Serological tests reveal significant cross-reactive human antibody responses to Zika and Dengue viruses in the Mexican population

https://doi.org/10.1016/j.actatropica.2019.105201 Get rights and content

Highlights

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    rNS1β-leader and rEDIII Zika recombinant viral proteins were evaluated as candidate antigens to develop an in-house indirect ELISA test.
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    In-house and commercial ELISA assays were tested in patients with PCR-confirmed Dengue and Zika virus (DENV and ZIKV) infections and in two groups (dengue exposed and dengue naive) of Zika-naive subjects with no signs or symptoms of acute febrile illness.
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    Extensive cross-reactive IgM and IgG antibody responses were observed in both commercial and in-house ELISA assays when testing patients with ZIKV and DENV infections.
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    DENV-exposed ZIKV patients presented lower anti-ZIKV IgM and higher anti-ZIKV IgG reactivity similar to a secondary dengue response.
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    Given the extensive cross-reactivity in current serological methods, future research should focus on the development of sensitive and specific serological tests to distinguish ZIKV from DENV infections.

Abstract

Zika virus (ZIKV) is a mosquito-borne flavivirus that has caused recent large outbreaks in the Americas. Given its association with severe congenital defects including microcephaly, distinguishing infections caused by ZIKV from those caused by dengue virus (DENV) is of primordial importance. The objectives of this study were to evaluate the recombinant proteins rEIII-ZIKV (Envelope protein domain III) and rNS1ß-leader-ZIKV (non-structural protein 1) for the serological diagnosis of ZIKV in the Mexican population. We also evaluated potential cross-reactivity in commercial enzyme-linked immunosorbent assays (ELISA) based on the ZIKV NS1 and DENV NS1 proteins. rEIII-ZIKV and rNS1ß-leader-ZIKV proteins were tested with sera from 30 PCR-confirmed ZIKV cases, 50 ZIKV-naive, DENV-exposed subjects with no acute febrile disease, (asymptomatic subjects, AS), and 50 ZIKV-naive and DENV naive AS. Commercial ELISA tests were evaluated with sera from 57 ZIKV and 20 DENV PCR-confirmed cases, and 50 ZIKV-naive, DENV-exposed AS. In-house ELISA assays showed that IgM antibody levels against rEIII-ZIKV and rNS1ß-ZIKV were higher in ZIKV naive, DENV-exposed AS than in acutely infected ZIKV individuals. IgG reactivity was highest for rEIII-ZIKV, and indistinguishable between acutely infected ZIKV cases and DENV exposed AS. Positivity for the Euroimmun Zika IgM assay at 7–10 days was considerably higher in DENV-naive ZIKV patients (86%) than in DENV-exposed ZIKV patients (33%), while 39% of the latter had false-negative anti-ZIKV IgG before 7 days of onset. DENV-exposed ZIKV patients presented lower anti-ZIKV IgM and higher IgG responses similar to a secondary dengue response. Forty-four percent of DENV- exposed acute ZIKV patients were DENV IgM positive with the Panbio Dengue assay, and two (15%) of the DENV-naive ZIKV patients presented false DENV IgG conversion. Given the extensive cross-reactivity to both the NS1 and EDIII proteins in current serological methods, the development of sensitive and specific serological tests to distinguish ZIKV from DENV infections is an urgent priority.

