Epitope Addition and Ablation via Manipulation of a Dengue Virus Serotype 1 Infectious Clone.
ABSTRACT: Despite the clinical relevance, dengue virus (DENV) research has been hampered by the absence of robust reverse genetic systems to manipulate the viral serotypes for propagation and generation of mutant viruses. In this article, we describe application of an infectious clone system for DENV serotype 1 (DENV1). Similar to previous clones in both flaviviruses and coronaviruses, the approach constructs a panel of contiguous cDNAs that span the DENV genome and can be systematically and directionally assembled to produce viable, full-length viruses. Comparison of the virus derived from the infectious clone with the original viral isolate reveals identical sequence, comparable endpoint titers, and similar focus staining. Both focus-forming assays and percent infection by flow cytometry revealed overlapping replication levels in two different cell types. Moreover, serotype-specific monoclonal antibodies (MAbs) bound similarly to infectious clone and the natural isolate. Using the clone, we were able to insert a DENV4 type-specific epitope recognized by primate MAb 5H2 into envelope (E) protein domain I (EDI) of DENV1 and recover a viable chimeric recombinant virus. The recombinant DENV1 virus was recognized and neutralized by the DENV4 type-specific 5H2 MAb. The introduction of the 5H2 epitope ablated two epitopes on DENV1 EDI recognized by human MAbs (1F4 and 14C10) that strongly neutralize DENV1. Together, the work demonstrates the utility of the infectious clone and provides a resource to rapidly manipulate the DENV1 serotype for generation of recombinant and mutant viruses. IMPORTANCE Dengue viruses (DENVs) are significant mosquito-transmitted pathogens that cause widespread infection and can lead to severe infection and complications. Here we further characterize a novel and robust DENV serotype 1 (DENV1) infectious clone system that can be used to support basic and applied research. We demonstrate how the system can be used to probe the antigenic relationships between strains by creating viable recombinant viruses that display or lack major antibody epitopes. The DENV1 clone system and recombinant viruses can be used to analyze existing vaccine immune responses and inform second-generation bivalent vaccine designs.
Project description:The four dengue virus (DENV) serotypes are mosquito-borne flaviviruses responsible for dengue fever and dengue hemorrhagic fever. People exposed to DENV develop antibodies (Abs) that strongly neutralize the serotype responsible for infection. Historically, infection with DENV serotype 4 (DENV4) has been less common and less studied than infections with the other three serotypes. However, DENV4 has been responsible for recent large and sustained epidemics in Asia and Latin America. The neutralizing antibody responses and the epitopes targeted against DENV4 have not been characterized in human infection. In this study, we mapped and characterized epitopes on DENV4 recognized by neutralizing antibodies in people previously exposed to DENV4 infections or to a live attenuated DENV4 vaccine. To study the fine specificity of DENV4 neutralizing human antibodies, B cells from two people exposed to DENV4 were immortalized and screened to identify DENV-specific clones. Two human monoclonal antibodies (MAbs) that neutralized DENV4 were isolated, and their epitopes were finely mapped using recombinant viruses and alanine scan mutation array techniques. Both antibodies bound to quaternary structure epitopes near the hinge region between envelope protein domain I (EDI) and EDII. In parallel, to characterize the serum neutralizing antibody responses, convalescence-phase serum samples from people previously exposed to primary DENV4 natural infections or a monovalent DENV4 vaccine were analyzed. Natural infection and vaccination also induced serum-neutralizing antibodies that targeted similar epitope domains at the EDI/II hinge region. These studies defined a target of neutralizing antigenic site on DENV4 targeted by human antibodies following natural infection or vaccination.IMPORTANCE The four serotypes of dengue virus are the causative agents of dengue fever and dengue hemorrhagic fever. People exposed to primary DENV infections develop long-term neutralizing antibody responses, but these principally recognize only the infecting serotype. An effective vaccine against dengue should elicit long-lasting protective antibody responses to all four serotypes simultaneously. We and others have defined antigenic sites on the envelope (E) protein of viruses of dengue virus serotypes 1, 2, and 3 targeted by human neutralizing antibodies. The epitopes on DENV4 E protein targeted by the human neutralizing antibodies and the mechanisms of serotype 4 neutralization are poorly understood. Here, we report the properties of human antibodies that neutralize dengue virus serotype 4. People exposed to serotype 4 infections or a live attenuated serotype 4 vaccine developed neutralizing antibodies that bound to similar sites on the viral E protein. These studies have provided a foundation for developing and evaluating DENV4 vaccines.
