Publications by authors named "Sylvie Alonso"

90 Publications

Maternal Immunity and Vaccination Influence Disease Severity in Progeny in a Novel Mast Cell-Deficient Mouse Model of Severe Dengue.

Viruses 2021 05 12;13(5). Epub 2021 May 12.

Program in Emerging Infectious Diseases, Duke-National University of Singapore Medical School, Singapore 169857, Singapore.

Sub-neutralizing concentrations of antibodies in dengue infected patients is a major risk factor for the development of dengue hemorrhagic fever and dengue shock syndrome. Here, we describe a mouse model with a deficiency in mast cells (MCs) in addition to a deficiency in Type-I and II IFN receptors for studying dengue virus (DENV) infection. We used this model to understand the influence of MCs in a maternal antibody-dependent model of severe dengue, where offspring born to DENV-immune mothers are challenged with a heterologous DENV serotype. Mice lacking both MCs and IFN receptors were found susceptible to primary DENV infection and showed morbidity and mortality. When these mice were immunized, pups born to DENV-immune mothers were found to be protected for a longer duration from a heterologous DENV challenge. In the absence of MCs and type-I interferon signaling, IFN-γ was found to protect pups born to naïve mothers but had the opposite effect on pups born to DENV-immune mothers. Our results highlight the complex interactions between MCs and IFN-signaling in influencing the role of maternal antibodies in DENV-induced disease severity.
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http://dx.doi.org/10.3390/v13050900DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8152039PMC
May 2021

Enterovirus-A71 exploits peripherin and Rac1 to invade the central nervous system.

EMBO Rep 2021 Jun 19;22(6):e51777. Epub 2021 Apr 19.

Infectious Diseases Translational Research Programme, Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.

Enterovirus-A71 (EV-A71) has been associated with severe neurological forms of hand, foot, and mouth disease (HFMD). EV-A71 infects motor neurons at neuromuscular junctions (NMJs) to invade the central nervous system (CNS). Here, we investigate the role of peripherin (PRPH) during EV-A71 infection, a type III intermediate neurofilament involved in neurodegenerative conditions. In mice infected with EV-A71, PRPH co-localizes with viral particles in the muscles at NMJs and in the spinal cord. In motor neuron-like and neuroblastoma cell lines, surface-expressed PRPH facilitates viral entry, while intracellular PRPH influences viral genome replication through interactions with structural and non-structural viral components. Importantly, PRPH does not play a role during infection with coxsackievirus A16, another causative agent of HFMD rarely associated with neurological complications, suggesting that EV-A71 ability to exploit PRPH represents a unique attribute for successful CNS invasion. Finally, we show that EV-A71 also exploits some of the many PRPH-interacting partners. Of these, small GTP-binding protein Rac1 represents a potential druggable host target to limit neuroinvasion of EV-A71.
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http://dx.doi.org/10.15252/embr.202051777DOI Listing
June 2021

Viral determinants that drive Enterovirus-A71 fitness and virulence.

Emerg Microbes Infect 2021 Dec;10(1):713-724

Infectious Diseases Translational Research Programme, Department of Microbiology&Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.

Hand, Foot and Mouth Disease (HFMD) is usually a self-limiting, mild childhood disease that is caused mainly by Coxsackie virus A16 (CVA16) and Enterovirus A71 (EV-A71), both members of the family. However, recurring HFMD outbreaks and epidemics due to EV-A71 infection in the Western Pacific region, and the propensity of EV-A71 strains to cause severe neurological complications have made this neurotropic virus a serious public health concern in afflicted countries. High mutation rate leading to viral quasispecies combined with frequent intra- and inter-typic recombination events amongst co-circulating EV-A71 strains have contributed to the great diversity and fast evolution of EV-A71 genomes, making impossible any accurate prediction of the next epidemic strain. Comparative genome sequence analyses and mutagenesis approaches have led to the identification of a number of viral determinants involved in EV-A71 fitness and virulence. These viral determinants include amino acid residues located in the structural proteins of the virus, affecting attachment to the host cell surface, receptor binding, and uncoating events. Critical residues in non-structural proteins have also been identified, including 2C, 3A, 3C proteases and the RNA-dependent RNA polymerase. Finally, mutations altering key secondary structures in the 5' untranslated region were also found to influence EV-A71 fitness and virulence. While our current understanding of EV-A71 pathogenesis remains fragmented, these studies may help in the rational design of effective treatments and broadly protective vaccine candidates.
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http://dx.doi.org/10.1080/22221751.2021.1906754DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8043536PMC
December 2021

HflX is a GTPase that controls hypoxia-induced replication arrest in slow-growing mycobacteria.

Proc Natl Acad Sci U S A 2021 Mar;118(12)

Infectious Diseases Translational Research Programme, Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, 117456 Singapore;

GTPase high requency of ysogenization X (HflX) is highly conserved in prokaryotes and acts as a ribosome-splitting factor as part of the heat shock response in Here we report that HflX produced by slow-growing bacillus Calmette-Guérin (BCG) is a GTPase that plays a critical role in the pathogen's transition to a nonreplicating, drug-tolerant state in response to hypoxia. Indeed, HflX-deficient BCG (KO) replicated markedly faster in the microaerophilic phase of a hypoxia model that resulted in premature entry into dormancy. The KO mutant displayed hallmarks of nonreplicating mycobacteria, including phenotypic drug resistance, altered morphology, low intracellular ATP levels, and overexpression of Dormancy (Dos) regulon proteins. Mice nasally infected with HflX KO mutant displayed increased bacterial burden in the lungs, spleen, and lymph nodes during the chronic phase of infection, consistent with the higher replication rate observed in vitro in microaerophilic conditions. Unlike fast growing mycobacteria, BCG HlfX was not involved in antibiotic resistance under aerobic growth. Proteomics, pull-down, and ribo-sequencing approaches supported that mycobacterial HflX is a ribosome-binding protein that controls translational activity of the cell. With HflX fully conserved between BCG and , our work provides further insights into the molecular mechanisms deployed by pathogenic mycobacteria to adapt to their hypoxic microenvironment.
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http://dx.doi.org/10.1073/pnas.2006717118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8000101PMC
March 2021

A single-dose live attenuated chimeric vaccine candidate against Zika virus.

