Compensating for cross-reactions using avidity and computation in a suspension multiplex immunoassay for serotyping of Zika versus other flavivirus infections.
ABSTRACT: The recent spread of Zika virus (ZIKV) in the Americas and Asia necessitates an increased preparedness for improved maternal and perinatal health and blood safety. However, serological cross-reactions, especially to Dengue virus (DENV), complicate ZIKV antibody serodiagnosis. A novel "pan-Flavi" suspension multiplex immunoassay (PFSMIA) using 25 antigens, whole virus (WV), non-structural protein 1 (NS1), and envelope (E) proteins, from 7 zoonotic flaviviruses for specific detection of ZIKV and DENV IgM and IgG was developed. Patterns of antibody cross-reactivity, avidity, and kinetics were established in 104 sera from returning travelers with known ZIKV and DENV infections. PFSMIA gave IgM- and IgG-sensitivities for both viruses of 96-100%, compared to an immunofluorescence assay. Main IgM cross-reactions were to NS1, for IgG to the E and WV antigens. Infecting virus yielded reactivity to several antigens of the homologous virus, while cross-reactions tended to occur only to a single antigen from heterologous virus(es). A specificity-enhancing computer procedure took into account antibody isotype, number of antibody-reactive antigens per virus, avidity, average degree of cross-reactivity to heterologous flavivirus antigens, and reactivity changes in serial sera. It classified all 50 cases correctly. Applied to sera from 200 pregnant women and 173 blood donors from Sweden, one blood donor was found ZIKV NS1 IgM positive, and another as ZIKV NS1 IgG positive. These samples did not react with other ZIKV antigens and were thereby judged as false-positives. PFSMIA provided sensitive and specific ZIKV and DENV serology, warranting high-throughput serological surveillance and a minimized need for laborious and expensive virus neutralization assays.
Project description:Background:The explosive spread of Zika virus (ZIKV) and associated microcephaly present an urgent need for sensitive and specific serodiagnostic tests, particularly for pregnant women in dengue virus (DENV)-endemic regions. Recent reports of enhanced ZIKV replication by dengue-immune sera have raised concerns about the role of previous DENV infection on the risk and severity of microcephaly and other ZIKV complications. Methods:Enzyme-linked immunosorbent assays (ELISAs) based on ZIKV and DENV nonstructural protein 1 (NS1) were established to test acute, convalescent phase, and post-convalescent phase serum/plasma samples from reverse-transcription polymerase chain reaction-confirmed cases including 20 primary ZIKV, 25 ZIKV with previous DENV, 58 secondary DENV, and 16 primary DENV1 infections. Results:ZIKV-NS1 immunoglobulin M (IgM) and immunoglobulin G (IgG) ELISAs combined can detect ZIKV infection with a sensitivity of 95% and specificity of 66.7%. The ZIKV-NS1 IgG cross-reactivity by samples from secondary DENV infection cases ranged from 66.7% to 28.1% (within 1 month to 1-2 years post-illness, respectively). Addition of DENV1-NS1 IgG ELISA can distinguish primary ZIKV infection; the ratio of absorbance of ZIKV-NS1 to DENV1-NS1 IgG ELISA can distinguish ZIKV with previous DENV and secondary DENV infections with a sensitivity of 87.5% and specificity of 81.3%. These findings were supported by analysis of sequential samples. Conclusions:An algorithm for ZIKV serodiagnosis based on 3 simple ELISAs is proposed to distinguish primary ZIKV, ZIKV with previous DENV, and secondary DENV infections; this could be applied to serodiagnosis for ZIKV, serosurveillance, and monitoring ZIKV infection during pregnancy to understand the epidemiology, pathogenesis, and complications of ZIKV in dengue-endemic regions.
