Mapping Putative B-Cell Zika Virus NS1 Epitopes Provides Molecular Basis for Anti-NS1 Antibody Discrimination between Zika and Dengue Viruses.
ABSTRACT: B-cell epitope sequences from Zika virus (ZIKV) NS1 protein have been identified using epitope prediction tools. Mapping these sequences onto the NS1 surface reveals two major conformational epitopes and a single linear one. Despite an overall average sequence identity of ca. 55% between the NS1 from ZIKV and the four dengue virus (DENV) serotypes, epitope sequences were found to be highly conserved. Nevertheless, nonconserved epitope-flanking residues are responsible for a dramatically divergent electrostatic surface potential on the epitope regions of ZIKV and DENV2 serotypes. These findings suggest that strategies for differential diagnostics on the basis of short linear NS1 sequences are likely to fail due to immunological cross-reactions. Overall, results provide the molecular basis of differential discrimination between Zika and DENVs by NS1 monoclonal antibodies.
Project description:Dengue nonstructural protein 1 (NS1) is a multi-functional glycoprotein with essential functions both in viral replication and modulation of host innate immune responses. NS1 has been established as a good surrogate marker for infection. In the present study, we generated four anti-NS1 monoclonal antibodies against recombinant NS1 protein from dengue virus serotype 2 (DENV2), which were used to map three NS1 epitopes. The sequence 193AVHADMGYWIESALNDT209 was recognized by monoclonal antibodies 2H5 and 4H1BC, which also cross-reacted with Zika virus (ZIKV) protein. On the other hand, the sequence 25VHTWTEQYKFQPES38 was recognized by mAb 4F6 that did not cross react with ZIKV. Lastly, a previously unidentified DENV2 NS1-specific epitope, represented by the sequence 127ELHNQTFLIDGPETAEC143, is described in the present study after reaction with mAb 4H2, which also did not cross react with ZIKV. The selection and characterization of the epitope, specificity of anti-NS1 mAbs, may contribute to the development of diagnostic tools able to differentiate DENV and ZIKV infections.
Project description:The recent outbreaks of Zika virus (ZIKV) and associated birth defects in regions of dengue virus (DENV) endemicity emphasize the need for sensitive and specific serodiagnostic tests. We reported previously that enzyme-linked immunosorbent assays (ELISAs) based on the nonstructural protein 1 (NS1) of DENV serotype 1 (DENV1) and ZIKV can distinguish primary DENV1, secondary DENV, and ZIKV infections. Whether ELISAs based on NS1 proteins of other DENV serotypes can discriminate various DENV and ZIKV infections remains unknown. We herein developed DENV2, DENV3, and DENV4 NS1 IgG ELISAs to test convalescent- and postconvalescent-phase samples from reverse transcription-PCR-confirmed cases, including 25 primary DENV1, 24 primary DENV2, 10 primary DENV3, 67 secondary DENV, 36 primary West Nile virus, 38 primary ZIKV, and 35 ZIKV with previous DENV infections as well as 55 flavivirus-naive samples. Each ELISA detected primary DENV infection with a sensitivity of 100% for the same serotype and 23.8% to 100% for different serotypes. IgG ELISA using a mixture of DENV1-4 NS1 proteins detected different primary and secondary DENV infections with a sensitivity of 95.6% and specificity of 89.5%. The ZIKV NS1 IgG ELISA detected ZIKV infection with a sensitivity of 100% and specificity of 82.9%. On the basis of the relative optical density ratio, the combination of DENV1-4 and ZIKV NS1 IgG ELISAs distinguished ZIKV with previous DENV and secondary DENV infections with a sensitivity of 91.7% to 94.1% and specificity of 87.0% to 95.0%. These findings have important applications to serodiagnosis, serosurveillance, and monitoring of both DENV and ZIKV infections in regions of endemicity.
