Antibody to the nonstructural protein NS1 of Japanese encephalitis virus: potential application of mAb-based 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.
Section snippets
Case definitions
JE infections were defined as febrile illness associated with clinical features of viral encephalitis and the presence of IgM to JE virus (IgM negative to dengue virus) in the cerebrospinal fluid [27] or a four-fold or greater rise in hemagglutination inhibition (HI) test against JE antigen in paired serum [28]. Dengue infections were defined as febrile illness associated with the isolation of a dengue virus, the detection of dengue-specific IgM, positive test of polymerase-chain-reaction [29],
Establishment of JE virus NS1-specific indirect IgM, IgA, IgG ELISA
To study the NS1-specific antibody responses in JE patients, we have established an indirect ELISA to measure the NS1-specific IgM, IgA, and IgG antibodies. Initially, a sandwich ELISA was performed to titrate the concentrations of NS1 antigens of JE, DEN-1, DEN-2, DEN-3, and DEN-4 viruses in the culture supernatants of Vero cells infected with each of these viruses. The results showed that the NS1 antigens captured by D2/8-1 mAb reached plateau at the 1:3 dilution of culture supernatants for
Discussion
The serodiagnosis of flavivirus is rather complicated for the following reasons: (1) The high cross-reactivity of IgG antibodies to homologous and heterologous viruses prohibits its use of definitive diagnosis; (2) Sequential flavivirus infection makes differential diagnosis difficult due to pre-existing antibodies and ‘original antigenic sin’ phenomenon in regions where two or more flaviviruses co-circulate; and (3) Antibodies to viral structural protein induced by JE vaccination complicate
Acknowledgements
We wish to thank Yun-Yih Chang for her expert technical assistance, Yaw-Hsiung Huang for providing postinfection sera of JE patients. This work was in part supported by grants DDO I-871X-CR-501 P from the National Health Research Institute, Department of Health, and NSC 88-2318-13-04313-001-M51 and NSC 89-2318-B-043B-001-M51 from the National Science Council, Taiwan, Republic of China.
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