Development of a novel NS1 competitive enzyme-linked immunosorbent assay for the early detection of Zika virus infection

Zika virus (ZIKV) is a flavivirus that has emerged as a global health threat after the 2015 outbreak in the Americas, where devastating congenital defects were documented. There are currently no vaccines to prevent ZIKV infections nor commercially available clinical diagnostic tests demonstrated to identify ZIKV without cross-reactive interference of related flaviviruses. Early diagnosis is critical when treating symptomatic patients and in preventing ZIKV transmission. In this context, the development of sensitive and accurate diagnostic methods are urgently needed for the detection of ZIKV acute infection. The aim of this study consisted of obtaining monoclonal antibodies (mAbs) against denatured monomeric ZIKV Nonstructural protein 1 (ZNS1), a useful diagnostic marker for flavivirus early detection, in order to develop a highly specific and sensitive ZNS1 indirect competitive ELISA (icELISA). The production of hybridomas secreting ZNS1 mAbs was carried out through immunizations with denatured monomeric ZNS1. We selected 1F5 and 6E2 hybridoma clones, which recognized the heat-denatured ZNS1 hexameric form by indirect ELISA. Cross-reaction studies indicated that these mAbs specifically bind to a ZNS1 linear epitope, and that they do not cross-react with the NS1 protein from other related flaviviruses. The 1F5 mAb enabled the development of a sensitive and reproducible icELISA to detect and quantify small amounts of ZNS1 disease marker in heat-denatured human sera. Here, we establish a reliable 1F5 based-icELISA that constitutes a promising diagnostic tool for control strategies and the prevention of ZIKV propagation.

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Development and Characterization of Double-Antibody Sandwich ELISA for Detection of Zika Virus Infection

Viruses ◽

10.3390/v10110634 ◽

2018 ◽

Vol 10 (11) ◽

pp. 634 ◽

Cited By ~ 10

Author(s):

Liding Zhang ◽

Xuewei Du ◽

Congjie Chen ◽

Zhixin Chen ◽

Li Zhang ◽

...

Keyword(s):

Zika Virus ◽

Nonstructural Protein ◽

Severe Disease ◽

Sandwich Elisa ◽

Diagnostic Methods ◽

Detection Methods ◽

Molecular Diagnostic ◽

Ns1 Protein ◽

Nonstructural Protein 1 ◽

Congenital Birth Defect

Zika virus (ZIKV) is an emerging mosquito-transmitted flavivirus that can cause severe disease, including congenital birth defect and Guillain−Barré syndrome during pregnancy. Although, several molecular diagnostic methods have been developed to detect the ZIKV, these methods pose challenges as they cannot detect early viral infection. Furthermore, these methods require the extraction of RNA, which is easy to contaminate. Nonstructural protein 1 (NS1) is an important biomarker for early diagnosis of the virus, and the detection methods associated with the NS1 protein have recently been reported. The aim of this study was to develop a rapid and sensitive detection method for the detection of the ZIKV based on the NS1 protein. The sensitivity of this method is 120 ng mL−1 and it detected the ZIKV in the supernatant and lysates of Vero and BHK cells, as well as the sera of tree shrews infected with the ZIKV. Without the isolation of the virus and the extraction of the RNA, our method can be used as a primary screening test as opposed to other diagnosis methods that detect the ZIKV.

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Vesicular Stomatitis Virus and DNA Vaccines Expressing Zika Virus Nonstructural Protein 1 Induce Substantial but Not Sterilizing Protection against Zika Virus Infection

Journal of Virology ◽

10.1128/jvi.00048-20 ◽

2020 ◽

Vol 94 (17) ◽

Author(s):

Anzhong Li ◽

Miaoge Xue ◽

Zayed Attia ◽

Jingyou Yu ◽

Mijia Lu ◽

...