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:ELISAs and rapid diagnostic tests (RDTs) are widely used for diagnosing dengue virus (DENV) infection. Using 138 single blood samples, we compared the ability to detect non-structural (NS)-1 antigen and anti-DENV IgM/IgG antibodies among (1) DENV Detect NS1 ELISA, DENV Detect IgM capture ELISA and DENV Detect IgG ELISA (InBios International, Inc.); (2) Anti-Dengue virus IgM Human ELISA and Anti-Dengue virus IgG Human ELISA (Abcam); (3) Dengue virus NS1 ELISA, Anti-Dengue virus ELISA (IgM) and Anti-Dengue virus ELISA (IgG) (Euroimmun); (4) Asan Easy Test Dengue NS1 Ag 100 and Asan Easy Test Dengue IgG/IgM (Asan Pharm); (5) SD BIOLINE Dengue Duo (Standard Diagnostics); and (6) Ichroma Dengue NS1 and Ichroma Dengue IgG/IgM (Boditech Med). For NS1 antigen detection, InBios and Euroimmun showed higher sensitivities (100%) than the RDTs (42.9-64.3%). All tests demonstrated variable sensitivities for IgM (38.1-90.5%) and IgG (65.7-100.0%). InBios and Boditech Med demonstrated higher sensitivity (95.6% and 88.2%, respectively) than the other tests for combined NS1 antigen and IgM antibody. Five NS1 antigen tests had good agreement (92.8-98.6%) without showing positivity for chikungunya. However, all IgG tests demonstrated potential false-positivity with variable ranges. Clinical laboratories should note performance variations across tests and potential cross-reactivity.
Project description:Zika virus (ZIKV) is a mosquito-borne pathogen causing a febrile illness with arthralgia, conjunctivitis and rash. The complications include Guillain-Barré syndrome, congenital brain and other abnormalities and miscarriage. The serodiagnosis of ZIKV infection is hampered by cross-reactivity with other members of the Flavivirus family, notably dengue (DENV). This report describes a novel serological platform for the diagnosis of ZIKV infection. The approach utilizes time-resolved Förster resonance energy transfer (TR-FRET) elicited by two chromophore-labeled proteins (a ZIKV antigen and a super-antigen) simultaneously binding to a given antibody molecule. The antigen used in the assay is ZIKV non-structural protein 1 (NS1) and the super-antigen is bacterial protein L. Three assay variants were developed: the first measuring all anti-ZIKV-NS1 antibodies (LFRET), the second measuring IgM and IgA (acute-LFRET) and the third measuring IgG (immunity-LFRET). The assays were evaluated with a panel of samples from clinical ZIKV cases in travelers (n = 25) and seronegative (n = 24) samples. DENV (n = 38), yellow fever (n = 16) and tick-borne-encephalitis (n = 20) seropositive samples were examined for assessment of flavivirus cross-reactivity. The diagnostic sensitivities of the respective LFRET assays were 92%, 100% and 83%, and the diagnostic specificities 88%, 95% and 100% for LFRET, acute-LFRET and immunity-LFRET. Furthermore, we evaluated the assays against a widely-used commercial ELISA. In conclusion, the new FRET-based serological approaches based on NS1 protein are applicable to diagnosing zika virus infections in travelers and differentiating them from other flavivirus infections.
Project description:Detection of antibodies is widely used for the diagnosis of infections with arthropod-borne flaviviruses including dengue (DENV) and Zika virus (ZIKV). Due to the emergence of ZIKV in areas endemic for DENV, massive co-circulation is observed and methods to specifically diagnose these infections and differentiate them from each other are mandatory. However, serological assays for flaviviruses in general, and for DENV and ZIKV in particular, are compromised by the high degree of similarities in their proteins which can lead to cross-reacting antibodies and false-positive test results. Cross-reacting flavivirus antibodies mainly target the highly conserved fusion loop (FL) domain in the viral envelope (E-) protein, and we and others have shown previously that recombinant E-proteins bearing FL-mutations strongly reduce cross-reactivity. Here we investigate whether such mutant E-proteins can be used to specifically detect antibodies against DENV and ZIKV in an ELISA-format. IgM antibodies against DENV and ZIKV virus were detected with 100% and 94.2% specificity and 90.7% and 87.5% sensitivity, respectively. For IgG the mutant E-proteins showed cross-reactivity, which was overcome by pre-incubation of the sera with the heterologous antigen. This resulted in specificities of 97.1% and 97.9% and in sensitivities of 100% and 100% for the DENV and ZIKV antigens, respectively. Our results suggest that E-proteins bearing mutations in the FL-domain have a high potential for the development of serological DENV and ZIKV tests with high specificity.
