Soluble and immunoreactive flaviviral ns1 polypeptides

#According to the World Health Organization tropical diseases transferred by flaviviruses such as Zika virus, Dengue virus and others, continue to spread worldwide (Olliaro et al. PLOS Neglected Tropical Diseases Feb. 1, 2018, 1-13). Flavivirus is a genus of the family Flaviviridae, including e.g. Zika virus, Dengue virus, West Nile virus, tick-borne encephalitis virus, yellow fever virus and Japanese encephalitis virus. Flaviviruses are transmitted by arthropod vectors such as insects, mosquitos or ticks, e.g. the Egyptian and the Asian mosquito strains Aedes aegypti and Aedes albopictus, and are therefore classified as arboviruses (arthropod-borne virus).

#Infection of an otherwise healthy person with a flavivirus may lead to mild symptoms like fever, fatigue, rash and body aches. However, depending on the person's general health and immune status the infection may also lead to severe and sometimes even lethal sequelae. While the mild symptoms share some similarity the serious complications are very different, depending on the respective flavivirus.

#Zika virus can be transmitted from an expectant mother to her fetus in utero and is suspected to cause severe brain malformations and defects such as microcephaly in the unborn child. 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-Barrd syndrome (muscle weakness caused by the immune system which attacks the peripheral nervous system).

#Commercially available immunoassays (both IgG and IgM immunoassays) for detecting flaviviral antibodies such as antibodies against Zika virus, Dengue virus and West Nile virus are based on the ELISA principle. However, the known immunoassays evidently use the full length NS1 antigen that on the one hand is highly immunoreactive but on the other hand shows a high immunological cross-reactivity with antibodies raised against the NS1 homologues of Zika, Dengue and other flaviviruses like West Nile virus, yellow fever virus or other flaviviruses. In addition, due to its complex quaternary structure it is not possible to provide NS1 in a soluble and stable monomeric form which would be a prerequisite in designing an immunoassay for specific IgG detection in the double-antigen sandwich format. Dedicated flaviviral NS1 antigens that would fulfill these requirements and would enable highly specific immunoassays suitable for automation have not been described in prior art. Surprisingly, by confining the flaviviral NS1 antigen to its wing domain and removing the so-called ß-ladder domain sequences of the NS1 antigen, a soluble and stable NS1 antigen variant is obtained that is able to specifically detect antibodies against a specific kind of flavivirus.

#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 showed 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 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.

#Similarly, for Dengue virus antibody detection we were able to identify NS1 wing domain antigens from all of the four Dengue virus serotypes that we overexpressed in E. coli with high yield, and which refold into an immunoreactive form after purification and functional solubilization. We found evidence that the individual Dengue wing domains of DENV 1-4 react very differently with a pre-characterized commercial set of DENV positive sera. The results indicate that an immunological differentiation of the four distinct Dengue virus serotypes is feasible using the Dengue NS1 wing domain as antigen in an immunoassay. Overall, the Dengue NS1 wing domain lacking the ß-ladder domain of NS1 is an antigen suitable for specific Dengue antibody detection.