RESUMEN
The Flavivirus E protein induces protective immunity, and its Abs cause serious problems for serodiagnosis because of the difficulty in differentiating cross-reactive Abs. Moreover, cross-reactive Abs may increase disease severity after secondary Flavivirus infections via Ab-dependent enhancement. Cross-reactive epitopes are therefore critical for understanding serodiagnosis and improving the general knowledge of Flavivirus infections. A minimal epitope, 227GSSAGTWQN235, was identified by a neutralizing mAb 1G2 against duck Tembusu virus (DTMUV), which recognized only monomer E protein under nonreducing conditions. It was unexpectedly found that mutations in the epitope residues G231 or W233 completely abolished reactivity to 1G2 and sera from mice infected with Japanese encephalitis virus, West Nile virus, and Zika virus. An immunofluorescence assay confirmed that mAb 1G2 could cross-react with the E proteins from Japanese encephalitis virus, West Nile virus, and Zika virus. Protein and virus modeling revealed that the epitope was surface accessible in the mature virus and located in the hi loop of domain II. The neutralization of DTMUV by 1G2 played a clear therapeutic role in mouse models. The passive transfer of 1G2 resulted in 100% survival, reduced weight loss, and the complete clearance of DTMUV from the blood of BALB/c mice. Our findings document, for the first time to our knowledge, that mAb 1G2 targets the cross-reactive epitope on the hi loop of domain II in the E protein and might be of potential therapeutic value in treating DTMUV infection and improve the understanding of the issues related to serodiagnosis.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Reacciones Cruzadas/inmunología , Epítopos/inmunología , Flavivirus/inmunología , Proteínas del Envoltorio Viral/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Línea Celular , Patos/virología , Infecciones por Flavivirus/inmunología , Ratones , Ratones Endogámicos BALB C , Virus del Nilo Occidental/inmunología , Virus Zika/inmunología , Infección por el Virus Zika/inmunologíaRESUMEN
Duck hepatitis A subtype 1 virus (DHAV-1) infection causes high mortality in ducklings, resulting in significant losses to duck industries. VP3 is a structural protein of DHAV-1. However, B-cell epitopes on VP3 have not been investigated. To stimulate VP3 antibody response, eukaryotic expression plasmid pCI-neo-VP3 was constructed and used as DNA immunogen to prepare mAbs. Western blot showed that 25.5 kDa VP3 could be detected by mAbs in duck embryo fibroblast (DEF) cells transfected with pCI-neo-VP3. Immunofluorescence assay showed that mAbs could specifically bind to DEF cells infected with DHAV-1. DAPI staining indicated that VP3 localizes to the cytoplasm and nucleus of DHAV-1 infected DEF. With neutralizing mAb 3B7, minimal epitope PSNI was mapped. Sequence alignment indicated that 205PSNI208 is highly conserved among DHAV-1, but different from those of DHAV-2 and DHAV-3. Epitope peptide reacted specifically with DHAV-1-positive duck sera by dot blotting, revealing PSNI is DHAV-1 type-specific epitope and the importance of these amino acids in antibody-epitope binding reactivity. These findings provided useful information for understanding the antigenicity of VP3 and might be valuable in the development of epitope-based vaccine or diagnostic kit for DHAV-1 infection and provide insights for understanding the pathogenesis of DHAV-1.
Asunto(s)
Mapeo Epitopo , Epítopos de Linfocito B/inmunología , Virus de la Hepatitis del Pato/inmunología , Proteínas Estructurales Virales/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/aislamiento & purificación , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/aislamiento & purificación , Núcleo Celular/química , Células Cultivadas , Citoplasma/química , Patos , Fibroblastos/virología , Técnica del Anticuerpo FluorescenteRESUMEN
BACKGROUND: The VP1 protein of duck hepatitis A virus (DHAV) is a major structural protein that induces neutralizing antibodies in ducks; however, B-cell epitopes on the VP1 protein of duck hepatitis A genotype 1 virus (DHAV-1) have not been characterized. METHODS AND RESULTS: To characterize B-cell epitopes on VP1, we used the monoclonal antibody (mAb) 2D10 against Escherichia coli-expressed VP1 of DHAV-1. In vitro, mAb 2D10 neutralized DHAV-1 virus. By using an array of overlapping 12-mer peptides, we found that mAb 2D10 recognized phages displaying peptides with the consensus motif LPAPTS. Sequence alignment showed that the epitope 173LPAPTS178 is highly conserved among the DHAV-1 genotypes. Moreover, the six amino acid peptide LPAPTS was proven to be the minimal unit of the epitope with maximal binding activity to mAb 2D10. DHAV-1-positive duck serum reacted with the epitope in dot blotting assay, revealing the importance of the six amino acids of the epitope for antibody-epitope binding. Competitive inhibition assays of mAb 2D10 binding to synthetic LPAPTS peptides and truncated VP1 protein fragments, detected by Western blotting, also verify that LPAPTS was the VP1 epitope. CONCLUSIONS AND SIGNIFICANCE: We identified LPAPTS as a VP1-specific linear B-cell epitope recognized by the neutralizing mAb 2D10. Our findings have potential applications in the development of diagnostic techniques and epitope-based marker vaccines against DHAV-1.