Design, development, and validation of multi-epitope proteins for serological diagnosis of Zika virus infections and discrimination from dengue virus seropositivity

Zika virus (ZIKV), an arbovirus from the Flaviviridae family, is the causative agent of Zika fever, a mild and frequent oligosymptomatic disease in humans. Nonetheless, on rare occasions, ZIKV infection can be associated with Guillain-Barr & eacute; Syndrome (GBS), and severe congenital complications, such as microcephaly. The oligosymptomatic disease, however, presents symptoms that are quite similar to those observed in infections caused by other frequent co-circulating arboviruses, including dengue virus (DENV). Moreover, the antigenic similarity between ZIKV and DENV, and even with other members of the Flaviviridae family, complicates serological testing due to the high cross-reactivity of antibodies. Here, we designed, produced in a prokaryotic expression system, and purified three multiepitope proteins (ZIKV-1, ZIKV-2, and ZIKV-3) for differential diagnosis of Zika. The proteins were evaluated as antigens in ELISA tests for the detection of anti-ZIKV IgG using ZIKV- and DENV-positive human sera. The recombinant proteins were able to bind and detect anti-ZIKV antibodies without cross-reactivity with DENV-positive sera and showed no reactivity with Chikungunya virus (CHIKV)- positive sera. ZIKV-1, ZIKV-2, and ZIKV-3 proteins presented 81.6%, 95%, and 66% sensitivity and 97%, 96%, and 84% specificity, respectively. Our results demonstrate the potential of the designed and expressed antigens in the development of specific diagnostic tests for the detection of IgG antibodies against ZIKV, especially in regions with the circulation of multiple arboviruses. From 2015 onward, Zika virus (ZKV) caused major epidemics in Brazil and other South American countries, where many children were born with congenital problems, such as microcephaly, as a result of infections in pregnant women. Additionally, many cases of Guillain Barr & eacute; Syndrome in adults were also caused by infections with the virus. Detection of ZKV infection is, therefore, very important; nonetheless, most patients infected by ZKV develop either no symptoms or symptoms that are not distinguishable from diseases caused by other co-circulating arboviruses, including dengue. To complicate matters, Zika, and dengue viruses show intense genetic resemblance, and serological diagnosis presents high levels of uncertainty due to extensive cross-reactivity. In this work, we developed proteins to serologically differentiate Zika from dengue infections with no or minimum cross-reactivity. We produced three multiepitope, recombinant proteins which were able to differentiate zika-positive samples from dengue-positive sera. The tests employing the recombinant proteins had high sensitivity and specificity, especially in the case of ZIKV-1 and ZIKV-2 proteins, whilst the ZIKV-3 protein was less efficient. The recombinant proteins showed clear potential to be used as diagnostic tools for detecting antibodies against ZIKV infections with no cross-reactivity to other prevalent arbovirus.