can augment flaviviral LAV efficacy such as that of dengue and yellow fever virus, especially when pre-existing antibodies are present at sub-neutralizing levels. The increased vaccine immunogenicity can be facilitated by antibody-dependent enhancement of virus infection, enhancing virus uptake in antigen-presenting cells, and robust induction of innate immune responses that promote vaccine immunogenicity. This review examines the literature on this topic and examines the circumstances where pre-existing antibodies can inhibit or enhance LAV efficacy. A better knowledge of the underlying mechanisms involved could allow us to better manage immunization in seropositive individuals and even identify possibilities that could allow us to exploit pre-existing antibodies to boost vaccine-induced responses for improved vaccine efficacy.

Dengue is caused by infection with any one of four dengue viruses (DENV); the risk of severe disease appears to be enhanced by the cross-reactive or subneutralizing levels of antibody from a prior DENV infection. These antibodies opsonize DENV entry through the activating Fc gamma receptors (FcγR), instead of infection through canonical receptor-mediated endocytosis, to result in higher levels of DENV replication. However, whether the enhanced replication is solely due to more efficient FcγR-mediated DENV entry or is also through FcγR-mediated alteration of the host transcriptome response to favor DENV infection remains unclear. Indeed, more efficient viral entry through activation of the FcγR can result in an increased viral antigenic load within target cells and confound direct comparisons of the host transcriptome response under antibody-dependent and antibody-independent conditions. Herein, we show that, despite controlling for the viral antigenic load in primary monocytes, the antibody-dependent and non-antibody-dependent routes of DENV entry induce transcriptome responses that are remarkably different. Notably, antibody-dependent DENV entry upregulated DENV host dependency factors associated with RNA splicing, mitochondrial respiratory chain complexes, and vesicle trafficking. Additionally, supporting findings from other studies, antibody-dependent DENV entry impeded the downregulation of ribosomal genes caused by canonical receptor-mediated endocytosis to increase viral translation. Collectively, our findings support the notion that antibody-dependent DENV entry alters host responses that support the viral life cycle and that host responses to DENV need to be defined in the context of its entry pathway. IMPORTANCE Dengue virus is the most prevalent mosquito-borne viral infection globally, resulting in variable manifestations ranging from asymptomatic viremia to life-threatening shock and multiorgan failure. Previous studies have indicated that the risk of severe dengue in humans can be increased by a specific range of preexisting anti-dengue virus antibody titers, a phenomenon termed antibody-dependent enhancement. There is hence a need to understand how antibodies augment dengue virus infection compared to the alternative canonical receptor-mediated viral entry route. Herein, we show that, besides facilitating viral uptake, antibody-mediated entry increases the expression of early host dependency factors to promote viral infection; these factors include RNA splicing, mitochondrial respiratory chain complexes, vesicle trafficking, and ribosomal genes. These findings will enhance our understanding of how differences in entry pathways can affect host responses and offer opportunities to design therapeutics that can specifically inhibit antibody-dependent enhancement of dengue virus infection., (Copyright © 2019 Chan et al.)

Flaviviral infections result in a wide spectrum of clinical outcomes, ranging from asymptomatic infection to severe disease. Although the correlates of severe disease have been explored 1-4 , the pathophysiology that differentiates symptomatic from asymptomatic infection remains undefined. To understand the molecular underpinnings of symptomatic infection, the blood transcriptomic and metabolomic profiles of individuals were examined before and after inoculation with the live yellow fever viral vaccine (YF17D). It was found that individuals with adaptive endoplasmic reticulum (ER) stress and reduced tricarboxylic acid cycle activity at baseline showed increased susceptibility to symptomatic outcome. YF17D infection in these individuals induced maladaptive ER stress, triggering downstream proinflammatory responses that correlated with symptomatic outcome. The findings of the present study thus suggest that the ER stress response and immunometabolism underpin symptomatic yellow fever and possibly even other flaviviral infections. Modulating either ER stress or metabolism could be exploited for prophylaxis against symptomatic flaviviral infection outcome.

