Soluble and immunoreactive zika virus ns1 polypeptides

#Zika virus is an arbovirus (arthropod-borne virus, i.e. virus transmitted by arthropod vectors such as insects) that belongs to the Flaviviridae family of viruses and is spread by the Egyptian and the Asian mosquito strains Aedes aegypti and Aedes albopictus. In the years 2013 and 2014, the incidence of Zika infections in French Polynesia escalated, followed by large outbreaks in Latin America since 2015, peaking epidemic-like at the time around the Olympic Games in Brazil in 2016. So far, efficacious medical treatment or vaccination is lacking. In healthy persons an infection with Zika virus usually leads to no or only mild symptoms. However, for pregnant women the situation is different. The Zika virus can be transmitted from a pregnant woman to her unborn child and is suspected to cause severe brain malformations and defects such as microcephaly. Microcephaly (derived from Greek for “small head”) is a condition in which a baby's brain does not develop properly, and thus its head has a smaller size than normal. Infection in adults may lead to the so-called Guillan-Barré syndrome (muscle weakness caused by the immune system which attacks the peripheral nervous system). Efficacious medical treatment after infection is lacking. In order to receive timely supportive treatment it is crucial to know if a patient has been infected with Zika virus. In particular, highly specific immunoassays are utterly needed in regions with high prevalence of multiple flaviviral infections. As a matter of course, it is a daunting task to reliably diagnose, e.g., a recent Zika infection in an individual that has undergone other flaviviral infections such as Dengue or yellow fever in the past and whose serum is characterized by polyclonal antibodies against the main immunogens of Dengue and yellow fever virus.

#Therefore, apart from a vaccination and medication development strategy, also highly sensitive and specific serological diagnostics need to be developed in order to reliably confirm or rule out an infection of a patient with Zika virus. Since 2016 a couple of ELISA-based immunoassays detecting antibodies against the Zika virus NS1 antigen are commercially available (e.g. Huzly et al., Euro Surveill. 2016; 21 (16) pii=30203, 1-4). However, these assays are suspected to cross-react with antibodies that have been raised against related viruses such as Dengue virus and other arboviruses that belong to the family of flaviviruses like West Nile virus, yellow fever virus, tick-borne encephalitis virus (FSME) or Japanese encephalitis virus. This cross-reactivity would lead to false positive results and erroneous interpretation of a patient's immune status and seems to be due to the structural and sequence homologies of Zika NS1 (non-structural antigen) and its counterparts in other flaviviruses such as West Nile and Dengue viruses (Hilgenfeld 27 Oct. 2016, Embo J. 1-3).

#It is conceivable that the incidence and prevalence data that have been reported for the Zika epidemic in Brazil in 2015/2016, have suffered the flaw of an exaggeration bias due to the limited specificity of the immunoassays that have been used at the time. In order to evaluate prevalence and incidence data and to assess the objective risk of an emerging epidemic, clear-cut diagnostics are an indispensable prerequisite. Serologic assays with poor specificity would overemphasize the extent of an epidemic and therefore lead to panic-driven decisions not only by the authorities, but also by unsettled and frightened individuals. For instance, it has been reported that the number of abortions in Brazil has significantly increased in the wake of the medial reporting on the true or ostensible Zika epidemic.

#Currently available ELISA format immunoassays for detecting anti-Zika virus antibodies employ an NS1 antigen as immunoreactive reagent. However, we find that these assays lack specificity, leading to a fairly high number of false positive results. Surprisingly, by confining the Zika NS1 antigen to its wing domain as explained further below, the number of erroneously reactive samples can be considerably reduced while the high sensitivity of the assay is maintained.

#When samples positive for anti-Zika virus antibodies were tested with two different fragments of the NS1 antigen of Zika, i.e. with the so-called “wing” domain antigen and the so-called “ß-ladder” domain antigen it became evident that both antigens are able to detect anti-Zika antibodies. However, we found out that the wing domain antigen does not cross-react with Dengue antibody positive samples whereas the Zika NS1 ß-ladder domain antigen does cross-react with Dengue antibody positive samples, leading to false positive results and to erroneous conclusions. Additional blocking experiments with anti-Zika positive sera and NS1 antigens of related arboviruses conclusively show that the Zika NS1 wing domain antigen signal can hardly be quenched by these related arboviral NS1 antigens whereas the Zika NS1 ß-ladder domain antigen signal is quenched significantly. We infer that the ß-ladder antigen is considerably blocked from binding to the immunoglobulins by the competing related arboviral NS1 antigens. Thus, we were able to show that the Zika NS1 wing domain antigen is less susceptible to immunological cross-reactivity with other arboviral NS1 homologues. As a consequence, the Zika NS1 wing domain enables an immunoassay for anti-Zika antibodies with superior specificity that is capable of diagnosing Zika virus infections in the presence of other (recent or past) arbovirus infections, in an embodiment discriminating Zika virus infections from Dengue virus infections.

#The invention therefore concerns a polypeptide suitable for detecting antibodies against Zika virus in an isolated biological sample comprising a Zika antigen with a Zika virus NS1 wing domain specific amino acid sequence wherein no amino acid sequences from the Zika virus NS1 ß-ladder domain are present in said polypeptide. In an embodiment, no further Zika virus NS1 specific amino acid sequences are present in this polypeptide, in an embodiment no further Zika virus specific amino acid sequences are present in said polypeptide. In an embodiment, said Zika virus NS1 domain specific amino acid sequence consists essentially of SEQ ID NOs. 1 or 2, in an embodiment consists of SEQ ID NOs 1 or 2. In particular, no amino acid sequences from the C-terminal so-called ß-ladder domain are present in the polypeptide of the invention. In an embodiment SEQ ID NO:4 is not present in said polypeptide.