Introduction

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus of significant public health concern. Previously considered a pathogen of low virulence with a negligible burden of disease, it began causing massive outbreaks in Oceania and the Pacific Islands in 2007. In 2015, it arrived for the first time in the Americas and rapidly disseminated throughout the continent (Musso and Gubler, 2016). Notably, an unprecedented number of newborns with microcephaly and other serious congenital malformations were born after the outbreak, which was subsequently associated with Zika infection during pregnancy (Franca et al., 2016). These complications, now known as Zika congenital syndrome, are an ongoing threat in endemic regions.
ZIKV is a single-stranded RNA virus transmitted by Aedes species mosquitoes, primary vectors for other members of the Flavivirus genus such as the dengue (DENV) and yellow fever viruses. All flaviviruses are antigenically related and many of them co-circulate in regions now endemic for ZIKV. ZIKV shows a 45–55% protein homology with DENV, and shares all of its essential structural features, including the capsid (C), envelope (E) and precursor of membrane (prM) proteins, as well as its quaternary structure (Heinz and Stiasny, 2017). During natural DENV and ZIKV infections, antibodies are induced against these proteins as well as against the non-structural proteins NS1, NS3 and NS5 (Heinz and Stiasny, 2017; Wahala and de Silva, 2011). Consequently, considerable immunological cross-reactivity has been observed among different viruses of the genus, particularly between DENV and ZIKV (Priyamvada et al., 2016; Felix et al., 2017; Tsai et al., 2017).
The ZIKV NS1 molecule exists predominantly in a dimeric form, and is associated with intracellular and cell surface membranes. Along with the E protein, it induces strong antibody responses during ZIKV infections. Recent studies with human monoclonal antibodies (mAb) to ZIKV NS1 showed these were largely specific for this virus, while mAb against the E protein domains I/II were cross-reactive with all four DENV serotypes (Stettler et al., 2016; Balmaseda et al., 2017).
Currently, detection of viral RNA with nucleic acid amplification tests is the preferred method of diagnosis for acute ZIKV infection. Diagnosis may also be established by detection of anti-ZIKV immunoglobulins with ELISA-based assays that are widely used in clinical settings. Given the serious complications of ZIKV infections, and the limited time frame for detection of viral RNA the development of an accurate serological test is of paramount importance. Several commercial ZIKV ELISA assays, based on the NS1 molecule and the E glycoprotein, have been developed for which good sensitivity and specificity have been reported (Steinhagen et al., 2016; Granger et al., 2017). These tests, however, have yet to be extensively evaluated in populations that have been repeatedly exposed to different DENV serotypes.
The Yucatan Peninsula, in southeast Mexico, is a highly endemic area for DENV, Chikungunya, and more recently, ZIKV. Over the last two decades, all four DENV serotypes have circulated throughout the region causing large outbreaks. Average DENV seroprevalence typically ranges from 70−85% (Pavia-Ruz et al., 2018), but reaches almost 100% in individuals over 30 years of age (MBZ author's unpublished data). The first ZIKV case in Mexico was detected in November 2015. By 2016, a major outbreak occurred, with 7560 confirmed cases reported by the Mexican Ministry of Health. The state of Yucatan had the second highest incidence rate, with a total of 820 confirmed cases, most of which were detected between July and October. (Boletin Epidemiologico, 2016).
Due to the concurrent and widespread circulation of both DENV and ZIKV in Mexico, we evaluated the rEIII-ZIKV (Envelope protein domain III) and rNS1ß-leader-ZIKV (non-structural protein 1) recombinant proteins as candidate antigens for the serological diagnosis of ZIKV. We also assessed the potential cross-reactivity in widely used commercial ELISA tests based on ZIKV and DENV NS1.

Section snippets

Serum samples

The sera used for this study was derived from three different collections. The first set included acute and convalescent sera collected from patients with acute febrile disease seen at the Hospital General O'Horán in Merida, Yucatan, for a prospective arbovirus study conducted during 2015 and 2016. In 2015, all patients had a sample each of whole blood and sera collected on the first day they sought medical care, and a second sample of sera at 21–28 days after onset of illness. During 2016,

Evaluation of commercial Zika and Dengue ELISA assays

Table 1 shows the results for the DENV and ZIKV commercial assays. Only two (5%) of the DENV-exposed acute ZIKV patients and two (14%) of the DENV-naive acute ZIKV patients had detectable anti-ZIKV IgM antibodies during the first week of illness with the Zika Euroimmun assay. By 7–10 days of illness, DENV-naive ZIKV cases presented more marked increases in ZIKV IgM (86%), when compared to DENV-exposed ZIKV cases (33%). Of note, 39% of the DENV-exposed acute ZIKV patients had anti-ZIKV IgG

Discussion

Our findings concur with numerous studies in the literature that report significant cross-reactivity in human antibody responses to DENV and ZIKV (Priyamvada et al., 2016; Felix et al., 2017; Tsai et al., 2017). In our study, 39% of acutely ill DENV-exposed ZIKV patients tested positive for anti-ZIKV IgG antibodies before 7 days of disease onset, likely explained by the presence of cross-reacting anti-DENV IgG. During the first week of illness, ZIKV patients that were previously exposed to DENV

Declaration of Competing Interest

None to declare.

Acknowledgments

This work was supported by the HRP/TDR/PAHO Joint Small Grants Programme [Project 2017-03-0030] and intramural funds from the Fundacion Mexicana para la Salud, Capitulo Peninsular and the CINVESTAV-IPN.
We wish to thank Dr. Leopoldo Flores-Romo for critical review of the manuscript and Dr. Martha Valtierra-Rosales, Dr. Karla Escobedo-Rodriguez and Dr. Yania Alvarado-Rodriguez for generously providing the samples from Zacatecas.

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