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

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.

Expression and purification of domain III proteins from Dengue and Zika viruses.

The envelope (E) protein from Dengue and Zika viruses comprises three functional and structural domains (DI, DII, and DIII). Domain III induces most of the neutralizing antibodies and, as such, is considered as having the highest antigenic potential for the evaluation of population-level surveillance and for detecting past infections in both Dengue and Zika patients. The present study aimed to clone and express recombinant proteins of domain III from Dengue virus serotype 2 and from Zika virus in a prokaryotic system, as well as evaluate their immunogenicity and cross-reactivity. Both antigens were successfully purified and their antigenicity was assessed in mice. The antibodies elicited by domain III of Zika and Dengue virus antigens recognized specifically the native proteins in infected cells. Furthermore, the antigens showed a more specific immunogenic response than that of domain III proteins from Dengue virus. The generated recombinant proteins can be potentially used in subunit vaccines or for surveillance studies.