Project description:The presence of dengue virus (DENV), Zika virus (ZIKV) and Chikungunya virus (CHIKV) in Brazil, may result in a difficult diagnosis due to the signs and symptoms shared by those. Moreover, as DENV and ZIKV belong to the same family, serological assays may show a high rate of cross-reactivity. Here, we evaluated a Dengue NS1 capture assay for early and differential diagnosis of dengue during the Zika epidemic occurred in Brazil in 2016. Samples (n?=?227) from 218 patients included sera, plasma and urine from previously confirmed acute cases of Zika, dengue and Zika/dengue co-infections. Nine of those patients presented two specimens. The Dengue NS1 test was very specific for dengue diagnosis (99.32%), even in the co-circulation with ZIKV, and exhibited a high accuracy in not detecting acute Zika infections (92.43%). Our findings showed that the dengue NS1 capture test analyzed here was not able to recognize the ZIKV NS1 and its potential for cross-reaction.
Project description:Current efforts to develop Zika virus (ZIKV) subunit vaccines have been focused on pre-membrane (prM) and envelope (E) proteins, but the role of NS1 in ZIKV-specific immune response and protection is poorly understood. Here, we develop an attenuated recombinant vesicular stomatitis virus (rVSV)-based vaccine expressing ZIKV prM-E-NS1 as a polyprotein. This vectored vaccine candidate is attenuated in mice, where a single immunization induces ZIKV-specific antibody and T cell immune responses that provide protection against ZIKV challenge. Co-expression of prM, E, and NS1 induces significantly higher levels of Th2 and Th17 cytokine responses than prM-E. In addition, NS1 alone is capable of conferring partial protection against ZIKV infection in mice even though it does not induce neutralizing antibodies. These results demonstrate that attenuated rVSV co-expressing prM, E, and NS1 is a promising vaccine candidate for protection against ZIKV infection and highlights an important role for NS1 in ZIKV-specific cellular immune responses.
Project description:The spread of dengue (DENV) and Zika virus (ZIKV) is a major public health concern. The primary target of antibodies that neutralize DENV and ZIKV is the envelope (E) glycoprotein, and there is interest in using soluble recombinant E (sRecE) proteins as subunit vaccines. However, the most potent neutralizing antibodies against DENV and ZIKV recognize epitopes on the virion surface that span two or more E proteins. Therefore, to create effective DENV and ZIKV vaccines, presentation of these quaternary epitopes may be necessary. The sRecE proteins from DENV and ZIKV crystallize as native-like dimers, but studies in solution suggest that these dimers are marginally stable. To better understand the challenges associated with creating stable sRecE dimers, we characterized the thermostability of sRecE proteins from ZIKV and three DENV serotypes, DENV2-4. All four proteins irreversibly unfolded at moderate temperatures (46-53 °C). At 23 °C and low micromolar concentrations, DENV2 and ZIKV were primarily dimeric, and DENV3-4 were primarily monomeric, whereas at 37 °C, all four proteins were predominantly monomeric. We further show that the dissociation constant for DENV2 dimerization is very temperature-sensitive, ranging from <1 ?m at 25 °C to 50 ?m at 41 °C, due to a large exothermic enthalpy of binding of -79 kcal/mol. We also found that quaternary epitope antibody binding to DENV2-4 and ZIKV sRecE is reduced at 37 °C. Our observation of reduced sRecE dimerization at physiological temperature highlights the need for stabilizing the dimer as part of its development as a subunit vaccine.
Project description:Zika virus (ZIKV) is a mosquito-borne flavivirus that recently emerged in the South Pacific, Americas, and Caribbean islands, where the larger epidemics were documented. ZIKV infection in humans is responsible for neurological disorders and microcephaly. Flavivirus NS1 is a non-structural glycoprotein that is expressed on the cell surface and secreted as a hexameric lipoprotein particle. Intracellular NS1 exists as a dimer that is required for viral replication, whereas the secreted NS1 hexamer interacts with host factors, leading to pathophysiological conditions. In an effort to dispose of specific anti-ZIKV NS1 immune serum, Vero cells were transduced with a lentiviral vector containing the NS1 gene from an epidemic strain of ZIKV. We showed that stably transduced Vero/ZIKV NS1 cell clone was efficient in the secretion of recombinant NS1 oligomer. Immunization of adult rat with purified extracellular NS1 developed anti-ZIKV antibodies that specifically react with the NS1 dimer produced in human cells infected with African and Asian strains of ZIKV. The rat antibody against ZIKV NS1 dimer is a reliable biological tool that enables the immunological detection of secreted NS1 from host-cells infected with ZIKV.