Neutralizing antibodies (nAbs) are a critical component for protection against dengue virus (DENV) infection, but little is known about the immune mechanisms governing their induction and whether such mechanisms can be harnessed for vaccine development. In this study, we profiled the early immune responses to flaviviruses in human peripheral blood mononuclear cells and screened a panel of toll-like receptor (TLR) agonists that stimulate the same immune signatures. Monocyte/macrophage-driven inflammatory responses and interferon responses were characteristics of flavivirus infection and associated with induction of nAbs in humans immunized with the yellow fever vaccine YF-17D. The signatures were best reproduced by the combination of TLR agonists Pam 3 CSK 4 and PolyI:C (PP). Immunization of both mice and macaques with a poorly immunogenic recombinant DENV-2 envelope domain III (EDIII) induced more consistent nAb and CD4 + T-cell responses with PP compared to alum plus monophosphoryl lipid A. Induction of nAbs by PP required interferon-mediated signals in macrophages in mice. However, EDIII + PP vaccination only provided partial protection against viral challenge. These results provide insights into mechanisms underlying nAb induction and a basis for further improving antigen/adjuvant combinations for dengue vaccine development., Competing Interests: Competing interestsThe authors declare no competing interests.

Following the recent emergence of Zika virus (ZIKV), many murine and human neutralizing anti-ZIKV antibodies have been reported. Given the risk of virus escape mutants, engineering antibodies that target mutationally constrained epitopes with therapeutically relevant potencies can be valuable for combating future outbreaks. Here, we applied computational methods to engineer an antibody, ZAb_FLEP, that targets a highly networked and therefore mutationally constrained surface formed by the envelope protein dimer. ZAb_FLEP neutralized a breadth of ZIKV strains and protected mice in distinct in vivo models, including resolving vertical transmission and fetal mortality in infected pregnant mice. Serial passaging of ZIKV in the presence of ZAb_FLEP failed to generate viral escape mutants, suggesting that its epitope is indeed mutationally constrained. A single-particle cryo-EM reconstruction of the Fab-ZIKV complex validated the structural model and revealed insights into ZAb_FLEP's neutralization mechanism. ZAb_FLEP has potential as a therapeutic in future outbreaks., (Copyright © 2018. Published by Elsevier Inc.)

Zika virus (ZIKV) is a flavivirus that can cause congenital disease and requires development of an effective long-term preventative strategy. A replicative ZIKV vaccine with properties similar to the yellow fever 17D (YF17D) live-attenuated vaccine (LAV) would be advantageous, as a single dose of YF17D produces lifelong immunity. However, a replicative ZIKV vaccine must also be safe from causing persistent organ infections. Here we report an approach to ZIKV LAV development. We identify a ZIKV variant that produces small plaques due to interferon (IFN)-restricted viral propagation and displays attenuated infection of endothelial cells. We show that these properties collectively reduce the risk of organ infections and vertical transmission in a mouse model but remain sufficiently immunogenic to prevent wild-type ZIKV infection. Our findings suggest a strategy for the development of a safe but efficacious ZIKV LAV.

The innate immune response shapes the development of adaptive immunity following infections and vaccination. However, it can also induce symptoms such as fever and myalgia, leading to the possibility that the molecular basis of immunogenicity and reactogenicity of vaccination are inseparably linked. To test this possibility, we used the yellow fever live-attenuated vaccine (YFLAV) as a model to study the molecular correlates of reactogenicity or adverse events (AEs). We analyzed the outcome of 68 adults who completed a YFLAV clinical trial, of which 43 (63.2%) reported systemic AEs. Through whole-genome profiling of blood collected before and after YFLAV dosing, we observed that activation of innate immune genes at day 1, but not day 3 after vaccination, was directly correlated with AEs. These findings contrast with the gene expression profile at day 3 that we and others have previously shown to be correlated with immunogenicity. We conclude that although the innate immune response is a double-edged sword, its expression that induces AEs is temporally distinct from that which engenders robust immunity. The use of genomic profiling thus provides molecular insights into the biology of AEs that potentially forms a basis for the development of safer vaccines.

Dengue virus (DENV) has been found to replicate in lymphoid organs such as the lymph nodes, spleen, and liver in post-mortem analysis. These organs are known to have low oxygen levels (~0.5-4.5% O 2 ) due to the vascular anatomy. However, how physiologically low levels of oxygen affect DENV infection via hypoxia-induced changes in the immune response remains unknown. Here, we show that monocytes adapted to 3% O 2 show greater susceptibility to antibody-dependent enhancement of DENV infection. Low oxygen level induces HIF1α-dependent upregulation of fragment crystallizable gamma receptor IIA (FcγRIIA) as well as HIF1α-independent alterations in membrane ether lipid concentrations. The increased FcγRIIA expression operates synergistically with altered membrane composition, possibly through increase membrane fluidity, to increase uptake of DENV immune complexes for enhanced infection. Our findings thus indicate that the increased viral burden associated with secondary DENV infection is antibody-dependent but hypoxia-induced and suggest a role for targeting hypoxia-induced factors for anti-dengue therapy., (© 2017 The Authors. Published under the terms of the CC BY 4.0 license.)

