Publications by authors named "Juthathip Mongkolsapaya"

63 Publications

Flavivirus maturation leads to the formation of an occupied lipid pocket in the surface glycoproteins.

Nat Commun 2021 02 23;12(1):1238. Epub 2021 Feb 23.

Division of Structural Biology, The Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK.

Flaviviruses such as Dengue (DENV) or Zika virus (ZIKV) assemble into an immature form within the endoplasmatic reticulum (ER), and are then processed by furin protease in the trans-Golgi. To better grasp maturation, we carry out cryo-EM reconstructions of immature Spondweni virus (SPOV), a human flavivirus of the same serogroup as ZIKV. By employing asymmetric localised reconstruction we push the resolution to 3.8 Å, enabling us to refine an atomic model which includes the crucial furin protease recognition site and a conserved Histidine pH-sensor. For direct comparison, we also solve structures of the mature forms of SPONV and DENV to 2.6 Å and 3.1 Å, respectively. We identify an ordered lipid that is present in only the mature forms of ZIKV, SPOV, and DENV and can bind as a consequence of rearranging amphipathic stem-helices of E during maturation. We propose a structural role for the pocket and suggest it stabilizes mature E.
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http://dx.doi.org/10.1038/s41467-021-21505-9DOI Listing
February 2021

Native-like SARS-CoV-2 spike glycoprotein expressed by ChAdOx1 nCoV-19/AZD1222 vaccine.

bioRxiv 2021 Jan 19. Epub 2021 Jan 19.

Vaccine development against the SARS-CoV-2 virus focuses on the principal target of the neutralizing immune response, the spike (S) glycoprotein. Adenovirus-vectored vaccines offer an effective platform for the delivery of viral antigen, but it is important for the generation of neutralizing antibodies that they produce appropriately processed and assembled viral antigen that mimics that observed on the SARS-CoV-2 virus. Here, we describe the structure, conformation and glycosylation of the S protein derived from the adenovirus-vectored ChAdOx1 nCoV-19/AZD1222 vaccine. We demonstrate native-like post-translational processing and assembly, and reveal the expression of S proteins on the surface of cells adopting the trimeric prefusion conformation. The data presented here confirms the use of ChAdOx1 adenovirus vectors as a leading platform technology for SARS-CoV-2 vaccines.
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http://dx.doi.org/10.1101/2021.01.15.426463DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7836103PMC
January 2021

Convalescent plasma therapy for the treatment of patients with COVID-19: Assessment of methods available for antibody detection and their correlation with neutralising antibody levels.

Transfus Med 2020 Dec 17. Epub 2020 Dec 17.

NHS Blood and Transplant, Oxford, John Radcliffe Hospital, Oxford, UK.

Introduction: The lack of approved specific therapeutic agents to treat coronavirus disease (COVID-19) associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has led to the rapid implementation of convalescent plasma therapy (CPT) trials in many countries, including the United Kingdom. Effective CPT is likely to require high titres of neutralising antibody (nAb) in convalescent donations. Understanding the relationship between functional neutralising antibodies and antibody levels to specific SARS-CoV-2 proteins in scalable assays will be crucial for the success of a large-scale collection. We assessed whether neutralising antibody titres correlated with reactivity in a range of enzyme-linked immunosorbent assays (ELISA) targeting the spike (S) protein, the main target for human immune response.

Methods: Blood samples were collected from 52 individuals with a previous laboratory-confirmed SARS-CoV-2 infection. These were assayed for SARS-CoV-2 nAbs by microneutralisation and pseudo-type assays and for antibodies by four different ELISAs. Receiver operating characteristic (ROC) analysis was used to further identify sensitivity and specificity of selected assays to identify samples containing high nAb levels.

Results: All samples contained SARS-CoV-2 antibodies, whereas neutralising antibody titres of greater than 1:20 were detected in 43 samples (83% of those tested) and >1:100 in 22 samples (42%). The best correlations were observed with EUROimmun immunoglobulin G (IgG) reactivity (Spearman Rho correlation coefficient 0.88; p < 0.001). Based on ROC analysis, EUROimmun would detect 60% of samples with titres of >1:100 with 100% specificity using a reactivity index of 9.1 (13/22).

Discussion: Robust associations between nAb titres and reactivity in several ELISA-based antibody tests demonstrate their possible utility for scaled-up production of convalescent plasma containing potentially therapeutic levels of anti-SARS-CoV-2 nAbs.
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http://dx.doi.org/10.1111/tme.12746DOI Listing
December 2020

SARS-CoV-2 RNA detected in blood products from patients with COVID-19 is not associated with infectious virus.

Wellcome Open Res 2020 12;5:181. Epub 2020 Oct 12.

Public Health England, 61 Colindale Ave, London, NW9 5EQ, UK.