Project description:BACKGROUND:Zika virus (ZIKV) had spread rapidly in the past few years in southern hemisphere where dengue virus (DENV) had caused epidemic problems for over half a century. The high degree of cross-reactivity of Envelope (E) protein specific antibody responses between ZIKV and DENV made it challenging to perform differential diagnosis between the two infections using standard ELISA method for E protein. METHODS:Using an IgG capture ELISA, we investigated the kinetics of nonstructural protein 1 (NS1) antibody response during natural ZIKV infection and the cross-reactivity to NS1 proteins using convalescent sera obtained from patients infected by either DENV or ZIKV. RESULTS:The analyses of the sequential serum samples from ZIKV infected individuals showed NS1 specific Abs appeared 2 weeks later than E specific Abs. Notably, human sera from ZIKV infected individuals did not contain cross-reactivity to NS1 proteins of any of the four DENV serotypes. Furthermore, four out of five NS1-specific monoclonal antibodies (mAbs) isolated from ZIKV infected individuals did not bind to DENV NS1 proteins. Only limited amount of cross-reactivity to ZIKV NS1 was displayed in 108 DENV1 immune sera at 1:100 dilution. CONCLUSIONS:The high degree of NS1-specific Abs in both ZIKV and DENV infection revealed here suggest that NS1-based diagnostics would significantly improve the differential diagnosis between DENV and ZIKV infections.
Project description:The causal association of Zika virus (ZIKV) with microcephaly, congenital malformations in infants, and Guillain-Barré syndrome in adults highlights the need for effective vaccines. Thus far, efforts to develop ZIKV vaccines have focused on the viral envelope. ZIKV NS1 as a vaccine immunogen has not been fully explored, although it can circumvent the risk of antibody-dependent enhancement of ZIKV infection, associated with envelope antibodies. Here, we describe a novel DNA vaccine encoding a secreted ZIKV NS1, that confers rapid protection from systemic ZIKV infection in immunocompetent mice. We identify novel NS1 T cell epitopes in vivo and show that functional NS1-specific T cell responses are critical for protection against ZIKV infection. We demonstrate that vaccine-induced anti-NS1 antibodies fail to confer protection in the absence of a functional T cell response. This highlights the importance of using NS1 as a target for T cell–based ZIKV vaccines.
Project description:The association of Zika virus (ZIKV) infections with microcephaly and neurological diseases has highlighted an emerging public health concern. Here, we report the crystal structure of the full-length ZIKV nonstructural protein 1 (NS1), a major host-interaction molecule that functions in flaviviral replication, pathogenesis, and immune evasion. Of note, a long intertwined loop is observed in the wing domain of ZIKV NS1, and forms a hydrophobic "spike", which can contribute to cellular membrane association. For different flaviviruses, the amino acid sequences of the "spike" are variable but their common characteristic is either hydrophobic or positively charged, which is a beneficial feature for membrane binding. Comparative studies with West Nile and Dengue virus NS1 structures reveal conserved features, but diversified electrostatic characteristics on both inner and outer faces. Our results suggest different mechanisms of flavivirus pathogenesis and should be considered during the development of diagnostic tools.
Project description:Virus-host interactions determine an infection outcome. The Asian lineage of Zika virus (ZIKV), responsible for the recent epidemics, has fixed a mutation in the NS1 gene after 2012 that enhances mosquito infection. Here we report that the same mutation confers NS1 to inhibit interferon-β induction. This mutation enables NS1 binding to TBK1 and reduces TBK1 phosphorylation. Engineering the mutation into a pre-epidemic ZIKV strain debilitates the virus for interferon-β induction; reversing the mutation in an epidemic ZIKV strain invigorates the virus for interferon-β induction; these mutational effects are lost in IRF3-knockout cells. Additionally, ZIKV NS2A, NS2B, NS4A, NS4B, and NS5 can also suppress interferon-β production through targeting distinct components of the RIG-I pathway; however, for these proteins, no antagonistic difference is observed among various ZIKV strains. Our results support the mechanism that ZIKV has accumulated mutation(s) that increases the ability to evade immune response and potentiates infection and epidemics.