NPJ Vaccines 2021 Jan 29;6(1):20. Epub 2021 Jan 29.

Laboratory of Molecular RNA Virology and Antiviral Strategies, Department of Microbiology and Immunology, Infectious Diseases Translational Research Programme, Yong Loo Lin School of Medicine, National University Health System, National University of Singapore, MD4 Level 5, 5 Science Drive 2, Singapore, 117597, Singapore.

The mosquito-borne Zika virus is an emerging pathogen from the Flavivirus genus for which there are no approved antivirals or vaccines. Using the clinically validated PDK-53 dengue virus vaccine strain as a backbone, we created a chimeric dengue/Zika virus, VacDZ, as a live attenuated vaccine candidate against Zika virus. VacDZ demonstrates key markers of attenuation: small plaque phenotype, temperature sensitivity, attenuation of neurovirulence in suckling mice, and attenuation of pathogenicity in interferon deficient adult AG129 mice. VacDZ may be administered as a traditional live virus vaccine, or as a DNA-launched vaccine that produces live VacDZ in vivo after delivery. Both vaccine formulations induce a protective immune response against Zika virus in AG129 mice, which includes neutralising antibodies and a strong Th1 response. This study demonstrates that VacDZ is a safe and effective vaccine candidate against Zika virus.
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http://dx.doi.org/10.1038/s41541-021-00282-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7846741PMC
January 2021

Changes in complement alternative pathway components, factor B and factor H during dengue virus infection in the AG129 mouse.

J Gen Virol 2021 Mar 7;102(3). Epub 2021 Jan 7.

Microbiology and Infectious Diseases, Flinders University, Bedford Park, Adelaide 5042, South Australia.

The complement alternative pathway (AP) is tightly regulated and changes in two important AP components, factor B (FB) and factor H (FH) are linked to severe dengue in humans. Here, a mouse model of dengue was investigated to define the changes in FB and FH and assess the utility of this model to study the role of the AP in severe dengue. Throughout the period of viremia in the AG129 IFN signalling-deficient mouse, an increase in FB and a decrease in FH was observed following dengue virus (DENV) infection, with the former only seen in a model of more severe disease associated with antibody-dependent enhancement (ADE). Terminal disease was associated with a decrease in FB and FH, with greater changes during ADE, and accompanied by increased C3 degradation consistent with complement activation. analysis of NFκΒ, signal transducer and activator of transcription (STAT) and IFN-driven FB and FH promoter elements to reflect the likely impact of the lack of IFN-responses in AG129 mice, demonstrated that these elements differed markedly between human and mouse, notably with mouse FH lacking NFκΒ and key IFN-stimulated response elements (ISRE), and FB with many more NFκΒ and STAT-responsive elements than human FB. Thus, the AG129 mouse offers utility in demonstrating changes in FB and FH that, similar to humans, are associated with severe disease, but lack predicted important human-specific and IFN-dependent responses of FB and FH to DENV-infection that are likely to regulate the subtleties of the overall AP response during dengue disease in humans.
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http://dx.doi.org/10.1099/jgv.0.001547DOI Listing
March 2021

Dual inhibition of the terminal oxidases eradicates antibiotic-tolerant Mycobacterium tuberculosis.

EMBO Mol Med 2021 Jan 7;13(1):e13207. Epub 2020 Dec 7.

School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.

The approval of bedaquiline has placed energy metabolism in the limelight as an attractive target space for tuberculosis antibiotic development. While bedaquiline inhibits the mycobacterial F F ATP synthase, small molecules targeting other components of the oxidative phosphorylation pathway have been identified. Of particular interest is Telacebec (Q203), a phase 2 drug candidate inhibitor of the cytochrome bcc:aa terminal oxidase. A functional redundancy between the cytochrome bcc:aa and the cytochrome bd oxidase protects M. tuberculosis from Q203-induced death, highlighting the attractiveness of the bd-type terminal oxidase for drug development. Here, we employed a facile whole-cell screen approach to identify the cytochrome bd inhibitor ND-011992. Although ND-011992 is ineffective on its own, it inhibits respiration and ATP homeostasis in combination with Q203. The drug combination was bactericidal against replicating and antibiotic-tolerant, non-replicating mycobacteria, and increased efficacy relative to that of a single drug in a mouse model. These findings suggest that a cytochrome bd oxidase inhibitor will add value to a drug combination targeting oxidative phosphorylation for tuberculosis treatment.
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http://dx.doi.org/10.15252/emmm.202013207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7799364PMC
January 2021

Sustainable, three-component, one-pot procedure to obtain active anti-flavivirus agents.

Eur J Med Chem 2021 Jan 4;210:112992. Epub 2020 Nov 4.