Introduction: Dengue is a prevalent disease in tropical and subtropical countries with an estimated 400 million people infected annually. While significant advancement has been made in the chase for an effective dengue vaccine, the recently licensed Sanofi vaccine was, in contrast to in vitro data, only partially protective. Areas covered: This suggests that our understanding of the serological correlates for dengue is currently inadequate. With growing evidence supporting the role of fragment crystalizable gamma receptors (FcγRs) in antibody-mediated neutralization or antibody-dependent enhancement (ADE) of dengue virus (DENV) infection, FcγR-expressing cells have been increasingly used for measuring neutralizing antibody responses elicited by dengue vaccines. Here, we review the mechanisms of how FcγRs modulates both DENV neutralization and enhanced infections via its interactions with antibodies. Expert commentary: This review provides insights on the importance of factoring FcγRs for in vitro neutralization assays. Bridging the gap between in vitro and clinical observations would allow researchers to more accurately predict in vivo vaccine efficacy.

Secondary infection with a heterologous dengue virus (DENV) serotype increases the risk of severe dengue, through a process termed antibody-dependent enhancement (ADE). During ADE, DENV is opsonized with non- or sub-neutralizing antibody levels that augment entry into monocytes and dendritic cells through Fc-gamma receptors (FcγRs). We previously reported that co-ligation of leukocyte immunoglobulin-like receptor-B1 (LILRB1) by antibody-opsonized DENV led to recruitment of SH2 domain-containing phosphatase-1 (SHP-1) to dephosphorylate spleen tyrosine kinase (Syk) and reduce interferon stimulated gene induction. Here, we show that LILRB1 also signals through SHP-1 to attenuate the otherwise rapid acidification for lysosomal enzyme activation following FcγR-mediated uptake of DENV. Reduced or slower trafficking of antibody-opsonized DENV to lytic phagolysosomal compartments, demonstrates how co-ligation of LILRB1 also permits DENV to overcome a cell-autonomous immune response, enhancing intracellular survival of DENV. Our findings provide insights on how antiviral drugs that modify phagosome acidification should be used for viruses such as DENV.

Vaccination has achieved remarkable successes in the control of childhood viral diseases. To control emerging infections, however, vaccines will need to be delivered to older individuals who, unlike infants, probably have had prior infection or vaccination with related viruses and thus have cross-reactive antibodies against the vaccines. Whether and how these cross-reactive antibodies impact live attenuated vaccination efficacy is unclear. Using an open-label randomized trial design, we show that subjects with a specific range of cross-reactive antibody titres from a prior inactivated Japanese encephalitis vaccination enhanced yellow fever (YF) immunogenicity upon YF vaccination. Enhancing titres of cross-reactive antibodies prolonged YF vaccine viraemia, provoked greater pro-inflammatory responses, and induced adhesion molecules intrinsic to the activating Fc-receptor signalling pathway, namely immune semaphorins, facilitating immune cell interactions and trafficking. Our findings clinically demonstrate antibody-enhanced infection and suggest that vaccine efficacy could be improved by exploiting cross-reactive antibodies.

Competing Interests: The authors have declared that no competing interests exist.