Keyword(s):

Vesicular Stomatitis Virus ◽

Zika Virus ◽

Neutralizing Antibodies ◽

Vaccine Development ◽

Nonstructural Protein ◽

Ns1 Protein ◽

Zikv Infection ◽

Partial Protection ◽

Nonstructural Protein 1 ◽

Vesicular Stomatitis

ABSTRACT The nonstructural protein 1 (NS1) of several flaviviruses, including West Nile, dengue, and yellow fever viruses, is capable of inducing variable degrees of protection against flavivirus infection in animal models. However, the immunogenicity of NS1 protein of Zika virus (ZIKV) is less understood. Here, we determined the efficacy of ZIKV NS1-based vaccine candidates using two delivery platforms, methyltransferase-defective recombinant vesicular stomatitis virus (mtdVSV) and a DNA vaccine. We first show that expression of ZIKV NS1 could be significantly enhanced by optimizing the signal peptide. A single dose of mtdVSV-NS1-based vaccine or two doses of DNA vaccine induced high levels of NS1-specfic antibody and T cell immune responses but provided only partial protection against ZIKV viremia in BALB/c mice. In Ifnar1−/− mice, neither NS1-based vaccine provided protection against a lethal high dose (105 PFU) ZIKV challenge, but mtdVSV-NS1-based vaccine prevented deaths from a low dose (103 PFU) challenge, though they experienced viremia and body weight loss. We conclude that ZIKV NS1 alone conferred substantial, but not complete, protection against ZIKV infection. Nevertheless, these results highlight the value of ZIKV NS1 for vaccine development. IMPORTANCE Most Zika virus (ZIKV) vaccine research has focused on the E or prM-E proteins and the induction of high levels of neutralizing antibodies. However, these ZIKV neutralizing antibodies cross-react with other flaviviruses, which may aggravate the disease via an antibody-dependent enhancement (ADE) mechanism. ZIKV NS1 protein may be an alternative antigen for vaccine development, since antibodies to NS1 do not bind to the virion, thereby eliminating the risk of ADE. Here, we show that recombinant VSV and DNA vaccines expressing NS1, alone, confer partial protection against ZIKV infection in both immunocompetent and immunodeficient mice, highlighting the value of NS1 as a potential vaccine candidate.

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Utility of a Commercial Nonstructural Protein 1 Antigen Capture Kit as a Dengue Virus Diagnostic Tool

Clinical and Vaccine Immunology ◽

10.1128/cvi.00041-10 ◽

2010 ◽

Vol 17 (6) ◽

pp. 949-953 ◽

Cited By ~ 27

Author(s):

Kovi Bessoff ◽

Elena Phoutrides ◽

Mark Delorey ◽

Luz N. Acosta ◽

Elizabeth Hunsperger

Keyword(s):

Dengue Virus ◽

Acute Phase ◽

Nonstructural Protein ◽

Acute Infection ◽

False Negative ◽

Diagnostic Tools ◽

Enzyme Linked Immunosorbent Assay ◽

Convalescent Phase ◽

Nonstructural Protein 1 ◽

Antigen Capture

ABSTRACT Annually, over 2.5 billion people are at risk for infection with dengue virus (DENV), while between 50 and 100 million people contract the infection. There is an urgent need for alternative diagnostic tools that can detect DENV during acute infection. Recent studies have shown that DENV nonstructural protein 1 (NS1) is detectable in the blood as early as the onset of symptoms and persists well into the convalescent phase of the infection. We evaluated the utility of the Bio-Rad Platelia DENV NS1 antigen capture kit in combination with real-time reverse transcriptase PCR (RT-PCR) and an IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) for refining a new algorithm for the diagnosis of acute- or convalescent-phase DENV infection with a single clinical sample. We tested the Bio-Rad kit with three panels of sera. These panels were designed to evaluate the sensitivities of the NS1 kit for (i) early-convalescent-phase samples, (ii) acute-phase samples with false-negative PCR results, and (iii) IgM-negative convalescent-phase samples from patients with confirmed secondary DENV infections. Results show that NS1 can be detected in 22% of serum samples collected more than 10 days after the onset of illness and in 22% of samples that did not elicit an IgM response. Additionally, NS1 was detected in 37% of the tested acute-phase samples with false-negative PCR results, suggesting that NS1 detection may be valuable in increasing the sensitivity of current acute-phase diagnostics. These results will improve diagnosis with a single acute-phase or early-convalescent-phase sample for disease surveillance and clinical diagnosis.