Dipartimento di Scienze Farmaceutiche, Università degli Studi di Perugia, Via Del Liceo, 1-06123, Perugia, Italy. Electronic address:

The mosquito-borne viruses belonging to the genus Flavivirus such as Dengue virus (DENV) and Zika virus (ZIKV) cause human infections ranging from mild flu-like symptoms to hemorrhagic fevers, hepatitis, and neuropathies. To date, there are vaccines only for few flaviviruses while no effective treatments are available. Pyridobenzothiazole (PBTZ) derivatives are a class of compounds endowed with a promising broad-spectrum anti-flavivirus activity and most of them have been reported as potent inhibitors of the flaviviral NS5 polymerase. However, synthesis of PBTZ analogues entails a high number of purification steps, the use of hazardous reagents and environmentally unsustainable generation of waste. Considering the promising antiviral activity of PBTZ analogues which require further exploration, in this work, we report the development of a new and sustainable three-component reaction (3CR) that can be combined with a basic hydrolysis in a one-pot procedure to obtain the PBTZ scaffold, thus reducing the number of synthetic steps, improving yields and saving time. 3CR was significantly explored in order to demonstrate its wide scope by using different starting materials. In addition, taking advantage of these procedures, we next designed and synthesized a new set of PBTZ analogues that were tested as anti-DENV-2 and anti-ZIKV agents. Compound 22 inhibited DENV-2 NS5 polymerase with an IC of 10.4 μM and represented the best anti-flavivirus compound of the new series by inhibiting DENV-2- and ZIKV-infected cells with EC values of 1.2 and 5.0 μM, respectively, that translates into attractive selectivity indexes (SI - 83 and 20, respectively). These results strongly reaffirm PBTZ derivatives as promising anti-flavivirus agents that now can be synthesized through a convenient and sustainable 3CR in order to obtain more potent compounds for further pre-clinical development studies.
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http://dx.doi.org/10.1016/j.ejmech.2020.112992DOI Listing
January 2021

Display of Native Antigen on cDC1 That Have Spatial Access to Both T and B Cells Underlies Efficient Humoral Vaccination.

J Immunol 2020 10 24;205(7):1842-1856. Epub 2020 Aug 24.

Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria 3800, Australia.

Follicular dendritic cells and macrophages have been strongly implicated in presentation of native Ag to B cells. This property has also occasionally been attributed to conventional dendritic cells (cDC) but is generally masked by their essential role in T cell priming. cDC can be divided into two main subsets, cDC1 and cDC2, with recent evidence suggesting that cDC2 are primarily responsible for initiating B cell and T follicular helper responses. This conclusion is, however, at odds with evidence that targeting Ag to Clec9A (DNGR1), expressed by cDC1, induces strong humoral responses. In this study, we reveal that murine cDC1 interact extensively with B cells at the border of B cell follicles and, when Ag is targeted to Clec9A, can display native Ag for B cell activation. This leads to efficient induction of humoral immunity. Our findings indicate that surface display of native Ag on cDC with access to both T and B cells is key to efficient humoral vaccination.
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http://dx.doi.org/10.4049/jimmunol.2000549DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7504891PMC
October 2020

An update on dengue vaccine development, challenges, and future perspectives.

Expert Opin Drug Discov 2021 Jan 25;16(1):47-58. Epub 2020 Aug 25.

Yong Loo Lin School of Medicine, National University of Singapore , Singapore, Singapore.

Introduction: From both a public health and economic perspective, vaccination is arguably the most effective approach to combat endemic and pandemic infectious diseases. Dengue affects more than 100 countries in the tropical and subtropical world, with 100-400 million infections every year. In the wake of the recent setback faced by Dengvaxia, the only FDA-approved dengue vaccine, safer and more effective dengue vaccines candidates are moving along the clinical pipeline.

Area Covered: This review provides an update of the latest outcomes of dengue vaccine clinical trials. In the light of recent progress made in our understanding of dengue pathogenesis and immune correlates of protection, novel vaccine strategies have emerged with promising second-generation dengue vaccine candidates. Finally, the authors discuss the dengue-specific challenges that remain to be addressed and overcome.

Expert Opinion: The authors propose to explore various adjuvants and delivery systems that may help improve the design of safe, effective, and affordable vaccines against dengue. They also challenge the concept of a 'universal' dengue vaccine as increasing evidence support that DENV strains have evolved different virulence mechanisms.
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http://dx.doi.org/10.1080/17460441.2020.1811675DOI Listing
January 2021

Relative contribution of nonstructural protein 1 in dengue pathogenesis.

J Exp Med 2020 09;217(9)

Infectious Disease Programme and Department of Microbiology & Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

Dengue is a major public health concern in the tropical and subtropical world, with no effective treatment. The controversial live attenuated virus vaccine Dengvaxia has boosted the pursuit of subunit vaccine approaches, and nonstructural protein 1 (NS1) has recently emerged as a promising candidate. However, we found that NS1 immunization or passive transfer of NS1 antibodies failed to confer protection in symptomatic dengue mouse models using two non-mouse-adapted DENV2 strains that are highly virulent. Exogenous administration of purified NS1 also failed to worsen in vivo vascular leakage in sublethally infected mice. Neither method of NS1 immune neutralization changed the disease outcome of a chimeric strain expressing a vascular leak-potent NS1. Instead, virus chimerization involving the prME structural region indicated that these proteins play a critical role in driving in vivo fitness and virulence of the virus, through induction of key proinflammatory cytokines. This work highlights that the pathogenic role of NS1 is DENV strain dependent, which warrants reevaluation of NS1 as a universal dengue vaccine candidate.
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http://dx.doi.org/10.1084/jem.20191548DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7478733PMC
September 2020

Harnessing the Immunomodulatory Properties of Bacterial Ghosts to Boost the Anti-mycobacterial Protective Immunity.

Front Immunol 2019 22;10:2737. Epub 2019 Nov 22.

Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.

