Antibody to the nonstructural protein NS1 of Japanese encephalitis virus: potential application of mAb-based indirect ELISA to differentiate infection from vaccination

Abstract

An indirect enzyme-linked immunosorbent assay (ELISA) was developed to detect and differentiate the antibody responses to Japanese encephalitis (JE) virus nonstructural protein NS1 between infected and vaccinated individuals. The results showed that all convalescent sera from JE patients contained NS1-specific IgG antibodies, while 65 and 40% of these sera showed detectable NS1-specific IgM and IgA antibodies, respectively. Specificity analysis showed that NS1-specific IgM and IgA antibodies from JE patients do not cross-react to dengue virus NS1 glycoprotein, while IgG antibodies from 10% of JE patients showed significant cross-reaction to dengue virus NS1 glycoprotein. To differentiate infection from vaccination, the immune sera from 24 children vaccinated with inactivated JE vaccine were analyzed. The data showed that none of these immune sera had detectable NS1-specific IgG antibodies. The results demonstrated the potential application of JE NS1-specific indirect ELISA to differentiate infection from vaccination.

Introduction

The family Flaviviridae consists of more than 60 viruses, which can be divided into at least eight antigenic subgroups, based on cross-neutralization tests with polyclonal antisera [1]. Among these, Japanese encephalitis (JE) virus and dengue virus are the two dominant flaviviruses found in tropical and subtropical regions of the world [2], [3]. In the regions, where two or more flaviviruses are co-circulating, the serodiagnosis is difficult due to high serological cross-reactivity between various flaviviruses [1], [4]. Makino et al. [5] had reported that the cross-reactivity among flaviviruses in different subgroups was quite often in the sequential flavivirus infection even by neutralization tests, which is the least cross-reactive among the available serological assays. Thus, in the endemic area where JE has been well controlled due to the successful use of inactivated JE vaccine [6], the immunization program will actually complicate the serodiagnosis due to cross-reactive antibodies induced by JE vaccination. In addition, since antibodies to the structural proteins, envelope (E) and membrane (M), are induced by both vaccination and infection, it is not possible to differentiate naturally infected individuals from vaccinees by detection of antibodies to structural proteins alone. Therefore, other antigenic marker should be sought to develop a new diagnostic assay to differentiate JE infection from vaccination.

Studies from various pathogens have demonstrated the usefulness of nonstructural protein-specific antibody assay to differentiate infection from vaccination [7], [8], [9]. A number of nonstructural proteins are rational candidates to identify antibodies against virus-specific proteins that are present only in the infected individuals. Among these, NS1 glycoprotein is an excellent target antigen in JE infection since high titer NS1-specific antibodies were reported in 56% sera of JE patients using Western blot analysis [10]. In addition, the inactivated JE vaccine produced from mouse brain homogenate should consist of purified preparation of inactivated virions with minor NS1 contaminants and therefore induces antibodies almost exclusively to the structural proteins of the JE virus. The flavivirus NS1 has been implicated in viral RNA replication, although its precise role has not been identified [11]. The NS1 glycoprotein has been shown to be an amphitropic homodimer of ∼90 kDa present both as an intracellular, membrane associated form and as an extracellular, secreted form in infected mammalian cells [12], [13].

NSI-specific antibodies have been demonstrated to provide protective immunity against Yellow Fever virus [14], [15], dengue viruses [16], [17], [18], [19], tick-borne encephalitis virus [20], and JE virus [21] in mouse and/or monkey animal models.

Limited information is available to the anti-NS1 antibody response from JE [10] and dengue [22], [23], [24] patients up to now. We have studied the NS1-specific antibody responses of JE and dengue infections for diagnostic and vaccine interests and reported the dengue virus NS1-specific antibody responses from dengue patients using synthetic peptides and monoclonal antibody (mAb) approaches [25], [26]. The results demonstrated that all of the dengue patients with dengue fever or dengue hemorrhagic fever produced significant NS1-specific antibodies. In this study, we analyzed and compared the NS1-specific antibody responses from naturally infected JE patients and JE vaccinee in Taiwan area. The results demonstrated that the potential application of JE virus NS1-specific indirect ELISA to differentiate infection from vaccination.