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The immunodominant antibody response to Zika virus NS1 protein is characterized by cross-reactivity to self

Journal of Experimental Medicine ◽

10.1084/jem.20210580 ◽

2021 ◽

Vol 218 (9) ◽

Author(s):

Cecilia B. Cavazzoni ◽

Vicente B.T. Bozza ◽

Tostes C.V. Lucas ◽

Luciana Conde ◽

Bruno Maia ◽

...

Keyword(s):

Immune Response ◽

B Cell ◽

Humoral Immune Response ◽

Zika Virus ◽

Nonstructural Protein ◽

Cross Reactivity ◽

Ns1 Protein ◽

Humoral Immune ◽

Nonstructural Protein 1 ◽

Autoreactive Antibodies

Besides antigen-specific responses to viral antigens, humoral immune response in virus infection can generate polyreactive and autoreactive antibodies. Dengue and Zika virus infections have been linked to antibody-mediated autoimmune disorders, including Guillain-Barré syndrome. A unique feature of flaviviruses is the secretion of nonstructural protein 1 (NS1) by infected cells. NS1 is highly immunogenic, and antibodies targeting NS1 can have both protective and pathogenic roles. In the present study, we investigated the humoral immune response to Zika virus NS1 and found NS1 to be an immunodominant viral antigen associated with the presence of autoreactive antibodies. Through single B cell cultures, we coupled binding assays and BCR sequencing, confirming the immunodominance of NS1. We demonstrate the presence of self-reactive clones in germinal centers after both infection and immunization, some of which present cross-reactivity with NS1. Sequence analysis of anti-NS1 B cell clones showed sequence features associated with pathogenic autoreactive antibodies. Our findings demonstrate NS1 immunodominance at the cellular level as well as a potential role for NS1 in ZIKV-associated autoimmune manifestations.

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Zika Virus Non-Structural Protein 1 Antigen-Capture Immunoassay

Viruses ◽

10.3390/v13091771 ◽

2021 ◽

Vol 13 (9) ◽

pp. 1771

Author(s):

Brandon J. Beddingfield ◽

Jessica N. Hartnett ◽

Russell B. Wilson ◽

Peter C. Kulakosky ◽

Kristian G. Andersen ◽

...

Keyword(s):

Zika Virus ◽

Dynamic Range ◽

Yellow Fever Virus ◽

Limit Of Detection ◽

Nonstructural Protein ◽

Ns1 Protein ◽

Capture Elisa ◽

Antigen Capture Elisa ◽

Nonstructural Protein 1 ◽

Antigen Capture

Infection with Zika virus (ZIKV), a member of the Flavivirus genus of the Flaviviridae family, typically results in mild self-limited illness, but severe neurological disease occurs in a limited subset of patients. In contrast, serious outcomes commonly occur in pregnancy that affect the developing fetus, including microcephaly and other major birth defects. The genetic similarity of ZIKV to other widespread flaviviruses, such as dengue virus (DENV), presents a challenge to the development of specific ZIKV diagnostic assays. Nonstructural protein 1 (NS1) is established for use in immunodiagnostic assays for flaviviruses. To address the cross-reactivity of ZIKV NS1 with proteins from other flaviviruses we used site-directed mutagenesis to modify putative epitopes. Goat polyclonal antibodies to variant ZIKV NS1 were affinity-purified to remove antibodies binding to the closely related NS1 protein of DENV. An antigen-capture ELISA configured with the affinity-purified polyclonal antibody showed a linear dynamic range between approximately 500 and 30 ng/mL, with a limit of detection of between 1.95 and 7.8 ng/mL. NS1 proteins from DENV, yellow fever virus, St. Louis encephalitis virus and West Nile virus showed significantly reduced reactivity in the ZIKV antigen-capture ELISA. Refinement of approaches similar to those employed here could lead to development of ZIKV-specific immunoassays suitable for use in areas where infections with related flaviviruses are common.