Tuberculosis (TB) pathogenesis is characterized by inadequate immune cell activation and delayed T cell response in the host. Recent immunotherapeutic efforts have been directed at stimulating innate immunity and enhancing interactions between antigen presenting cells and T cells subsets to improve the protective immunity against TB. In this study, we investigated the immunostimulatory properties of bacterial ghosts (BG) as a novel approach to potentiate the host immunity against mycobacterial infection. BG are intact cytoplasm-free envelopes and have been developed as bacterial vaccines and adjuvant/delivery system in cancer immunotherapy. However, BG have yet to be exploited as immunopotentiators in the context of infectious diseases. Here, we showed that BG are potent inducers of dendritic cells (DC), which led to enhanced T cell proliferation and differentiation into effector cells. BG also induced macrophage activation, which was associated with enhanced nitric oxide production, a key anti-mycobacterial weapon. We further demonstrated that the immunostimulatory capability of BG far exceeds that of LPS and involves both TLR4-dependent and independent pathways. Consistently, BG treatment, but not LPS treatment, reduced the bacterial burden in infected mice, which correlated with increased influx of innate and adaptive effector immune cells and increased production of key cytokines in the lungs. Finally and importantly, enhanced bacilli killing was seen in mice co-administered with BG and second-line TB drugs bedaquiline and delamanid. Overall, this work paves the way for BG as potent immunostimulators that may be harnessed to improve mycobacteria killing at the site of infection.
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http://dx.doi.org/10.3389/fimmu.2019.02737DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6883722PMC
November 2020

Dengue virus infects the mouse eye following systemic or intracranial infection and induces inflammatory responses.

J Gen Virol 2020 01;101(1):79-85

Microbiology and Infectious Diseases, College of Medicine and Public Health, Flinders University, Bedford Park, Adelaide, 5042, Australia.

Dengue virus (DENV) infection is associated with clinical ocular presentations and here DENV infection of the eye was assessed in mice. In an AG129 mouse model of antibody-dependent enhancement of DENV infection, DENV RNA was detected in the eye and vascular changes were present in the retinae. Intraocular CD8 and IFN-γ mRNA were increased in mice born to DENV-naïve, but not DENV-immune mothers, while TNF-α mRNA was induced and significantly higher in mice born to DENV-immune than DENV-naïve mothers. DENV RNA was detected in the eye following intracranial DENV infection and CD8 mRNA but not IFN-γ nor TNF-α were induced. In all models, viperin was increased following DENV infection. Thus, DENV in the circulation or the brain can infect the eye and stimulate innate immune responses, with induction of viperin as one response that consistently occurs in multiple DENV eye-infection models in both an IFN-dependent and independent manner.
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http://dx.doi.org/10.1099/jgv.0.001354DOI Listing
January 2020

Recent progress and challenges in drug development to fight hand, foot and mouth disease.

Expert Opin Drug Discov 2020 03 30;15(3):359-371. Epub 2019 Aug 30.

Department of Microbiology&Immunology, Yong Loo Lin School of Medicine, Immunology program, Life Sciences Institute, National University of Singapore, Singapore, Singapore.

: Hand, foot and mouth disease (HFMD) is a serious public health concern in the Asia-Pacific region with recurrent cyclical outbreaks. Enterovirus 71 (EV-A71) and coxsackievirus type A are the main causative agents of HFMD. While majority of HFMD cases are mild and self-limiting, neurological complications have been reported for EV-A71 associated HFMD. There is currently no effective treatment against HFMD and monovalent vaccines against EV-A71 are currently limited to the Chinese market.: As of today, HFMD antiviral development has focused on EV-A71 and involves conventional screening of drug libraries. In recent years, attention has shifted toward identifying druggable host factors to avoid drug resistance and identify drug candidates with broader antiviral activity across EV-A71 genogroups and other HFMD causative agents.: The effective development of HFMD interventions requires us to address the gaps in our understanding of its pathogenesis at the molecular level. The limited resources devoted to the development of HFMD treatment strategies worldwide also contribute to the slow progress of promising drug and vaccine candidates along the clinical pipeline.
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http://dx.doi.org/10.1080/17460441.2019.1659241DOI Listing
March 2020

Microneedle-based intradermal delivery of stabilized dengue virus.

Bioeng Transl Med 2019 May 25;4(2):e10127. Epub 2019 Feb 25.

Infectious Diseases IRG Singapore-MIT Alliance for Research and Technology Singapore.

Current live-attenuated dengue vaccines require strict cold chain storage. Methods to preserve dengue virus (DENV) viability, which enable vaccines to be transported and administered at ambient temperatures, will be decisive towards the implementation of affordable global vaccination schemes with broad immunization coverage in resource-limited areas. We have developed a microneedle (MN)-based vaccine platform for the stabilization and intradermal delivery of live DENV from minimally invasive skin patches. Dengue virus-stabilized microneedle arrays (VSMN) were fabricated using saccharide-based formulation of virus and could be stored dry at ambient temperature up to 3 weeks with maintained virus viability. Following intradermal vaccination, VSMN-delivered DENV was shown to elicit strong neutralizing antibody responses and protection from viral challenge, comparable to that of the conventional liquid vaccine administered subcutaneously. This work supports the potential for MN-based dengue vaccine technology and the progression towards cold chain-independence. Dengue virus can be stabilized using saccharide-based formulations and coated on microneedle array vaccine patches for storage in dry state with preserved viability at ambient temperature (VSMN; virus-stabilized microneedle arrays).
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http://dx.doi.org/10.1002/btm2.10127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6584444PMC
May 2019

A T164S mutation in the dengue virus NS1 protein is associated with greater disease severity in mice.

Sci Transl Med 2019 06;11(498)

Program in Emerging Infectious Diseases, Duke-NUS Medical School, 8 College Road, Singapore 169857, Singapore.