Dengue is the most common vector-borne viral disease, causing nearly 400 million infections yearly. Currently there are no approved therapies. Antibody epitopes that elicit weak humoral responses may not be accessible by conventional B cell panning methods. To demonstrate an alternative strategy to generating a therapeutic antibody, we employed a non-immunodominant, but functionally relevant, epitope in domain III of the E protein, and engineered by structure-guided methods an antibody directed to it. The resulting antibody, Ab513, exhibits high-affinity binding to, and broadly neutralizes, multiple genotypes within all four serotypes. To assess therapeutic relevance of Ab513, activity against important human clinical features of dengue was investigated. Ab513 mitigates thrombocytopenia in a humanized mouse model, resolves vascular leakage, reduces viremia to nearly undetectable levels, and protects mice in a maternal transfer model of lethal antibody-mediated enhancement. The results demonstrate that Ab513 may reduce the public health burden from dengue., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Dengue virus (DENV) infection is a major emerging disease widely distributed throughout the tropical and subtropical regions of the world affecting several millions of people. Despite constants efforts, no specific treatment or effective vaccine is yet available. Here we show a novel design of a DNA immunisation strategy that resulted in the induction of strong antibody responses with high neutralisation titres in mice against all four viral serotypes. The immunogenic molecule is an engineered version of the domain III (DIII) of the virus E protein fused to the dimerising CH3 domain of the IgG immunoglobulin H chain. The DIII sequences were also codon-optimised for expression in mammalian cells. While DIII alone is very poorly secreted, the codon-optimised fusion protein is rightly expressed, folded and secreted at high levels, thus inducing strong antibody responses. Mice were immunised using gene-gun technology, an efficient way of intradermal delivery of the plasmid DNA, and the vaccine was able to induce neutralising titres against all serotypes. Additionally, all sera showed reactivity to a recombinant DIII version and the recombinant E protein produced and secreted from mammalian cells in a mono-biotinylated form when tested in a conformational ELISA. Sera were also highly reactive to infective viral particles in a virus-capture ELISA and specific for each serotype as revealed by the low cross-reactive and cross-neutralising activities. The serotype specific sera did not induce antibody dependent enhancement of infection (ADE) in non-homologous virus serotypes. A tetravalent immunisation protocol in mice showed induction of neutralising antibodies against all four dengue serotypes as well.

The lack of vaccines against several important viral diseases necessitates the development of therapeutics to save lives and control epidemics. In recent years, therapeutic antibodies have received considerable attention due to their good safety profiles and clinical success when used against viruses such as respiratory syncytial virus, Ebola virus and Hendra virus. The binding affinity of these antibodies can directly impact their therapeutic efficacy. However, we and others have also demonstrated that the subtype of Fc-gamma receptors (FcγRs) engaged influences the stoichiometric requirement for virus neutralization. Hence, the development of therapeutic antibodies against infectious diseases should consider the FcγRs engaged and Fc-effector functions involved. This review highlights the current state of knowledge about FcγRs and FcγR effector functions involved in virus neutralization, with emphasis on factors that can affect FcγR engagement. A better understanding of Fc-FcγR interactions during virus neutralization will allow development of therapeutic antibodies that are efficacious and can be administered with minimal side effects.

Viruses must evade the host innate defenses for replication and dengue is no exception. During secondary infection with a heterologous dengue virus (DENV) serotype, DENV is opsonized with sub- or nonneutralizing antibodies that enhance infection of monocytes, macrophages, and dendritic cells via the Fc-gamma receptor (FcγR), a process termed antibody-dependent enhancement of DENV infection. However, this enhancement of DENV infection is curious as cross-linking of activating FcγRs signals an early antiviral response by inducing the type-I IFN-stimulated genes (ISGs). Entry through activating FcγR would thus place DENV in an intracellular environment unfavorable for enhanced replication. Here we demonstrate that, to escape this antiviral response, antibody-opsonized DENV coligates leukocyte Ig-like receptor-B1 (LILRB1) to inhibit FcγR signaling for ISG expression. This immunoreceptor tyrosine-based inhibition motif-bearing receptor recruits Src homology phosphatase-1 to dephosphorylate spleen tyrosine kinase (Syk). As Syk is a key intermediate of FcγR signaling, LILRB1 coligation resulted in reduced ISG expression for enhanced DENV replication. Our findings suggest a unique mechanism for DENV to evade an early antiviral response for enhanced infection.

The early events of the dengue virus life cycle involve virus binding, internalization, trafficking, and fusion. Fluorescently labeled viruses can be used to visualize these early processes. As dengue virus has 180 identical copies of the envelope protein attached to the membrane surface and is surrounded by a lipid membrane, amine-reactive (Alexa Fluor) or lipophilic (DiD) dyes can be used for virus labeling. These dyes are highly photostable and are ideal for studies involving cellular uptake and endosomal transport. To improve virus labeling efficiency and minimize the nonspecific labeling of nonviral proteins, virus concentration and purification precede fluorescent labeling of dengue viruses. Besides using these viruses for single-particle tracking, DiD-labeled viruses can also be used to distinguish serotype-specific from cross-neutralizing antibodies. Here the details of virus concentration, purification, virus labeling, applications, and hints of troubleshooting are described.