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Development of a Flow-Free Automated Colorimetric Detection Assay Integrated with Smartphone for Zika NS1

Diagnostics ◽

10.3390/diagnostics10010042 ◽

2020 ◽

Vol 10 (1) ◽

pp. 42 ◽

Cited By ~ 7

Author(s):

Md Alamgir Kabir ◽

Hussein Zilouchian ◽

Mazhar Sher ◽

Waseem Asghar

Keyword(s):

Zika Virus ◽

Magnetic Beads ◽

Nonstructural Protein ◽

Point Of Care ◽

Colorimetric Detection ◽

Great Promise ◽

Enzyme Linked Immunosorbent Assay ◽

Magnetic Actuation ◽

Superparamagnetic Beads ◽

Nonstructural Protein 1

The Zika virus (ZIKV) is an emerging flavivirus transmitted to humans by Aedes mosquitoes that can potentially cause microcephaly, Guillain–Barré Syndrome, and other birth defects. Effective vaccines for Zika have not yet been developed. There is a necessity to establish an easily deployable, high-throughput, low-cost, and disposable point-of-care (POC) diagnostic platform for ZIKV infections. We report here an automated magnetic actuation platform suitable for a POC microfluidic sandwich enzyme-linked immunosorbent assay (ELISA) using antibody-coated superparamagnetic beads. The smartphone integrated immunoassay is developed for colorimetric detection of ZIKV nonstructural protein 1 (NS1) antigen using disposable chips to accommodate the reactions inside the chip in microliter volumes. An in-house-built magnetic actuator platform automatically moves the magnetic beads through different aqueous phases. The assay requires a total of 9 min to automatically control the post-capture washing, horseradish peroxidase (HRP) conjugated secondary antibody probing, washing again, and, finally, color development. By measuring the saturation intensity of the developed color from the smartphone captured video, the presented assay provides high sensitivity with a detection limit of 62.5 ng/mL in whole plasma. These results advocate a great promise that the platform would be useful for the POC diagnosis of Zika virus infection in patients and can be used in resource-limited settings.

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Highly Sensitive Detection of Zika Virus Nonstructural Protein 1 in Serum Samples by a Two-Site Nanobody ELISA

Biomolecules ◽

10.3390/biom10121652 ◽

2020 ◽

Vol 10 (12) ◽

pp. 1652

Author(s):

Triana Delfin-Riela ◽

Martín Rossotti ◽

Romina Alvez-Rosado ◽

Carmen Leizagoyen ◽

Gualberto González-Sapienza

Keyword(s):

Zika Virus ◽

High Throughput Screening ◽

Nonstructural Protein ◽

Low Cost ◽

Blood Stream ◽

Ns1 Protein ◽

Serum Samples ◽

Nonstructural Protein 1 ◽

Highly Sensitive ◽

Highly Sensitive Detection

The Zika virus was introduced in Brazil in 2015 and, shortly after, spread all over the Americas. Nowadays, it remains present in more than 80 countries and represents a major threat due to some singularities among other flaviviruses. Due to its easy transmission, high percentage of silent cases, the severity of its associated complications, and the lack of prophylactic methods and effective treatments, it is essential to develop reliable and rapid diagnostic tests for early containment of the infection. Nonstructural protein 1 (NS1), a glycoprotein involved in all flavivirus infections, is secreted since the beginning of the infection into the blood stream and has proven to be a valuable biomarker for the early diagnosis of other flaviviral infections. Here, we describe the development of a highly sensitive nanobody ELISA for the detection of the NS1 protein in serum samples. Nanobodies were selected from a library generated from a llama immunized with Zika NS1 (ZVNS1) by a two-step high-throughput screening geared to identify the most sensitive and specific nanobody pairs. The assay was performed with a sub-ng/mL detection limit in the sera and showed excellent reproducibility and accuracy when validated with serum samples spiked with 0.80, 1.60, or 3.10 ng/mL of ZVNS1. Furthermore, the specificity of the developed ELISA was demonstrated using a panel of flavivirus’ NS1 proteins; this is of extreme relevance in countries endemic for more than one flavivirus. Considering that the nanobody sequences are provided, the assay can be reproduced in any laboratory at low cost, which may help to strengthen the diagnostic capacity of the disease even in low-resource countries.