Dengue viruses cause severe and sudden human epidemics worldwide. The secreted form of the nonstructural protein 1 (sNS1) of dengue virus causes vascular leakage, a hallmark of severe dengue disease. Here, we reverse engineered the T164S mutation of NS1, associated with the severity of dengue epidemics in the Americas, into a dengue virus serotype 2 mildly infectious strain. The T164S mutant virus decreased infectious virus production and increased sNS1 production in mammalian cell lines and human peripheral blood mononuclear cells (PBMCs) without affecting viral RNA replication. Gene expression profiling of 268 inflammation-associated human genes revealed up-regulation of genes induced in response to vascular leakage. Infection of the mosquito vector with the T164S mutant virus resulted in increased viral load in the mosquito midgut and higher sNS1 production compared to wild-type virus infection. Infection of type 1 and 2 interferon receptor-deficient AG129 mice with the T164S mutant virus resulted in severe disease coupled with increased complement activation, tissue inflammation, and more rapid mortality compared to AG129 mice infected with wild-type virus. Molecular dynamics simulations predicted that mutant sNS1 formed stable dimers similar to the wild-type protein, whereas the hexameric mutant sNS1 was predicted to be unstable. Immunoaffinity-purified sNS1 from T164S mutant virus-infected mammalian cells was associated with different lipid classes compared to wild-type sNS1. Treatment of human PBMCs with sNS1 purified from T164S mutant virus resulted in a twofold higher production of proinflammatory cytokines, suggesting a mechanism for how mutant sNS1 may cause more severe dengue disease.
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http://dx.doi.org/10.1126/scitranslmed.aat7726DOI Listing
June 2019

Studies on B Cells in the Fruit-Eating Black Flying Fox (.

Front Immunol 2019 14;10:489. Epub 2019 Mar 14.

Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.

The ability of bats to act as reservoir for viruses that are highly pathogenic to humans suggests unique properties and functional characteristics of their immune system. However, the lack of bat specific reagents, in particular antibodies, has limited our knowledge of bat's immunity. Here, we report a panel of cross-reactive antibodies against MHC-II, NK1.1, CD3, CD21, CD27, and immunoglobulin (Ig), that allows flow cytometry analysis of B, T and NK cell populations in two different fruit-eating bat species namely, and . Results confirmed predominance of T cells in the spleen and blood of bats, as previously reported by us. However, the percentages of B cells in bone marrow and NK cells in spleen varied greatly between wild caught bats and colony bats, which may reflect inherent differences of their immune system or different immune status. Other features of bat B cells were investigated. A significant increase in sIg B cell population was observed in the spleen and blood from LPS-injected bats but not from poly I:C-injected bats, supporting T-independent polyclonal B cell activation by LPS. Furthermore, using an calcium release assay, B cells exhibited significant calcium release upon cross-linking of their B cell receptor. Together, this work contributes to improve our knowledge of bat adaptive immunity in particular B cells.
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http://dx.doi.org/10.3389/fimmu.2019.00489DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6428034PMC
September 2020

Structure mapping of dengue and Zika viruses reveals functional long-range interactions.

Nat Commun 2019 03 29;10(1):1408. Epub 2019 Mar 29.

Stem Cell and Regenerative Biology, Genome Institute of Singapore, Singapore, 138672, Singapore.

Dengue (DENV) and Zika (ZIKV) viruses are clinically important members of the Flaviviridae family with an 11 kb positive strand RNA genome that folds to enable virus function. Here, we perform structure and interaction mapping on four DENV and ZIKV strains inside virions and in infected cells. Comparative analysis of SHAPE reactivities across serotypes nominates potentially functional regions that are highly structured, conserved, and contain low synonymous mutation rates. Interaction mapping by SPLASH identifies many pair-wise interactions, 40% of which form alternative structures, suggesting extensive structural heterogeneity. Analysis of shared interactions between serotypes reveals a conserved macro-organization whereby interactions can be preserved at physical locations beyond sequence identities. We further observe that longer-range interactions are preferentially disrupted inside cells, and show the importance of new interactions in virus fitness. These findings deepen our understanding of Flavivirus genome organization and serve as a resource for designing therapeutics in targeting RNA viruses.
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http://dx.doi.org/10.1038/s41467-019-09391-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6441010PMC
March 2019

Invasion of a murine in vitro blood-brain barrier co-culture model by dengue virus serotypes 1 to 4.

Arch Virol 2019 Apr 19;164(4):1069-1083. Epub 2019 Feb 19.

Pengiran Anak Puteri Rashidah Sa'adatul Bolkiah Institute of Health Sciences, Universiti Brunei Darussalam, Jalan Tungku Link, Gadong, BE1410, Brunei Darussalam.

The blood-brain barrier (BBB) is a physical barrier that restricts the passage of cells and molecules as well as pathogens into the central nervous system (CNS). Some viruses enter the CNS by disrupting the BBB, while others can reach the CNS without altering the integrity of the BBB. Even though dengue virus (DENV) is not a distinctive neurotropic virus, the virus is considered to be one of the leading causes of neurological manifestations. In this study, we found that DENV is able to compromise the integrity of a murine in vitro blood-brain barrier (BBB) model, resulting in hyperpermeability, as shown by a significant increase in sucrose and albumin permeability. Infection of brain endothelial cells (ECs) was facilitated by the presence of glycans, in particular, mannose and N-acetyl glucosamine residues, on cell surfaces and viral envelope proteins, and the requirement for glycan moieties for cell infection was serotype-specific. Direct viral disruption of brain ECs was observed, leading to a significant decrease in tight-junction protein expression and peripheral localization, which contributed to the changes in BBB permeability. In conclusion, the hyperpermeability and breaching mechanism of BBB by DENV are primarily due to direct consequences of viral infection of ECs, as shown in this in vitro study.
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http://dx.doi.org/10.1007/s00705-019-04175-3DOI Listing
April 2019

Investing in gender equity in health and biomedical research: a Singapore perspective.