Project description:Diagnostic testing for Zika virus (ZIKV) or dengue virus (DENV) infection can be accomplished by a nucleic acid detection method; however, a negative result does not exclude infection due to the low virus titer during infection depending on the timing of sample collection. Therefore, a ZIKV- or DENV-specific serological assay is essential for the accurate diagnosis of patients and to mitigate potential severe health outcomes. A retrospective study design with dual approaches of collecting human serum samples for testing was developed. All serum samples were extensively evaluated by using both noninfectious wild-type (wt) virus-like particles (VLPs) and soluble nonstructural protein 1 (NS1) in the standard immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA). Both ZIKV-derived wt-VLP- and NS1-MAC-ELISAs were found to have similar sensitivities for detecting anti-premembrane/envelope and NS1 antibodies from ZIKV-infected patient sera, although lower cross-reactivity to DENV2/3-NS1 was observed. Furthermore, group cross-reactive (GR)-antibody-ablated homologous fusion peptide-mutated (FP)-VLPs consistently showed higher positive-to-negative values than homologous wt-VLPs. Therefore, we used DENV-2/3 and ZIKV FP-VLPs to develop a novel, serological algorithm for differentiating ZIKV from DENV infection. Overall, the sensitivity and specificity of the FP-VLP-MAC-ELISA and the NS1-MAC-ELISA were each higher than 80%, with no statistical significance. The accuracy can reach up to 95% with the combination of FP-VLP and NS1 assays. In comparison to current guidelines using neutralization tests to measure ZIKV antibody, this approach can facilitate laboratory screening for ZIKV infection, especially in regions where DENV infection is endemic and capacity for neutralization testing does not exist.
Project description:BACKGROUND:Zika virus (ZIKV) and Dengue virus (DENV) are often co-endemic. The high protein-sequence homology of flaviviruses renders IgG induced by and directed against them highly cross-reactive against their antigen(s), as observed on a large set of sera, leading to poorly reliable sero-diagnosis. METHODS:We selected Domain III of the ZIKV Envelope (ZEDIII) sequence, which is virus specific. This recombinant domain was expressed and purified for the specific detection of ZEDIII-induced IgG by ELISA from ZIKV-RT-PCR-positive, ZIKV-IgM-positive, flavivirus-positive but ZIKV-negative, or flavivirus-negative sera. We also assessed the reactivity of ZEDIII-specific human antibodies against EDIII of DENV serotype 4 (D4EDIII) as a specific control. Sera from ZEDIII-immunized mice were also tested. RESULTS:Cross-reactivity of IgG from 5,600 sera against total inactivated DENV or ZIKV was high (71.0% [69.1; 72.2]), whereas the specificity and sensitivity calculated using a representative cohort (242 sera) reached 90% [84.0; 95.8] and 92% [84.5; 99.5], respectively, using a ZEDIII-based ELISA. Moreover, purified human IgG against D2EDIII or D4EDIII did not bind to ZEDIII and we observed no D4EDIII reactivity with ZIKV-induced mouse polyclonal IgGs. CONCLUSIONS:We developed a ZEDIII-based ELISA that can discriminate between past or current DENV and ZIKV infections, allowing the detection of a serological scar from other flaviviruses. This could be used to confirm exposure of pregnant women or to follow the spread of an endemic disease.