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Monoclonal Antibodies against Zika Virus NS1 Protein Confer Protection via Fcγ Receptor-Dependent and -Independent Pathways

mBio ◽

10.1128/mbio.03179-20 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Lei Yu ◽

Xinglong Liu ◽

Xianmiao Ye ◽

Wan Su ◽

Xiaoyan Zhang ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Zika Virus ◽

Nonstructural Protein ◽

Effector Function ◽

Protective Effects ◽

Ns1 Protein ◽

Fcγ Receptor ◽

Zikv Infection ◽

Effector Cells ◽

Nonstructural Protein 1

ABSTRACT Zika virus (ZIKV) infection during pregnancy causes congenital defects such as fetal microcephaly. Monoclonal antibodies (MAbs) against the nonstructural protein 1 (NS1) have the potential to suppress ZIKV pathogenicity without enhancement of disease, but the pathways through which they confer protection remain obscure. Here, we report two types of NS1-targeted human MAbs that inhibit ZIKV infection through distinct mechanisms. MAbs 3G2 and 4B8 show a better efficacy than MAb 4F10 in suppressing ZIKV infection in C57BL/6 neonatal mice. Unlike MAb 4F10 that mainly triggers antibody-dependent cell-mediated cytotoxicity (ADCC), MAbs 3G2 and 4B8 not only trigger ADCC but inhibit ZIKV infection without Fcγ receptor-bearing effector cells, possibly at postentry stages. Destroying the Fc-mediated effector function of MAbs 3G2 and 4B8 reduces but does not abolish their protective effects, whereas destroying the effector function of MAb 4F10 eliminates the protective effects, suggesting that MAbs 3G2 and 4B8 engage both Fcγ receptor-dependent and -independent pathways. Further analysis reveals that MAbs 3G2 and 4B8 target the N-terminal region of NS1 protein, whereas MAb 4F10 targets the C-terminal region, implying that the protective efficacy of an NS1-targeted MAb may be associated with its epitope recognition. Our results illustrate that NS1-targeted MAbs have multifaceted protective effects and provide insights for the development of NS1-based vaccines and therapeutics. IMPORTANCE Zika virus (ZIKV) is a mosquito-borne flavivirus that has been linked to congenital microcephaly during recent epidemics. No licensed antiviral drug or vaccine is available. Monoclonal antibodies (MAbs) against the nonstructural protein 1 (NS1) inhibit ZIKV pathogenicity but do not enhance the disease as envelope protein-targeted MAbs do. However, the protection mechanisms are not fully understood. Here, we show that in the presence or absence of Fcγ receptor-bearing effector cells, NS1-targeted human MAbs 3G2 and 4B8 inhibit ZIKV infection. Compared to MAb 4F10 that has no inhibitory effects without effector cells, 3G2 and 4B8 confer better protection in ZIKV-infected neonatal mice. Destroying the Fc-mediated effector function reduces but does not abolish the protection of 3G2 and 4B8, suggesting that they engage both Fcγ receptor-dependent and -independent pathways. The protective efficacy of NS1-targeted MAbs may be associated with their epitope recognition. Our findings will help to develop NS1-based vaccines and therapeutics.

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Zika Virus Non-Structural Protein 1 Antigen-Capture Immunoassay

10.20944/preprints202108.0130.v1 ◽

2021 ◽

Author(s):

Brandon J. Beddingfield ◽

Jessica N. Hartnett ◽

Russell B. Wilson ◽

Peter C. Kulakosky ◽

Kristian G. Andersen ◽

...