Laboratory diagnosis of SARS-CoV-2 infection (the cause of COVID-19) uses PCR to detect viral RNA (vRNA) in respiratory samples. SARS-CoV-2 RNA has also been detected in other sample types, but there is limited understanding of the clinical or laboratory significance of its detection in blood. We undertook a systematic literature review to assimilate the evidence for the frequency of vRNA in blood, and to identify associated clinical characteristics. We performed RT-PCR in serum samples from a UK clinical cohort of acute and convalescent COVID-19 cases (n=212), together with convalescent plasma samples collected by NHS Blood and Transplant (NHSBT) (n=462 additional samples). To determine whether PCR-positive blood samples could pose an infection risk, we attempted virus isolation from a subset of RNA-positive samples. We identified 28 relevant studies, reporting SARS-CoV-2 RNA in 0-76% of blood samples; pooled estimate 10% (95%CI 5-18%). Among serum samples from our clinical cohort, 27/212 (12.7%) had SARS-CoV-2 RNA detected by RT-PCR. RNA detection occurred in samples up to day 20 post symptom onset, and was associated with more severe disease (multivariable odds ratio 7.5). Across all samples collected ≥28 days post symptom onset, 0/494 (0%, 95%CI 0-0.7%) had vRNA detected. Among our PCR-positive samples, cycle threshold (ct) values were high (range 33.5-44.8), suggesting low vRNA copy numbers. PCR-positive sera inoculated into cell culture did not produce any cytopathic effect or yield an increase in detectable SARS-CoV-2 RNA. There was a relationship between RT-PCR negativity and the presence of total SARS-CoV-2 antibody (p=0.02). vRNA was detectable at low viral loads in a minority of serum samples collected in acute infection, but was not associated with infectious SARS-CoV-2 (within the limitations of the assays used). This work helps to inform biosafety precautions for handling blood products from patients with current or previous COVID-19.
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http://dx.doi.org/10.12688/wellcomeopenres.16002.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7689603PMC
October 2020

Detection of neutralising antibodies to SARS-CoV-2 to determine population exposure in Scottish blood donors between March and May 2020.

Euro Surveill 2020 10;25(42)

Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom.

BackgroundThe progression and geographical distribution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in the United Kingdom (UK) and elsewhere is unknown because typically only symptomatic individuals are diagnosed. We performed a serological study of blood donors in Scotland in the spring of 2020 to detect neutralising antibodies to SARS-CoV-2 as a marker of past infection and epidemic progression.AimOur objective was to determine if sera from blood bank donors can be used to track the emergence and progression of the SARS-CoV-2 epidemic.MethodsA pseudotyped SARS-CoV-2 virus microneutralisation assay was used to detect neutralising antibodies to SARS-CoV-2. The study comprised samples from 3,500 blood donors collected in Scotland between 17 March and 18 May 2020. Controls were collected from 100 donors in Scotland during 2019.ResultsAll samples collected on 17 March 2020 (n = 500) were negative in the pseudotyped SARS-CoV-2 virus microneutralisation assay. Neutralising antibodies were detected in six of 500 donors from 23 to 26 March. The number of samples containing neutralising antibodies did not significantly rise after 5-6 April until the end of the study on 18 May. We found that infections were concentrated in certain postcodes, indicating that outbreaks of infection were extremely localised. In contrast, other areas remained comparatively untouched by the epidemic.ConclusionAlthough blood donors are not representative of the overall population, we demonstrated that serosurveys of blood banks can serve as a useful tool for tracking the emergence and progression of an epidemic such as the SARS-CoV-2 outbreak.
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http://dx.doi.org/10.2807/1560-7917.ES.2020.25.42.2000685DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7651873PMC
October 2020

Antibodies targeting epitopes on the cell-surface form of NS1 protect against Zika virus infection during pregnancy.

Nat Commun 2020 10 19;11(1):5278. Epub 2020 Oct 19.

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, 63110, USA.

There are no licensed therapeutics or vaccines available against Zika virus (ZIKV) to counteract its potential for congenital disease. Antibody-based countermeasures targeting the ZIKV envelope protein have been hampered by concerns for cross-reactive responses that induce antibody-dependent enhancement (ADE) of heterologous flavivirus infection. Nonstructural protein 1 (NS1) is a membrane-associated and secreted glycoprotein that functions in flavivirus replication and immune evasion but is absent from the virion. Although some studies suggest that antibodies against ZIKV NS1 are protective, their activity during congenital infection is unknown. Here we develop mouse and human anti-NS1 monoclonal antibodies that protect against ZIKV in both non-pregnant and pregnant mice. Avidity of antibody binding to cell-surface NS1 along with Fc effector functions engagement correlate with protection in vivo. Protective mAbs map to exposed epitopes in the wing domain and loop face of the β-platform. Anti-NS1 antibodies provide an alternative strategy for protection against congenital ZIKV infection without causing ADE.
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http://dx.doi.org/10.1038/s41467-020-19096-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7572419PMC
October 2020

Autoantibody-dependent amplification of inflammation in SLE.

Cell Death Dis 2020 09 9;11(9):729. Epub 2020 Sep 9.

Department of Medicine, Centre for Immunology & Vaccinology, Chelsea and Westminster Hospital, Imperial College London, London, SW10 9NH, UK.