Lancet 2019 02;393(10171):e21-e22

Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597; National University Hospital, National University Health System, Singapore.

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http://dx.doi.org/10.1016/S0140-6736(18)32096-8DOI Listing
February 2019

Neutralization of antibody-enhanced dengue infection by VIS513, a pan serotype reactive monoclonal antibody targeting domain III of the dengue E protein.

PLoS Negl Trop Dis 2018 02 9;12(2):e0006209. Epub 2018 Feb 9.

Infectious Diseases Interdisciplinary Research Group, Singapore-MIT Alliance for Research and Technology, Singapore, Singapore.

Dengue virus (DENV) infection imposes enormous health and economic burden worldwide with no approved treatment. Several small molecules, including lovastatin, celgosivir, balapiravir and chloroquine have been tested for potential anti-dengue activity in clinical trials; none of these have demonstrated a protective effect. Recently, based on identification and characterization of cross-serotype neutralizing antibodies, there is increasing attention on the potential for dengue immunotherapy. Here, we tested the ability of VIS513, an engineered cross-neutralizing humanized antibody targeting the DENV E protein domain III, to overcome antibody-enhanced infection and high but brief viremia, which are commonly encountered in dengue patients, in various in vitro and in vivo models. We observed that VIS513 efficiently neutralizes DENV at clinically relevant viral loads or in the presence of enhancing levels of DENV immune sera. Single therapeutic administration of VIS513 in mouse models of primary infection or lethal secondary antibody-enhanced infection, reduces DENV titers and protects from lethal infection. Finally, VIS513 administration does not readily lead to resistance, either in cell culture systems or in animal models of dengue infection. The findings suggest that rapid viral reduction during acute DENV infection with a monoclonal antibody is feasible.
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http://dx.doi.org/10.1371/journal.pntd.0006209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5823465PMC
February 2018

Prohibitin plays a critical role in Enterovirus 71 neuropathogenesis.

PLoS Pathog 2018 01 11;14(1):e1006778. Epub 2018 Jan 11.

Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

A close relative of poliovirus, enterovirus 71 (EV71) is regarded as an important neurotropic virus of serious public health concern. EV71 causes Hand, Foot and Mouth Disease and has been associated with neurological complications in young children. Our limited understanding of the mechanisms involved in its neuropathogenesis has hampered the development of effective therapeutic options. Here, using a two-dimensional proteomics approach combined with mass spectrometry, we have identified a unique panel of host proteins that were differentially and dynamically modulated during EV71 infection of motor-neuron NSC-34 cells, which are found at the neuromuscular junctions where EV71 is believed to enter the central nervous system. Meta-analysis with previously published proteomics studies in neuroblastoma or muscle cell lines revealed minimal overlapping which suggests unique host-pathogen interactions in NSC-34 cells. Among the candidate proteins, we focused our attention on prohibitin (PHB), a protein that is involved in multiple cellular functions and the target of anti-cancer drug Rocaglamide (Roc-A). We demonstrated that cell surface-expressed PHB is involved in EV71 entry into neuronal cells specifically, while membrane-bound mitochondrial PHB associates with the virus replication complex and facilitates viral replication. Furthermore, Roc-A treatment of EV71-infected neuronal cells reduced significantly virus yields. However, the inhibitory effect of Roc-A on PHB in NSC-34 cells was not through blocking the CRAF/MEK/ERK pathway as previously reported. Instead, Roc-A treated NSC-34 cells had lower mitochondria-associated PHB and lower ATP levels that correlated with impaired mitochondria integrity. In vivo, EV71-infected mice treated with Roc-A survived longer than the vehicle-treated animals and had significantly lower virus loads in their spinal cord and brain, whereas virus titers in their limb muscles were comparable to controls. Together, this study uncovers PHB as the first host factor that is specifically involved in EV71 neuropathogenesis and a potential drug target to limit neurological complications.
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http://dx.doi.org/10.1371/journal.ppat.1006778DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5764453PMC
January 2018

Enhancing vaccine antibody responses by targeting Clec9A on dendritic cells.

NPJ Vaccines 2017 6;2:31. Epub 2017 Nov 6.

Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Melbourne, VIC Australia.

Targeting model antigens (Ags) to Clec9A on DC has been shown to induce, not only cytotoxic T cells, but also high levels of Ab. In fact, Ab responses against immunogenic Ag were effectively generated even in the absence of DC-activating adjuvants. Here we tested if targeting weakly immunogenic putative subunit vaccine Ags to Clec9A could enhance Ab responses to a level likely to be protective. The proposed "universal" influenza Ag, M2e and the enterovirus 71 Ag, SP70 were linked to anti-Clec9A Abs and injected into mice. Targeting these Ags to Clec9A greatly increased Ab titres. For optimal responses, a DC-activating adjuvant was required. For optimal responses, a boost injection was also needed, but the high Ab titres against the targeting construct blocked Clec9A-targeted boosting. Heterologous prime-boost strategies avoiding cross-reactivity between the priming and boosting targeting constructs overcame this limitation. In addition, targeting small amounts of Ag to Clec9A served as an efficient priming for a conventional boost with higher levels of untargeted Ag. Using this Clec9A-targeted priming, conventional boosting strategy, M2e immunisation protected mice from infection with lethal doses of influenza H1N1 virus.
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http://dx.doi.org/10.1038/s41541-017-0033-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5674066PMC
November 2017

Dengue vaccine-induced CD8+ T cell immunity confers protection in the context of enhancing, interfering maternal antibodies.