Project description:Laboratory confirmation of Zika virus (ZIKV) infection during pregnancy is challenging due to cross-reactivity with dengue virus (DENV) and limited knowledge about the kinetics of anti-Zika antibody responses during pregnancy. We described ZIKV and DENV serological markers and the maternal-fetal transfer of antibodies among mothers and neonates after the ZIKV microcephaly outbreak in Northeast Brazil (2016). We included 89 microcephaly cases and 173 neonate controls at time of birth and their mothers. Microcephaly cases were defined as newborns with a particular head circumference (2 SD below the mean). Two controls without microcephaly were matched by the expected date of delivery and area of residence. We tested maternal serum for recent (ZIKV genome, IgM and IgG3 anti-NS1) and previous (ZIKV and DENV neutralizing antibodies [NAbs]) markers of infection. Multiple markers of recent or previous ZIKV and DENV infection in mothers were analyzed using principal component analysis (PCA). At delivery, 5.6% of microcephaly case mothers and 1.7% of control mothers were positive for ZIKV IgM. Positivity for ZIKV IgG3 anti-NS1 was 8.0% for case mothers and 3.5% for control mothers. ZIKV NAbs was slightly higher among mothers of cases (69.6%) than that of mothers of controls (57.2%; p = 0.054). DENV exposure was detected in 85.8% of all mothers. PCA discriminated two distinct components related to recent or previous ZIKV infection and DENV exposure. ZIKV NAbs were higher in newborns than in their corresponding mothers (p<0.001). We detected a high frequency of ZIKV exposure among mothers after the first wave of the ZIKV outbreak in Northeast Brazil. However, we found low sensitivity of the serological markers to recent infection (IgM and IgG3 anti-NS1) in perinatal samples of mothers of microcephaly cases. Since the neutralization test cannot precisely determine the time of infection, testing for ZIKV immune status should be performed as early as possible and throughout pregnancy to monitor acute Zika infection in endemic areas.
Project description:Dengue virus (DENV) and Zika virus (ZIKV) are flaviviruses of public health relevance. Both viruses circulate in the same endemic settings and acute infections generally manifest similar symptoms. This highlights the importance of accurate diagnosis for clinical management and outbreak control. One of the commonly used acute diagnostic markers for flaviviruses is nonstructural protein 1 (NS1). However, false positives due to antigenic cross-reactivity have been reported between DENV and ZIKV infections when using DENV NS1 antigen (NS1 Ag) detection assays in acute cases. Therefore, we investigated the lowest detectable virus titres and cross-reactivity of three commercial dengue NS1 Ag rapid assays and two ELISAs for different flaviviruses. Our results showed that substantially high viral titres of ZIKV, Kunjin virus (KUNV) and yellow fever virus (YFV) are required to give false-positive results when using DENV NS1 rapid detection assays. Commercial DENV NS1 ELISAs did not react with ZIKV and YFV. In comparison, tested assays detected DENV at a significantly low virus titre. Given the relatively low viral loads reported in clinical samples, our findings suggest that commercially available dengue NS1 Ag detection assays are less likely to generate false-positive results among clinical samples in areas where multiple flaviviruses cocirculate.
Project description:Zika virus (ZIKV) causes rash, moderate fever, conjunctivitis, and arthralgia, and has serious connection with neurological complications; therefore, it is a major threat to public health. A rapid and supersensitive method for detecting anti-ZIKV antibodies in humans and animals is thus urgently required. Here, we report an NS1-based luciferase immunosorbent assay (LISA), developed to detect ZIKV-specific IgG. Fusion proteins including a reporter Nano-luciferase (NLuc) and various fragments of ZIKV NS1 protein were expressed in 293 T cells. LISA was performed using the above cell lysates containing the expressed fusion proteins. Sample panels of humans and animals infected with ZIKV were examined for sensitivity of LISA, relative to those of ZIKV RT-PCR, commercial NS1-based ELISA, and micro-neutralization (MN) assays. Specificity and potential cross-reactivity were also evaluated using various convalescent serum samples derived from patients infected with dengue virus (DENV), Japanese encephalitis virus (JEV), and hepatitis C virus (HCV). Results indicated the optimal antigenic domain for anti-ZIKV IgG detection was located within 172-352 amino acids (aa) of ZIKV NS1 protein. NS1-based LISA performs better than commercial ELISA in anti-ZIKV IgG detection. LISA was shown to be at least fourfold more sensitive than commercial ELISA, and could detect anti-ZIKV IgG in various animal hosts without the need of species-specific labeled antibody. This novel assay is potentially useful for the rapid and sensitive detection of anti-ZIKV IgG in human and animal samples.