Anti-double stranded DNA antibodies (anti-dsDNA) are a hallmark of SLE but their role in disease pathogenesis is not fully resolved. Anti-dsDNA in serum are highly heterogeneous therefore in this study, we aimed to dissect the functional specificities of anti-dsDNA using a panel of human monoclonal antibodies (humAbs) generated from patients with active lupus nephritis. A total of 46 ANA reactive humAbs were isolated and divided into four broad classes based on their reactivity to histones, DNA and Crithidia. Functional analysis indicated that one subclass of antibodies bound strongly to decondensed DNA areas in neutrophil extracellular traps (NETs) and protected NETs from nuclease digestion, similar to the sera from active SLE patients. In addition, these anti-dsDNA antibodies could stimulate type I interferon responses in mononuclear phagocytic cells, or NF-kB activity in endothelial cells, by uptake of NETs-anti-NETs immune complexes and subsequently trigging inflammatory responses in an Fc-gamma receptor (Fcg-R)-dependant manner. Together our data suggest that only a subset of anti-dsDNA antibodies is capable to amplify inflammatory responses by deposit in the nephritic kidney in vivo, protecting NETs digestion as well as uptake of NETs immune complexes into Fcg-R-expressing cells in vitro.
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http://dx.doi.org/10.1038/s41419-020-02928-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7481301PMC
September 2020

Broad and strong memory CD4 and CD8 T cells induced by SARS-CoV-2 in UK convalescent individuals following COVID-19.

Nat Immunol 2020 11 4;21(11):1336-1345. Epub 2020 Sep 4.

MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, UK.

The development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines and therapeutics will depend on understanding viral immunity. We studied T cell memory in 42 patients following recovery from COVID-19 (28 with mild disease and 14 with severe disease) and 16 unexposed donors, using interferon-γ-based assays with peptides spanning SARS-CoV-2 except ORF1. The breadth and magnitude of T cell responses were significantly higher in severe as compared with mild cases. Total and spike-specific T cell responses correlated with spike-specific antibody responses. We identified 41 peptides containing CD4 and/or CD8 epitopes, including six immunodominant regions. Six optimized CD8 epitopes were defined, with peptide-MHC pentamer-positive cells displaying the central and effector memory phenotype. In mild cases, higher proportions of SARS-CoV-2-specific CD8 T cells were observed. The identification of T cell responses associated with milder disease will support an understanding of protective immunity and highlights the potential of including non-spike proteins within future COVID-19 vaccine design.
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http://dx.doi.org/10.1038/s41590-020-0782-6DOI Listing
November 2020

Structural basis for the neutralization of SARS-CoV-2 by an antibody from a convalescent patient.

Nat Struct Mol Biol 2020 10 31;27(10):950-958. Epub 2020 Jul 31.

Research Center for Emerging Viral Infections, College of Medicine, Chang Gung University, Taoyuan, Taiwan.

The COVID-19 pandemic has had an unprecedented health and economic impact and there are currently no approved therapies. We have isolated an antibody, EY6A, from an individual convalescing from COVID-19 and have shown that it neutralizes SARS-CoV-2 and cross-reacts with SARS-CoV-1. EY6A Fab binds the receptor binding domain (RBD) of the viral spike glycoprotein tightly (K of 2 nM), and a 2.6-Å-resolution crystal structure of an RBD-EY6A Fab complex identifies the highly conserved epitope, away from the ACE2 receptor binding site. Residues within this footprint are key to stabilizing the pre-fusion spike. Cryo-EM analyses of the pre-fusion spike incubated with EY6A Fab reveal a complex of the intact spike trimer with three Fabs bound and two further multimeric forms comprising the destabilized spike attached to Fab. EY6A binds what is probably a major neutralizing epitope, making it a candidate therapeutic for COVID-19.
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http://dx.doi.org/10.1038/s41594-020-0480-yDOI Listing
October 2020

Neutralization of SARS-CoV-2 by Destruction of the Prefusion Spike.

Cell Host Microbe 2020 09 19;28(3):445-454.e6. Epub 2020 Jun 19.

Division of Structural Biology, University of Oxford, The Wellcome Centre for Human Genetics, Headington, Oxford, OX3 7BN, UK; Diamond Light Source Ltd, Harwell Science & Innovation Campus, Didcot, OX11 0DE, UK; Instruct-ERIC, Oxford House, Parkway Court, John Smith Drive, Oxford, OX4 2JY, UK. Electronic address:

There are as yet no licensed therapeutics for the COVID-19 pandemic. The causal coronavirus (SARS-CoV-2) binds host cells via a trimeric spike whose receptor binding domain (RBD) recognizes angiotensin-converting enzyme 2, initiating conformational changes that drive membrane fusion. We find that the monoclonal antibody CR3022 binds the RBD tightly, neutralizing SARS-CoV-2, and report the crystal structure at 2.4 Å of the Fab/RBD complex. Some crystals are suitable for screening for entry-blocking inhibitors. The highly conserved, structure-stabilizing CR3022 epitope is inaccessible in the prefusion spike, suggesting that CR3022 binding facilitates conversion to the fusion-incompetent post-fusion state. Cryogenic electron microscopy (cryo-EM) analysis confirms that incubation of spike with CR3022 Fab leads to destruction of the prefusion trimer. Presentation of this cryptic epitope in an RBD-based vaccine might advantageously focus immune responses. Binders at this epitope could be useful therapeutically, possibly in synergy with an antibody that blocks receptor attachment.
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http://dx.doi.org/10.1016/j.chom.2020.06.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7303615PMC
September 2020

Broad and strong memory CD4 and CD8 T cells induced by SARS-CoV-2 in UK convalescent COVID-19 patients.

bioRxiv 2020 Jun 8. Epub 2020 Jun 8.

COVID-19 is an ongoing global crisis in which the development of effective vaccines and therapeutics will depend critically on understanding the natural immunity to the virus, including the role of SARS-CoV-2-specific T cells. We have conducted a study of 42 patients following recovery from COVID-19, including 28 mild and 14 severe cases, comparing their T cell responses to those of 16 control donors. We assessed the immune memory of T cell responses using IFNγ based assays with overlapping peptides spanning SARS-CoV-2 apart from ORF1. We found the breadth, magnitude and frequency of memory T cell responses from COVID-19 were significantly higher in severe compared to mild COVID-19 cases, and this effect was most marked in response to spike, membrane, and ORF3a proteins. Total and spike-specific T cell responses correlated with the anti-Spike, anti-Receptor Binding Domain (RBD) as well as anti-Nucleoprotein (NP) endpoint antibody titre (p<0.001, <0.001 and =0.002). We identified 39 separate peptides containing CD4 and/or CD8 epitopes, which strikingly included six immunodominant epitope clusters targeted by T cells in many donors, including 3 clusters in spike (recognised by 29%, 24%, 18% donors), two in the membrane protein (M, 32%, 47%) and one in the nucleoprotein (Np, 35%). CD8+ responses were further defined for their HLA restriction, including B*4001-restricted T cells showing central memory and effector memory phenotype. In mild cases, higher frequencies of multi-cytokine producing M- and NP-specific CD8 T cells than spike-specific CD8 T cells were observed. They furthermore showed a higher ratio of SARS-CoV-2-specific CD8 to CD4 T cell responses. Immunodominant epitope clusters and peptides containing T cell epitopes identified in this study will provide critical tools to study the role of virus-specific T cells in control and resolution of SARS-CoV-2 infections. The identification of T cell specificity and functionality associated with milder disease, highlights the potential importance of including non-spike proteins within future COVID-19 vaccine design.
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http://dx.doi.org/10.1101/2020.06.05.134551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7302222PMC
June 2020

Immunogenicity and Efficacy of Zika Virus Envelope Domain III in DNA, Protein, and ChAdOx1 Adenoviral-Vectored Vaccines.

Vaccines (Basel) 2020 Jun 16;8(2). Epub 2020 Jun 16.

The Jenner Institute, Nuffield Department of Medicine, University of Oxford, The Henry Wellcome Building for Molecular Physiology, Roosevelt Drive, Oxford OX3 7BN, UK.

The flavivirus envelope protein domain III (EDIII) was an effective immunogen against dengue virus (DENV) and other related flaviviruses. Whether this can be applied to the Zika virus (ZIKV) vaccinology remains an open question. Here, we tested the efficacy of ZIKV-EDIII against ZIKV infection, using several vaccine platforms that present the antigen in various ways. We provide data demonstrating that mice vaccinated with a ZIKV-EDIII as DNA or protein-based vaccines failed to raise fully neutralizing antibodies and did not control viremia, following a ZIKV challenge, despite eliciting robust antibody responses. Furthermore, we showed that ZIKV-EDIII encoded in replication-deficient Chimpanzee adenovirus (ChAdOx1-EDIII) elicited anti-ZIKV envelope antibodies in vaccinated mice but also provided limited protection against ZIKV in two physiologically different mouse challenge models. Taken together, our data indicate that contrary to what was shown for other flaviviruses like the dengue virus, which has close similarities with ZIKV-EDIII, this antigen might not be a suitable vaccine candidate for the correct induction of protective immune responses against ZIKV.
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http://dx.doi.org/10.3390/vaccines8020307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7350260PMC
June 2020

Author Correction: Human antibodies to the dengue virus E-dimer epitope have therapeutic activity against Zika virus infection.

Nat Immunol 2020 Mar;21(3):354

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41590-019-0560-5DOI Listing
March 2020

A protective Zika virus E-dimer-based subunit vaccine engineered to abrogate antibody-dependent enhancement of dengue infection.

Nat Immunol 2019 10 2;20(10):1291-1298. Epub 2019 Sep 2.

Division of Medical Sciences, University of Oxford, Oxford, UK.

Infections with dengue virus (DENV) and Zika virus (ZIKV) can induce cross-reactive antibody responses. Two immunodominant epitopes-one to precursor membrane protein and one to the fusion loop epitope on envelope (E) protein-are recognized by cross-reactive antibodies that are not only poorly neutralizing, but can also promote increased viral replication and disease severity via Fcγ receptor-mediated infection of myeloid cells-a process termed antibody-dependent enhancement (ADE). ADE is a significant concern for both ZIKV and DENV vaccines as the induction of poorly neutralizing cross-reactive antibodies may prime an individual for ADE on natural infection. In this report, we describe the design and production of covalently stabilized ZIKV E dimers, which lack precursor membrane protein and do not expose the immunodominant fusion loop epitope. Immunization of mice with ZIKV E dimers induces dimer-specific antibodies, which protect against ZIKV challenge during pregnancy. Importantly, the ZIKV E-dimer-induced response does not cross-react with DENV or induce ADE of DENV infection.
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http://dx.doi.org/10.1038/s41590-019-0477-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6839414PMC
October 2019

Dengue and Zika Virus Cross-Reactive Human Monoclonal Antibodies Protect against Spondweni Virus Infection and Pathogenesis in Mice.

Cell Rep 2019 02;26(6):1585-1597.e4

Department of Medicine, Washington University School of Medicine, Saint Louis, MO 63110, USA; Department of Pathology & Immunology, Washington University School of Medicine, Saint Louis, MO 63110, USA; Department of Molecular Microbiology, Washington University School of Medicine, Saint Louis, MO 63110, USA; The Andrew M. and Jane M. Bursky Center for Human Immunology and Immunotherapy Programs, Washington University School of Medicine, Saint Louis, MO 63110, USA. Electronic address:

Spondweni virus (SPOV) is the flavivirus that is most closely related to Zika virus (ZIKV). Although SPOV causes sporadic human infections in Africa, recently it was found in Culex mosquitoes in Haiti. To investigate the pathogenic spectrum of SPOV, we developed infection models in mice. Although two SPOV strains failed to cause disease in immunocompetent mice, each accumulated in the brain, spleen, eye, testis, and kidney when type I interferon signaling was blocked and unexpectedly caused infection, immune cell infiltration, and swelling in the ankle. In pregnant mice, SPOV replicated in the placenta and fetus but did not cause placental insufficiency or microcephaly. We identified human antibodies from ZIKV or DENV immune subjects that neutralized SPOV infection and protected against lethal challenge. Our experiments describe similarities and differences in clinical syndromes between SPOV and ZIKV and suggest that their serological relatedness has implications for antibody therapeutics and flavivirus vaccine development.
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http://dx.doi.org/10.1016/j.celrep.2019.01.052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6420780PMC
February 2019

Neutrophil Activation and Early Features of NET Formation Are Associated With Dengue Virus Infection in Human.

Front Immunol 2018 11;9:3007. Epub 2019 Jan 11.

Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, Thailand.

The involvement of the immune system in the protection and pathology of natural dengue virus (DENV) has been extensively studied. However, despite studies that have referred to activation of neutrophils in DENV infections, the exact roles of neutrophils remain elusive. Here, we explored the phenotypic and functional responses of neutrophils in a cohort of adult dengue patients. Results indicated that during an acute DENV infection, neutrophils up-regulate CD66b expression, and produce a more robust respiratory response as compared with that in convalescent or healthy individuals; this confirmed neutrophil activation during DENV infection. Spontaneous decondensation of nuclei, an early event of neutrophil extracellular trap (NET) formation, was also markedly increased in cells isolated from DENV-infected patients during the acute phase of the infection. incubation of NETs with DENV-2 virus significantly decreased DENV infectivity. Interestingly, increased levels of NET components were found in the serum of patients with more severe disease form-dengue hemorrhagic fever (DHF), but not uncomplicated dengue fever, during the acute phase of the infection. Levels of pro-inflammatory cytokines IL-8 and TNFα were also increased in DHF patients as compared with those in healthy and DF subjects. This suggested that NETs may play dual roles during DENV infection. The increased ability for NET formation during acute DENV infection appeared to be independent of PAD4-mediated histone H3 hyper-citrullination. Our study suggests that neutrophils are involved in immunological responses to DENV infection.
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http://dx.doi.org/10.3389/fimmu.2018.03007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6336714PMC
October 2019

The immune response against flaviviruses.

Nat Immunol 2018 11 17;19(11):1189-1198. Epub 2018 Oct 17.

Division of Medical Sciences, John Radcliffe Hospital, University of Oxford, Oxford, UK.

Arthropod-borne flaviviruses are important human pathogens that cause a diverse range of clinical conditions, including severe hemorrhagic syndromes, neurological complications and congenital malformations. Consequently, there is an urgent need to develop safe and effective vaccines, a process requiring better understanding of the immunological mechanisms involved during infection. Decades of research suggest a paradoxical role of the immune response against flaviviruses: although the immune response is crucial for the control, clearance and prevention of infection, poor clinical outcomes are commonly associated with virus-specific immunity and immunopathogenesis. This relationship is further complicated by the high homology among viruses and the implication of cross-reactive immune responses in protection and pathogenesis. This Review examines the dual role of the adaptive immune response against flaviviruses, particularly emphasizing the most recent findings regarding cross-reactive T cell and antibody responses, and the effects that these concepts have on vaccine-development endeavors.
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http://dx.doi.org/10.1038/s41590-018-0210-3DOI Listing
November 2018

Characterization of a potent and highly unusual minimally enhancing antibody directed against dengue virus.

Nat Immunol 2018 11 15;19(11):1248-1256. Epub 2018 Oct 15.

Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK.

Dengue virus is a major pathogen, and severe infections can lead to life-threatening dengue hemorrhagic fever. Dengue virus exists as four serotypes, and dengue hemorrhagic fever is often associated with secondary heterologous infections. Antibody-dependent enhancement (ADE) may drive higher viral loads in these secondary infections and is purported to result from antibodies that recognize dengue virus but fail to fully neutralize it. Here we characterize two antibodies, 2C8 and 3H5, that bind to the envelope protein. Antibody 3H5 is highly unusual as it not only is potently neutralizing but also promotes little if any ADE, whereas antibody 2C8 has strong capacity to promote ADE. We show that 3H5 shows resilient binding in endosomal pH conditions and neutralizes at low occupancy. Immunocomplexes of 3H5 and dengue virus do not efficiently interact with Fcγ receptors, which we propose is due to the binding mode of 3H5 and constitutes the primary mechanism of how ADE is avoided.
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http://dx.doi.org/10.1038/s41590-018-0227-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7051845PMC
November 2018

Potent Neutralizing Human Monoclonal Antibodies Preferentially Target Mature Dengue Virus Particles: Implication for Novel Strategy for Dengue Vaccine.

J Virol 2018 12 12;92(23). Epub 2018 Nov 12.

Department of Tropical Medicine, Medical Microbiology and Pharmacology, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, Hawaii, USA

The four serotypes of dengue virus (DENV) cause the most important mosquito-borne viral disease in humans. The envelope (E) protein is the major target of neutralizing antibodies and contains 3 domains (domain I [DI], DII, and DIII). Recent studies reported that human monoclonal antibodies (MAbs) recognizing DIII, the D1/DII hinge, the E-dimer epitope, or a quaternary epitope involving DI/DII/DIII are more potently neutralizing than those recognizing the fusion loop (FL) of DII. Due to inefficient cleavage of the premembrane protein, DENV suspensions consist of a mixture of mature, immature, and partially immature particles. We investigated the neutralization and binding of 22 human MAbs to DENV serotype 1 (DENV1) virions with differential maturation status. Compared with FL MAbs, DIII, DI/DII hinge, and E-dimer epitope MAbs showed higher maximum binding and avidity to mature particles relative to immature particles; this feature may contribute to the strong neutralizing potency of such MAbs. FL-specific MAbs required 57 to 87% occupancy on mature particles to achieve half-maximal neutralization (NT), whereas the potently neutralizing MAbs achieved NT states at 20 to 38% occupancy. Analysis of the MAb repertoire and polyclonal sera from patients with primary DENV1 infection supports the immunodominance of cross-reactive anti-E antibodies over type-specific antibodies. After depletion with viral particles from a heterologous DENV serotype, the type-specific neutralizing antibodies remained and showed binding features shared by potent neutralizing MAbs. Taken together, these findings suggest that the use of homogeneous mature DENV particles as an immunogen may induce more potent neutralizing antibodies against DENV than the use of immature or mixed particles. With an estimated 390 million infections per year, the four serotypes of dengue virus (DENV) cause the most important mosquito-borne viral disease in humans. The dengue vaccine Dengvaxia was licensed; however, its low efficacy among dengue-naive individuals and increased risk of causing severe dengue in children highlight the need for a better understanding of the role of human antibodies in immunity against DENV. DENV suspensions contain mature, immature, and partially immature particles. We investigated the binding of 22 human monoclonal antibodies (MAbs) to the DENV envelope protein on particles with different maturation states. Potently neutralizing MAbs had higher relative maximum binding and avidity to mature particles than weakly neutralizing MAbs. This was supported by analysis of MAb repertoires and polyclonal sera from patients with primary DENV infection. Together, these findings suggest that mature particles may be the optimal form of presentation of the envelope protein to induce more potent neutralizing antibodies against DENV.
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http://dx.doi.org/10.1128/JVI.00556-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6232466PMC
December 2018

Rational Zika vaccine design via the modulation of antigen membrane anchors in chimpanzee adenoviral vectors.

Nat Commun 2018 06 22;9(1):2441. Epub 2018 Jun 22.

The Jenner Institute, Nuffield Department of Medicine, University of Oxford, The Henry Wellcome Building for Molecular Physiology, Roosevelt Drive, Oxford, OX3 7BN, UK.

Zika virus (ZIKV) emerged on a global scale and no licensed vaccine ensures long-lasting anti-ZIKV immunity. Here we report the design and comparative evaluation of four replication-deficient chimpanzee adenoviral (ChAdOx1) ZIKV vaccine candidates comprising the addition or deletion of precursor membrane (prM) and envelope, with or without its transmembrane domain (TM). A single, non-adjuvanted vaccination of ChAdOx1 ZIKV vaccines elicits suitable levels of protective responses in mice challenged with ZIKV. ChAdOx1 prME ∆TM encoding prM and envelope without TM provides 100% protection, as well as long-lasting anti-envelope immune responses and no evidence of in vitro antibody-dependent enhancement to dengue virus. Deletion of prM and addition of TM reduces protective efficacy and yields lower anti-envelope responses. Our finding that immunity against ZIKV can be enhanced by modulating antigen membrane anchoring highlights important parameters in the design of viral vectored ZIKV vaccines to support further clinical assessments.
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http://dx.doi.org/10.1038/s41467-018-04859-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6015009PMC
June 2018

Therapeutic and protective efficacy of a dengue antibody against Zika infection in rhesus monkeys.

Nat Med 2018 06 4;24(6):721-723. Epub 2018 Jun 4.

Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.

Strategies to treat Zika virus (ZIKV) infection in dengue virus (DENV)-endemic areas are urgently needed. Here we show that a DENV-specific antibody against the E-dimer epitope (EDE) potently cross-neutralizes ZIKV and provides robust therapeutic efficacy as well as prophylactic efficacy against ZIKV in rhesus monkeys. Viral escape was not detected, suggesting a relatively high bar to escape. These data demonstrate the potential for antibody-based therapy and prevention of ZIKV.
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http://dx.doi.org/10.1038/s41591-018-0056-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5992501PMC
June 2018

Longitudinal Analysis of Antibody Cross-neutralization Following Zika Virus and Dengue Virus Infection in Asia and the Americas.

J Infect Dis 2018 07;218(4):536-545

Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley.

Background: The 4 dengue virus serotypes (DENV1-4) and Zika virus (ZIKV) are related mosquito-borne flaviviruses of major importance globally. While monoclonal antibodies and plasma from DENV-immune donors can neutralize or enhance ZIKV in vitro and in small-animal models, and vice versa, the extent, duration, and significance of cross-reactivity in humans remains unknown, particularly in flavivirus-endemic regions.

Methods: We studied neutralizing antibodies to ZIKV and DENV1-4 in longitudinal serologic specimens collected through 3 years after infection from people in Latin America and Asia with laboratory-confirmed DENV infections. We also evaluated neutralizing antibodies to ZIKV and DENV1-4 in patients with Zika through 6 months after infection.

Results: In patients with Zika, the highest neutralizing antibody titers were to ZIKV, with low-level cross-reactivity to DENV1-4 that was greater in DENV-immune individuals. We found that, in primary and secondary DENV infections, neutralizing antibody titers to ZIKV were markedly lower than to the infecting DENV and heterologous DENV serotypes. Cross-neutralization was greatest in early convalescence, then ZIKV neutralization decreased, remaining at low levels over time.

Conclusions: Patterns of antibody cross-neutralization suggest that ZIKV lies outside the DENV serocomplex. Neutralizing antibody titers can distinguish ZIKV from DENV infections when all viruses are analyzed simultaneously. These findings have implications for understanding natural immunity and vaccines.
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http://dx.doi.org/10.1093/infdis/jiy164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6047418PMC
July 2018

The immunology of Zika Virus.

F1000Res 2018 19;7:203. Epub 2018 Feb 19.

Medical Sciences Division, University of Oxford, Level 3, John Radcliffe Hospital, Oxford, OX3 9DU, UK.

Zika virus (ZIKV) was initially thought to cause only mild, self-limiting symptoms. However, recent outbreaks have been associated with the autoimmune disease Guillain-Barré syndrome and causally linked to a congenital malformation known as microcephaly. This has led to an urgent need for a safe and effective vaccine. A comprehensive understanding of the immunology of ZIKV infection is required to aid in the design of such a vaccine. Whilst details of both innate and adaptive immune responses to ZIKV are emerging, further research is needed. As immunopathogenesis has been implicated in poor outcomes following infection with the related dengue virus, identification of cross-reactive immune responses between flaviviruses and the impact they may have on disease progression is also of high importance.
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http://dx.doi.org/10.12688/f1000research.12271.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5820595PMC
February 2018

Evolution of neurovirulent Zika virus.

Science 2017 11;358(6365):863-864

Department of Medicine, Imperial College London, Commonwealth Building, Hammersmith Campus, Du Cane Road, London W12 0NN, UK.

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http://dx.doi.org/10.1126/science.aaq1297DOI Listing
November 2017

Human antibodies to the dengue virus E-dimer epitope have therapeutic activity against Zika virus infection.

Nat Immunol 2017 Nov 25;18(11):1261-1269. Epub 2017 Sep 25.

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA.

The Zika virus (ZIKV) epidemic has resulted in congenital abnormalities in fetuses and neonates. Although some cross-reactive dengue virus (DENV)-specific antibodies can enhance ZIKV infection in mice, those recognizing the DENV E-dimer epitope (EDE) can neutralize ZIKV infection in cell culture. We evaluated the therapeutic activity of human monoclonal antibodies to DENV EDE for their ability to control ZIKV infection in the brains, testes, placentas, and fetuses of mice. A single dose of the EDE1-B10 antibody given 3 d after ZIKV infection protected against lethality, reduced ZIKV levels in brains and testes, and preserved sperm counts. In pregnant mice, wild-type or engineered LALA variants of EDE1-B10, which cannot engage Fcg receptors, diminished ZIKV burden in maternal and fetal tissues, and protected against fetal demise. Because neutralizing antibodies to EDE have therapeutic potential against ZIKV, in addition to their established inhibitory effects against DENV, it may be possible to develop therapies that control disease caused by both viruses.
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http://dx.doi.org/10.1038/ni.3849DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5679314PMC
November 2017

Germline bias dictates cross-serotype reactivity in a common dengue-virus-specific CD8 T cell response.