JCI Insight 2017 12 21;2(24). Epub 2017 Dec 21.

Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, and.

Declining levels of maternal antibodies were shown to sensitize infants born to dengue-immune mothers to severe disease during primary infection, through the process of antibody-dependent enhancement of infection (ADE). With the recent approval for human use of Sanofi-Pasteur's chimeric dengue vaccine CYD-TDV and several vaccine candidates in clinical development, the scenario of infants born to vaccinated mothers has become a reality. This raises 2 questions: will declining levels of maternal vaccine-induced antibodies cause ADE; and, will maternal antibodies interfere with vaccination efficacy in the infant? To address these questions, the above scenario was modeled in mice. Type I IFN-deficient female mice were immunized with live attenuated DENV2 PDK53, the core component of the tetravalent DENVax candidate currently under clinical development. Pups born to PDK53-immunized dams acquired maternal antibodies that strongly neutralized parental strain 16681, but not the heterologous DENV2 strain D2Y98P-PP1, and instead caused ADE during primary infection with this strain. Furthermore, pups failed to seroconvert after PDK53 vaccination, owing to maternal antibody interference. However, a cross-protective multifunctional CD8+ T cell response did develop. Thus, our work advocates for the development of dengue vaccine candidates that induce protective CD8+ T cells despite the presence of enhancing, interfering maternal antibodies.
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http://dx.doi.org/10.1172/jci.insight.94500DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5752305PMC
December 2017

Dual-Specificity Phosphatase 12 Targets p38 MAP Kinase to Regulate Macrophage Response to Intracellular Bacterial Infection.

Front Immunol 2017 9;8:1259. Epub 2017 Oct 9.

Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, Singapore, Singapore.

The mitogen-activated protein kinase (MAPK) cascades are activated in innate immune cells such as macrophages upon the detection of microbial infection, critically regulating the expression of proinflammatory cytokines and chemokines such as TNF-α, IL-6, and MCP-1. As a result, activation of MAPKs is tightly regulated to ensure appropriate and adequate immune responses. Dual-specificity phosphatases (DUSPs) are a family of proteins which specifically dephosphorylates threonine and tyrosine residues essential for MAPK activation to negatively regulate their activation. DUSP12 is a member of atypical DUSPs that lack MAPK-binding domain. Its substrate and function in immune cells are unknown. In this study, we demonstrated that DUSP12 is able to interact with all the three groups of MAPKs, including extracellular signal-regulated protein kinase, JNK, and p38. To investigate the function of DUSP12 in macrophages in response to TLR activation and microbial infection, we established RAW264.7 cell lines stably overexpressing DUSP12 and found that overexpression of DUSP12 inhibited proinflammatory cytokine and chemokine production in response to TLR4 activation, heat-inactivated stimulation as well as infections by intracellular bacteria including and BCG by specifically inhibiting p38 and JNK. In addition, a scaffold protein known as signal transducing adaptor protein 2 (STAP2), was found to mediate the interaction between DUSP12 and p38. Thus, DUSP12 is a bona fide MAPK phosphatase, playing an important role in MAPK-regulated responses to bacterial infection. Our study provides a model where atypical DUSPs regulate MAPKs scaffold, thereby regulating immune responses to microbial infection.
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http://dx.doi.org/10.3389/fimmu.2017.01259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5640881PMC
October 2017

Dengue Virus Glycosylation: What Do We Know?

Front Microbiol 2017 25;8:1415. Epub 2017 Jul 25.

Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, and Immunology program, Life Sciences Institute, National University of SingaporeSingapore, Singapore.

In many infectious diseases caused by either viruses or bacteria, pathogen glycoproteins play important roles during the infection cycle, ranging from entry to successful intracellular replication and host immune evasion. Dengue is no exception. glycoproteins, envelope protein (E) and non-structural protein 1 (NS1) are two popular sub-unit vaccine candidates. E protein on the virion surface is the major target of neutralizing antibodies. NS1 which is secreted during DENV infection has been shown to induce a variety of host responses through its binding to several host factors. However, despite their critical role in disease and protection, the glycosylated variants of these two proteins and their biological importance have remained understudied. In this review, we seek to provide a comprehensive summary of the current knowledge on protein glycosylation in DENV, and its role in virus biogenesis, host cell receptor interaction and disease pathogenesis.
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http://dx.doi.org/10.3389/fmicb.2017.01415DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5524768PMC
July 2017

Exploiting the synthetic lethality between terminal respiratory oxidases to kill and clear host infection.

Proc Natl Acad Sci U S A 2017 07 26;114(28):7426-7431. Epub 2017 Jun 26.

Lee Kong Chian School of Medicine and School of Biological Sciences, Nanyang Technological University, Singapore 636921;

The recent discovery of small molecules targeting the cytochrome : in triggered interest in the terminal respiratory oxidases for antituberculosis drug development. The mycobacterial cytochrome : consists of a menaquinone:cytochrome reductase ( ) and a cytochrome -type oxidase. The clinical-stage drug candidate Q203 interferes with the function of the subunit b of the menaquinone:cytochrome reductase. Despite the affinity of Q203 for the : complex, the drug is only bacteriostatic and does not kill drug-tolerant persisters. This raises the possibility that the alternate terminal -type oxidase (cytochrome oxidase) is capable of maintaining a membrane potential and menaquinol oxidation in the presence of Q203. Here, we show that the electron flow through the cytochrome oxidase is sufficient to maintain respiration and ATP synthesis at a level high enough to protect from Q203-induced bacterial death. Upon genetic deletion of the cytochrome oxidase-encoding genes , Q203 inhibited mycobacterial respiration completely, became bactericidal, killed drug-tolerant mycobacterial persisters, and rapidly cleared infection in vivo. These results indicate a synthetic lethal interaction between the two terminal respiratory oxidases that can be exploited for anti-TB drug development. Our findings should be considered in the clinical development of drugs targeting the cytochrome : , as well as for the development of a drug combination targeting oxidative phosphorylation in .
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http://dx.doi.org/10.1073/pnas.1706139114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5514758PMC
July 2017

ZnO Nano-Rod Devices for Intradermal Delivery and Immunization.