Keyword(s):

Zika Virus ◽

Dynamic Range ◽

Yellow Fever Virus ◽

Limit Of Detection ◽

Nonstructural Protein ◽

Ns1 Protein ◽

Capture Elisa ◽

Antigen Capture Elisa ◽

Nonstructural Protein 1 ◽

Antigen Capture

Infection with Zika virus (ZIKV), a member of the Flavivirus genus of the Flaviviridae family, typically results in mild self-limited illness, but severe neurological disease occurs in a limited subset of patients. In contrast, serious outcomes commonly occur in pregnancy that affect the developing fetus, including microcephaly and other major birth defects. The genetic similarity of ZIKV to other widespread flaviviruses, such as dengue virus (DENV), presents a challenge to the development of specific ZIKV diagnostic assays. Nonstructural protein 1 (NS1) is established for use in immunodiagnostic assays for flaviviruses. To address the cross-reactivity of ZIKV NS1 with proteins from other flaviviruses we used site-directed mutagenesis to modified putative epitopes. Goat polyclonal antibodies to variant ZIKV NS1 were affinity-purified to remove antibodies binding to the closely related NS1 protein of DENV. An antigen-capture ELISA configured with the affinity-purified polyclonal antibody showed a linear dynamic range between approximately 500 to 30 ng/mL, with a limit of detection of between 1.95 and 7.8 ng/mL. NS1 proteins from DENV, yellow fever virus, St. Louis encephalitis virus and West Nile virus showed significantly reduced reactivity in the ZIKV antigen-capture ELISA. Refinement of approaches similar to those employed here could lead to development of ZIKV-specific immunoassays suitable for use in areas where infections with related flaviviruses are common.

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Serological Differences after Acute Zika Virus Infections between Children and Adults: Implication for Use of a Serological Test

American Journal of Tropical Medicine and Hygiene ◽

10.4269/ajtmh.21-0134 ◽

2021 ◽

Author(s):

Jurai Wongsawat ◽

Patama Suttha ◽

Sumalee Chanama ◽

Somkid Srisopa ◽

Nichapa Yonchoho ◽

...

Keyword(s):

Zika Virus ◽

Nonstructural Protein ◽

Serological Test ◽

Cross Reactivity ◽

Virus Infections ◽

Positive Real ◽

Nonstructural Protein 1 ◽

Age Related ◽

Polymerase Chain ◽

Post Onset

Information is limited regarding differential serological responses after acute Zika virus (ZIKV) infections and prevalence of cross-reactivity with anti-dengue virus (DENV) assays comparing children and adults. Early convalescent sera from a cohort of suspected mild DENV cases between December 2016 and September 2018 at Bamrasnaradura Infectious Diseases Institute in Thailand were tested for nonstructural protein 1 (NS1)–based anti-ZIKV IgM and IgG ELISAs (Euroimmun), and in-house anti-DENV IgM- and IgG-capture ELISAs. ZIKV cases were identified by positive real-time reverse transcriptase-polymerase chain reaction on urine. Sera from 26 (10 children and 16 adults) ZIKV and 237 (153 children and 74 adults) non-ZIKA cases collected at the median duration of 18 days (interquartile range [IQR] 18,19) post-onset of symptoms were tested. Comparing pediatric ZIKV to adult ZIKV cases, the mean anti-ZIKV IgM ratio was higher (2.12 versus 1.27 units, respectively; P = 0.07), whereas mean anti-ZIKV IgG ratio was lower (3.13 versus 4.24 units, respectively; P = 0.03). Sensitivity of anti-ZIKV IgM and specificity of anti-ZIKV IgG in pediatric ZIKV were higher than in adult ZIKV cases (80.0% versus 43.7% and 79.1% versus 43.2%, respectively). No cross-reactivity with anti-DENV IgM- and IgG-capture ELISA were reported in pediatric ZIKV cases in our study, whereas 25% and 12.5% were found in adult ZIKV cases, respectively. Age-related ZIKV serological differences have been observed. Positive NS1-based anti-ZIKV IgM and IgG ELISA at the early convalescent phase could be useful for ZIKV diagnosis in children, even in a dengue endemic setting.

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