Generation and Characterization of Monoclonal Antibodies against Dengue Virus Type 1 for Epitope Mapping and Serological Detection by Epitope-Based Peptide Antigens

Abstract

Dengue virus (DEN), the pathogen behind dengue hemorrhagic fever, remains a public health problem in Asia and South America. In this study, monoclonal antibodies (MAbs) against DEN serotype 1 (DEN-1) were generated by fusing NSI/1-Ag4-1 mouse myeloma cells with lymphocytes from BALB/c mice immunized with DEN-1. Twelve MAbs were found to react specifically to the DENs by enzyme-linked immunosorbent assay, immunofluorescence analysis, and immunoblotting analysis. Five MAbs, namely, DA4-7, DA6-7, DA9-5, DA10-2, and DA11-13, were found to react with envelope proteins of DEN-1. Two serotype-specific MAbs of DEN-1, DA6-7 and DA11-13, were further shown to neutralize DEN-1 infection by a plaque reduction neutralization test. The neutralizing epitopes of these MAbs were further identified from a random peptide library displayed on phage. Immunopositive phage clones reacted specifically with these MAbs and did not react with normal mouse serum. Epitope-based peptide antigens were proved able to detect antibodies in serum samples collected from DEN-1-infected patients but not in those taken from DEN-2-infected patients or healthy controls. We believe that these MAbs and neutralizing epitopes will provide information that will lead to the development of DEN-1 serotype-specific diagnostic reagents and vaccines.Dengue virus (DEN) causes a variety of illnesses that range in severity from mild, in such syndromes as dengue fever (DF), to severe, in the syndromes dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) (18,19). DF is manifested as a typical biphasic fever, headache, body pain, and rash. DHF, however, is characterized by abnormalities of hemostasis and vascular permeability and is often fatal. Two-fifths of the world's population is at risk of infection, and it is estimated that 50 million people a year are infected with this virus. One percent of people infected with this virus will develop DHF (59).Until now, no effective method has been reported to be capable of preventing the development of DHF/DSS because the pathogenic mechanisms of this disease are unclear (5, 6, 41). However, several relevant hypotheses exist, including antibody-dependent enhancement of infection and virus variation (20,35,37). DENs are divided into four serotypes, DEN-1, -2, -3, and -4, which have very similar genome sequences and envelope protein (E protein) antigenic properties. A secondary infection with a different DEN serotype may increase the risk for DHF (20). One possibility is that monocytes/macrophages take up the virus complexes by binding to nonneutralizing antibodies or subneutralizing cross-reactive antibodies (6,19,20). Antibody-dependent enhancement, in conjunction with activation of memory T-cell responses, is believed to contribute to the immunopathogenic disease process (50).Virus variation may also account for differences in severity of dengue-related diseases (32,47,49). Moreover, since DEN infection is often accompanied by the production of cytokines or chemokines and the activation of complement or immune cel...

Epithelial Cell Adhesion Molecule Regulation Is Associated with the Maintenance of the Undifferentiated Phenotype of Human Embryonic Stem Cells

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Analysis of Epitopes on Dengue Virus Envelope Protein Recognized by Monoclonal Antibodies and Polyclonal Human Sera by a High Throughput Assay

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DNA methylation and histone modification regulate silencing of OPG during tumor progression

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