Project description:The four dengue viruses (DENV1-4) are rapidly reemerging infectious RNA viruses. These positive-strand viral genomes contain structured 3' untranslated regions (UTRs) that interact with various host RNA binding proteins (RBPs). These RBPs are functionally important in viral replication, pathogenesis, and defense against host immune mechanisms. Here, we combined RNA chromatography and quantitative mass spectrometry to identify proteins interacting with DENV1-4 3' UTRs. As expected, RBPs displayed distinct binding specificity. Among them, we focused on quaking (QKI) because of its preference for the DENV4 3' UTR (DENV-4/SG/06K2270DK1/2005). RNA immunoprecipitation experiments demonstrated that QKI interacted with DENV4 genomes in infected cells. Moreover, QKI depletion enhanced infectious particle production of DENV4. On the contrary, QKI did not interact with DENV2 3' UTR, and DENV2 replication was not affected consistently by QKI depletion. Next, we mapped the QKI interaction site and identified a QKI response element (QRE) in DENV4 3' UTR. Interestingly, removal of QRE from DENV4 3' UTR abolished this interaction and increased DENV4 viral particle production. Introduction of the QRE to DENV2 3' UTR led to QKI binding and reduced DENV2 infectious particle production. Finally, reporter assays suggest that QKI reduced translation efficiency of viral RNA. Our work describes a novel function of QKI in restricting viral replication.
Project description:Dengue virus (DENV), a mosquito-borne virus, is responsible for millions of cases of infections worldwide. There are four DENV serotypes (DENV1 to -4). After a primary DENV infection, the antibodies elicited confer lifetime protection against that DENV serotype. However, in a secondary infection with another serotype, the preexisting antibodies may cause antibody-dependent enhancement (ADE) of infection of macrophage cells, leading to the development of the more severe form of disease, dengue hemorrhagic fever. Thus, a safe vaccine should stimulate protection against all dengue serotypes simultaneously. To facilitate the development of a vaccine, good knowledge of different DENV serotype structures is crucial. Structures of DENV1 and DENV2 had been solved previously. Here we present a near-atomic resolution cryo-electron microscopy (cryo-EM) structure of mature DENV4. Comparison of the DENV4 structure with similar-resolution cryo-EM structures of DENV1 and DENV2 showed differences in surface charge distribution, which may explain their differences in binding to cellular receptors, such as heparin. Also, observed variations in amino acid residues involved in interactions between envelope and membrane proteins on the virus surface correlate with their ability to undergo structural changes at higher temperatures.
Project description:BACKGROUND: Dengue fever is the most important vector-borne viral disease. Four serotypes of dengue virus, DENV1 to DENV4, coexist. Infection by one serotype elicits long-lasting immunity to that serotype but not the other three. Subsequent infection by a different serotype is a risk factor for severe dengue. Domain III (ED3) of the viral envelope protein interacts with cell receptors and contains epitopes recognized by neutralizing antibodies. We determined the serotype specificity and cross-reactivity of human IgMs directed against ED3 by using a well-characterized collection of 90 DENV-infected and 89 DENV-uninfected human serums. METHODS: The recognitions between the four serotypes of ED3 and the serums were assayed with an IgM antibody-capture ELISA (MAC-ELISA) and artificial homodimeric antigens. The results were analyzed with Receiving Operator Characteristic (ROC) curves. RESULTS: The DENV-infected serums contained IgMs that reacted with one or several ED3 serotypes. The discrimination by ED3 between serums infected by the homotypic DENV and uninfected serums varied with the serotype in the decreasing order DENV1?>?DENV2?>?DENV3?>?DENV4. The ED3 domain of DENV1 gave the highest discrimination between DENV-infected and DENV-uninfected serums, whatever the infecting serotype, and thus behaved like a universal ED3 domain for the detection of IgMs against DENV. Some ED3 serotypes discriminated between IgMs directed against the homotypic and heterotypic DENVs. The patterns of cross-reactivities and discriminations varied with the serotype. CONCLUSIONS: The results should help better understand the IgM immune response and protection against DENV since ED3 is widely used as an antigen in diagnostic assays and an immunogen in vaccine candidates.
Project description:Background. ?Recent trials of recombinant, live-attenuated chimeric yellow fever-dengue tetravalent dengue vaccine (CYD-TDV) demonstrated efficacy against symptomatic, virologically confirmed dengue disease with higher point estimates of efficacy toward dengue virus (DENV)3 and DENV4 and moderate levels toward DENV1 and DENV2. It is interesting to note that serotype-specific efficacy did not correlate with absolute neutralizing antibody (nAb) geometric mean titer (GMT) values measured in a Vero-based plaque reduction neutralization test assay. The absence of Fc? receptors on Vero cells may explain this observation. Methods. ?We performed parallel seroneutralization assays in Vero cells and CV-1 cells that express Fc?RIIa (CV-1-Fc) to determine the neutralizing and enhancing capacity of serotype-specific DENV Abs present in CYD-TDV clinical trial sera. Results. ?Enhancement of DENV infection was observed in CV-1-Fc cells in naturally exposed nonvaccine sera, mostly for DENV3 and DENV4, at high dilutions. The CYD-TDV-vaccinated sera showed similar enhancement patterns. The CV-1-Fc nAb GMT values were 2- to 9-fold lower than Vero for all serotypes in both naturally infected individuals and CYD-TDV-vaccinated subjects with and without previous dengue immunity. The relative (CV-1-Fc/Vero) GMT decrease for anti-DENV1 and anti-DENV2 responses was not greater than for the other serotypes. Conclusions. ?In vitro neutralization assays utilizing Fc?RIIa-expressing cells provide evidence that serotype-specific Ab enhancement may not be a primary factor in the serotype-specific efficacy differences exhibited in the CYD-TDV trials.
Project description:4 one-step, real-time, reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays were developed for the detection of dengue virus (DENV) serotypes by considering 2,056 full genome DENV sequences. DENV1 and DENV2 RT-LAMP assays were validated with 31 blood and 11 serum samples from Tanzania, Senegal, Sudan and Mauritania. DENV3 and DENV4 RT-LAMP assays were validated with 25 serum samples from Cambodia.4 final reaction primer mixes were obtained by using a combination of Principal Component Analysis of the full DENV genome sequences, and LAMP primer design based on sequence alignments using the LAVA software. These mixes contained 14 (DENV1), 12 (DENV2), 8 (DENV3) and 3 (DENV4) LAMP primer sets. The assays were evaluated with an External Quality Assessment panel from Quality Control for Molecular Diagnostics. The assays were serotype-specific and did not cross-detect with other flaviviruses. The limits of detection, with 95% probability, were 22 (DENV1), 542 (DENV2), 197 (DENV3) and 641 (DENV4) RNA molecules, and 100% reproducibility in the assays was obtained with up to 102 (DENV1) and 103 RNA molecules (DENV2, DENV3 and DENV4). Validation of the DENV2 assay with blood samples from Tanzania resulted in 23 samples detected by RT-LAMP, demonstrating that the assay is 100% specific and 95.8% sensitive (positive predictive value of 100% and a negative predictive value of 85.7%). All serum samples from Senegal, Sudan and Mauritania were detected and 3 untyped as DENV1. The sensitivity of RT-LAMP for DENV4 samples from Cambodia did not quite match qRT-PCR.We have shown a novel approach to design LAMP primers that makes use of fast growing sequence databases. The DENV1 and DENV2 assays were validated with viral RNA extracted clinical samples, showing very good performance parameters.
Project description:Dengue viruses (DENV) are mosquito-borne flaviviruses of global importance. DENV exist as four serotypes, DENV1-DENV4. Following a primary infection, individuals produce DENV-specific antibodies that bind only to the serotype of infection and other antibodies that cross-react with two or more serotypes. People exposed to a secondary DENV infection with another serotype are at greater risk of developing more severe forms of dengue disease. The increased risk of severe dengue in people experiencing repeat DENV infections appear to be due, at least in part, to the ability of pre-existing serotype cross-reactive antibodies to form virus-antibody complexes that can productively infect Fc? receptor-bearing target cells. While the theory of antibody-dependent enhancement (ADE) is supported by several human and small animal model studies, the specific viral antigens and epitopes recognized by enhancing human antibodies after natural infections have not been fully defined. We used antibody-depletion techniques to remove DENV-specific antibody sub-populations from primary DENV-immune human sera. The effects of removing specific antibody populations on ADE were tested both in vitro using K562 cells and in vivo using the AG129 mouse model. Removal of serotype cross-reactive antibodies ablated enhancement of heterotypic virus infection in vitro and antibody-enhanced mortality in vivo. Further depletion studies using recombinant viral antigens showed that although the removal of DENV E-specific antibodies using recombinant E (rE) protein resulted in a partial reduction in DENV enhancement, there was a significant residual enhancement remaining. Competition ADE studies using prM-specific Fab fragments in human immune sera showed that both rE-specific and prM-specific antibodies in primary DENV-immune sera significantly contribute to enhancement of heterotypic DENV infection in vitro. Identification of the targets of DENV-enhancing antibodies should contribute to the development of safe, non-enhancing vaccines against dengue.
Project description:The purpose of the study was to classify, through phylogenetic analyses, the main arboviruses that have been isolated in the metropolitan region of Porto Velho, Rondônia, Brazil. Serum samples from patients with symptoms suggesting arboviruses were collected and tested by One Step RT-qPCR for Zika, Dengue (serotypes 1-4), Chikungunya, Mayaro and Oropouche viruses. Positive samples were amplified by conventional PCR and sequenced utilizing the Sanger method. The obtained sequences were aligned, and an evolutionary analysis was carried out using Bayesian inference. A total of 308 samples were tested. Of this total, 20 had a detectable viral load for Dengue, being detected DENV1 (18/20), co-infection DENV1 and DENV2 (1/20) and DENV4 (1/20). For Dengue serotype 3 and for the CHIKV, ZIKV, MAYV and OROV viruses, no individuals with a detectable viral load were found. A total of 9 of these samples were magnified by conventional PCR for sequencing. Of these, 6 were successfully sequenced and, according to the evolutionary profile, 5 corresponded to serotype DENV-1 genotype V, and 1 to serotype DENV-4 genotype II. In the study, we demonstrate co-circulation of the DENV-1 genotype V and the DENV-4 genotype II. Co-circulation of several DENV serotypes in the same city poses a risk to the population and is correlated with the increase of the most severe forms of the disease. Similarly, co-circulation of genetically distinct DENV and the occurrence of simultaneous infections can affect recombination events and lead to the emergence of more virulent isolates.