Nanomaterials (Basel) 2017 Jun 15;7(6). Epub 2017 Jun 15.

Department of Pharmacy, National University of Singapore, Singapore 117583, Singapore.

Intradermal delivery of antigens for vaccination is a very attractive approach since the skin provides a rich network of antigen presenting cells, which aid in stimulating an immune response. Numerous intradermal techniques have been developed to enhance penetration across the skin. However, these methods are invasive and/or affect the skin integrity. Hence, our group has devised zinc oxide (ZnO) nano-rods for non-destructive drug delivery. Chemical vapour deposition was used to fabricate aligned nano-rods on ZnO pre-coated silicon chips. The nano-rods' length and diameter were found to depend on the temperature, time, quality of sputtered silicon chips, etc. Vertically aligned ZnO nano-rods with lengths of 30-35 µm and diameters of 200-300 nm were selected for in vitro human skin permeation studies using Franz cells with Albumin-fluorescein isothiocyanate (FITC) absorbed on the nano-rods. Fluorescence and confocal studies on the skin samples showed FITC penetration through the skin along the channels formed by the nano-rods. Bradford protein assay on the collected fluid samples indicated a significant quantity of Albumin-FITC in the first 12 h. Low antibody titres were observed with immunisation on Balb/c mice with ovalbumin (OVA) antigen coated on the nano-rod chips. Nonetheless, due to the reduced dimensions of the nano-rods, our device offers the additional advantage of excluding the simultaneous entrance of microbial pathogens. Taken together, these results showed that ZnO nano-rods hold the potential for a safe, non-invasive, and painless intradermal drug delivery.
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http://dx.doi.org/10.3390/nano7060147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5485794PMC
June 2017

EthA/R-Independent Killing of by Ethionamide.

Front Microbiol 2017 25;8:710. Epub 2017 Apr 25.

Department of Microbiology and Immunology, Yong Loo Lin School of MedicineSingapore, Singapore.

Ethionamide (ETH) is part of the drug arsenal available to treat multi-drug resistant tuberculosis. The current paradigm of this pro-drug activation involves the mycobacterial enzyme EthA and the transcriptional repressor, EthR. However, several lines of evidence suggest the involvement of additional players. The locus was deleted in BCG and three (MTB) strains. While complete resistance to ETH was observed with BCG KO, drug susceptibility and dose-dependent killing were retained in the KO MTB mutants, suggesting the existence of an alternative pathway of ETH bio-activation in MTB. We further demonstrated that this alternative pathway is EthR-independent, whereby re-introduction of in KO MTB did not lead to increased resistance to ETH. Consistently, KO MTB (with intact expression) displayed similar ETH susceptibility profile as their KO counterparts. To identify the alternative ETH bio-activator, spontaneous ETH-resistant mutants were obtained from KO MTB and whole genome sequencing identified single nucleotide polymorphisms in , involved in mycothiol biosynthesis and previously linked to ETH resistance. Deletion of in KO MTB led to complete ETH resistance, supporting that the role of MshA in ETH killing is EthA/R-independent. Furthermore single KO MTB displayed levels of ETH resistance similar or greater than those obtained with KO strains, supporting that is as critical as for ETH killing efficacy.
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http://dx.doi.org/10.3389/fmicb.2017.00710DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5403819PMC
April 2017

Increased Serum Hyaluronic Acid and Heparan Sulfate in Dengue Fever: Association with Plasma Leakage and Disease Severity.

Sci Rep 2017 04 10;7:46191. Epub 2017 Apr 10.

Communicable Disease Centre, Institute of Infectious Diseases and Epidemiology, Tan Tock Seng Hospital, Singapore.

Plasma leakage is a major pathogenic mechanism of severe dengue, but the etiology remains unclear. The association between endothelial glycocalyx integrity and vascular permeability in older adults with dengue has not been evaluated. A prospective cohort study of adults with undifferentiated fever screened for dengue by RT-PCR or NS1 antigen testing was performed. Patients were assessed daily while symptomatic and at convalescence. Serum hyaluronic acid (HA), heparan sulfate (HS) and selected cytokines (TNF-α, IL-6, IL-10) were measured on enrollment and convalescence. Patients were diagnosed as dengue fever (DF, n = 30), dengue hemorrhagic fever (DHF, n = 20) and non-dengue (ND) febrile illness (n = 11). Acute HA and HS levels were significantly higher in all dengue patients compared to ND (p = 0.0033 and p = 0.0441 respectively), but not different between DF and DHF (p = 0.3426 and p = 0.9180 respectively). Enrolment HA inversely correlated with serum albumin, protein and platelets in all dengue and DHF (p < 0.05). HA and HS in all dengue patients decreased significantly at convalescence. Serum IL-10 was significantly associated with HA in all dengue patients (p = 0.002). Serum HA and HS levels were increased in adult dengue and HA was associated with markers of disease severity. Endothelial glycocalyx damage may have a role in vascular leakage in dengue.
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http://dx.doi.org/10.1038/srep46191DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5385535PMC
April 2017