Nat Immunol 2017 Nov 25;18(11):1228-1237. Epub 2017 Sep 25.

Department of Medicine, Imperial College London, London, UK.

Adaptive immune responses protect against infection with dengue virus (DENV), yet cross-reactivity with distinct serotypes can precipitate life-threatening clinical disease. We found that clonotypes expressing the T cell antigen receptor (TCR) β-chain variable region 11 (TRBV11-2) were 'preferentially' activated and mobilized within immunodominant human-leukocyte-antigen-(HLA)-A*11:01-restricted CD8 T cell populations specific for variants of the nonstructural protein epitope NS3 that characterize the serotypes DENV1, DENV3 and DENV4. In contrast, the NS3-DENV2-specific repertoire was largely devoid of such TCRs. Structural analysis of a representative TRBV11-2 TCR demonstrated that cross-serotype reactivity was governed by unique interplay between the variable antigenic determinant and germline-encoded residues in the second β-chain complementarity-determining region (CDR2β). Extensive mutagenesis studies of three distinct TRBV11-2 TCRs further confirmed that antigen recognition was dependent on key contacts between the serotype-defined peptide and discrete residues in the CDR2β loop. Collectively, these data reveal an innate-like mode of epitope recognition with potential implications for the outcome of sequential exposure to heterologous DENVs.
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http://dx.doi.org/10.1038/ni.3850DOI Listing
November 2017

The immunopathology of dengue and Zika virus infections.

Curr Opin Immunol 2017 10 21;48:1-6. Epub 2017 Jul 21.

Department of Medicine, Imperial College London, London, United Kingdom. Electronic address:

A large proportion of the world's population live in areas with dengue virus (DENV) transmission resulting in tens of millions of symptomatic dengue cases each year. Serious complications following DENV infection occur more frequently in those suffering from a second or subsequent infection implicating virus-specific immunity as having a role in pathogenesis. In recent years outbreaks of the related Zika virus (ZIKV) have been associated with birth defects and neurological complications. As DENV and ZIKV share a viral vector sequential infections can occur. Given the sequence homology between the two viruses, the generation of cross-reactive immune responses is highly likely. This review examines the role immunopathogenesis plays during DENV infection as well as highlighting recent studies that demonstrate DENV immunity may have an effect on the outcome of ZIKV infection.
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http://dx.doi.org/10.1016/j.coi.2017.07.001DOI Listing
October 2017

Which Dengue Vaccine Approach Is the Most Promising, and Should We Be Concerned about Enhanced Disease after Vaccination? The Challenges of a Dengue Vaccine.

Cold Spring Harb Perspect Biol 2018 06 1;10(6). Epub 2018 Jun 1.

Faculty of Medicine, Imperial College London, London SW7 2AZ, United Kingdom.

A dengue vaccine has been pursued for more than 50 years and, unlike other flaviviral vaccines such as that against yellow fever, progress has been slow. In this review, we describe progress toward the first licensed dengue vaccine Dengvaxia, which does not give complete protection against disease. The antibody response to the dengue virion is reviewed, highlighting immunodominant yet poorly neutralizing responses in the context of a highly dynamic structurally flexible dengue virus particle. Finally, we review recent evidence for cross-reactivity between antibody responses to Zika and dengue viruses, which may further complicate the development of broadly protective dengue virus vaccines.
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http://dx.doi.org/10.1101/cshperspect.a029520DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5983192PMC
June 2018

Covalently linked dengue virus envelope glycoprotein dimers reduce exposure of the immunodominant fusion loop epitope.

Nat Commun 2017 05 23;8:15411. Epub 2017 May 23.

Division of Immunology and Inflammation, Department of Medicine, Hammersmith Hospital Campus, Imperial College London, w12 0NN London, UK.

A problem in the search for an efficient vaccine against dengue virus is the immunodominance of the fusion loop epitope (FLE), a segment of the envelope protein E that is buried at the interface of the E dimers coating mature viral particles. Anti-FLE antibodies are broadly cross-reactive but poorly neutralizing, displaying a strong infection enhancing potential. FLE exposure takes place via dynamic 'breathing' of E dimers at the virion surface. In contrast, antibodies targeting the E dimer epitope (EDE), readily exposed at the E dimer interface over the region of the conserved fusion loop, are very potent and broadly neutralizing. We here engineer E dimers locked by inter-subunit disulfide bonds, and show by X-ray crystallography and by binding to a panel of human antibodies that these engineered dimers do not expose the FLE, while retaining the EDE exposure. These locked dimers are strong immunogen candidates for a next-generation vaccine.
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http://dx.doi.org/10.1038/ncomms15411DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5457521PMC
May 2017