Project description:Dengue is one of the leading causes of morbidity in tropical and subtropical regions and infection with any of the four dengue virus serotypes (DENV1-4) result in a wide range of clinical manifestations. Given the geographic expansion of DENV1-4, assays for serotyping are needed to be able to perform surveillance and epidemiological studies. In this study, we describe the design and validation of one-step real-time serotype-specific DENV RT-PCR assays.The DENV1, DENV2, DENV3, and DENV4 RT-PCR assays were designed using all available whole genome DENV sequences in the NCBI nucleotide collection. Because of the high mutation rates of RNA viruses, the assays were performed in singleplex format to enable quick modifications to the primer and probe sequences when new genetic variants emerge. The analytical performance of the RT-PCR assays were evaluated using in vitro transcribed RNA and their specificity was determined by testing 24 DENV isolates, external DENV control panels and RNA preparation of non-DENV flaviviruses and non-dengue clinical samples. Additionally, the clinical performance of the serotype-specific DENV RT-PCR were compared to that of the CDC DENV-1-4 RT-PCR using 85 clinical samples collected from patients presenting with acute dengue.The RT-PCR assays were found to be specific for their respective serotype and did not cross-react with other flaviviruses or human mRNA. All assays had a linear dynamic range of 10(2) to 10(6) copies/reaction with detection limits between 12 and 44 copies/reaction. When testing sera from 85 confirmed acute dengue cases, the serotype-specific DENV RT-PCR assays had 100 % positive agreement with the FDA-approved CDC DENV-1-4 RT-PCR assay performed in a singleplex format. Additionally 15 samples that tested negative in the CDC DENV-1-4- RT-PCR assay were found positive using the serotype-specific DENV RT-PCR assays.Our results suggest that these RT-PCR assays are useful alternatives to existing methods for serotyping DENV in clinical sera.
Project description:Dengue virus (DENV) replication is inhibited by the prior addition of type I interferon or by RIG-I agonists that elicit RIG-I/MAVS/TBK1/IRF3-dependent protective responses. DENV infection of primary human endothelial cells (ECs) results in a rapid increase in viral titer, which suggests that DENV inhibits replication-restrictive RIG-I/interferon beta (IFN-β) induction pathways within ECs. Our findings demonstrate that DENV serotype 4 (DENV4) nonstructural (NS) proteins NS2A and NS4B inhibited RIG-I-, MDA5-, MAVS-, and TBK1/IKKε-directed IFN-β transcription (>80%) but failed to inhibit IFN-β induction directed by STING or constitutively active IRF3-5D. Expression of NS2A and NS4B dose dependently inhibited the phosphorylation of TBK1 and IRF3, which suggests that they function at the level of TBK1 complex activation. NS2A and NS4B from DENV1/2/4, as well as the West Nile virus NS4B protein, commonly inhibited TBK1 phosphorylation and IFN-β induction. A comparative analysis of NS4A proteins across DENVs demonstrated that DENV1, but not DENV2 or DENV4, NS4A proteins uniquely inhibited TBK1. These findings indicate that DENVs contain conserved (NS2A/NS4B) and DENV1-specific (NS4A) mechanisms for inhibiting RIG-I/TBK1-directed IFN responses. Collectively, our results define DENV NS proteins that restrict IRF3 and IFN responses and thereby facilitate DENV replication and virulence. Unique DENV1-specific NS4A regulation of IFN induction has the potential to be a virulence determinant that contributes to the increased severity of DENV1 infections and the immunodominance of DENV1 responses during tetravalent DENV1-4 vaccination.Our findings demonstrate that NS2A and NS4B proteins from dengue virus serotypes 1, 2, and 4 are inhibitors of RIG-I/MDA5-directed interferon beta (IFN-β) induction and that they accomplish this by blocking TBK1 activation. We determined that IFN inhibition is functionally conserved across NS4B proteins from West Nile virus and DENV1, -2, and -4 viruses. In contrast, DENV1 uniquely encodes an extra IFN regulating protein, NS4A, that inhibits TBK1-directed IFN induction. DENV1 is associated with an increase in severe patient disease, and added IFN regulation by the DENV1 NS4A protein may contribute to increased DENV1 replication, immunodominance, and virulence. The regulation of IFN induction by nonstructural (NS) proteins suggests their potential roles in enhancing viral replication and spread and as potential protein targets for viral attenuation. DENV1-specific IFN regulation needs to be considered in vaccine strategies where enhanced DENV1 replication may interfere with DENV2-4 seroconversion within coadministered tetravalent DENV1-4 vaccines.