title: Evaluation of recombinant dengue viral envelope B domain protein antigens for the detection of dengue complex-specific antibodies.
abstract: To increase the specificity of dengue (DEN) diagnosis based on antibody detection, we have evaluated recombinant proteins as antigens that incorporate most of the B domain of the DEN virus envelope protein fused to the trpE protein of Escherichia coli (trpE-DEN). A pooled antigen consisting of trpE-DEN proteins representing all four serotypes of DEN virus was used in an indirect ELISA for the detection of IgG or IgM antibody. This assay was compared with a standard IgG indirect ELISA and an IgM-capture ELISA using DEN virus-infected cell culture pooled antigens. The results indicated that the trpE-DEN antigens and the cell culture antigens were equally sensitive for detecting IgM and IgG antibodies in convalescent sera from Peru and Indonesia representing virus isolation-confirmed primary and secondary DEN infections, respectively. Fourteen day postinfection IgG antibody-positive sera obtained from individuals infected with DEN-1 virus who had been vaccinated with other flaviviruses were more strongly reactive with the cell culture antigen than with the recombinant antigen, but by day 21 postinfection, a strong antibody response to the trpE-DEN antigens was present. These results suggested that the early antibody response was directed predominantly towards shared flavivirus group antigens that were not detected with the trpE-DEN antigens. Comparison of the trpE-DEN-1 recombinant antigen with a DEN-1 virus-infected cell lysate antigen for the detection of IgG antibody in sera from a cohort of 55 individuals from Peru who seroconverted over a one-year period indicated greater specificity for the recombinant antigens. Also, sera from individuals with no known DEN infections that had been sequentially vaccinated with yellow fever and Japanese encephalitis reacted with the DEN virus cell culture antigen in the IgG ELISA, but did not react with the trpE-DEN pooled antigens. Similarly, YF IgM antibody positive samples that showed cross-reactivity with the DEN virus cell culture antigens, did not react with the trpE-DEN pooled antigens. These results indicated that the trpE-DEN pooled antigen provided a more specific diagnosis of dengue infections than DEN virus-infected cell culture antigen and avoided the biohazards associated with handling live virus during the preparation of diagnostic reagents. The trpE-DEN pooled antigen should permit a better approach to distinguish between past DEN and other flavivirus infections in epidemiologic surveys, and also increase the specificity of serologic diagnosis of acute DEN infections.
authors.affiliation: U.S. Naval Medical Research Institute, Bethesda, Maryland 20889-5607, USA.
authors.forename: M
authors.initials: M
authors.lastname: Simmons
authors.forename: K R
authors.initials: KR
authors.lastname: Porter
authors.forename: J
authors.initials: J
authors.lastname: Escamilla
authors.forename: R
authors.initials: R
authors.lastname: Graham
authors.forename: D M
authors.initials: DM
authors.lastname: Watts
authors.forename: K H
authors.initials: KH
authors.lastname: Eckels
authors.forename: C G
authors.initials: CG
authors.lastname: Hayes
mesh_terms: D000914:Antibodies, Viral / Q000097:blood*; D000956:Antigens, Viral / Q000276:immunology*; D015331:Cohort Studies; D003429:Cross Reactions; D003715:Dengue / Q000175:diagnosis* / Q000276:immunology; D003716:Dengue Virus / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D007075:Immunoglobulin M / Q000097:blood; D009500:Neutralization Tests; D011993:Recombinant Fusion Proteins / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology; D012680:Sensitivity and Specificity; D014759:Viral Envelope Proteins / Q000276:immunology*
chemical_list: D000914:Antibodies, Viral; D000956:Antigens, Viral; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins