Abstract
ABSTRACTJapanese encephalitis virus (JEV) remains a leading cause of viral encephalitis worldwide. Although JEV-specific antibodies have been described, an assessment of their ability to neutralize multiple genotypes of JEV has been limited. Here, we describe the development of a panel of mouse and human neutralizing monoclonal antibodies (MAbs) that inhibit infection in cell culture of four different JEV genotypes tested. Mechanism-of-action studies showed that many of these MAbs inhibited infection at a postattachment step, including blockade of virus fusion. Mapping studies using site-directed mutagenesis and hydrogen-deuterium exchange with mass spectrometry revealed that the lateral ridge on domain III of the envelope protein was a primary recognition epitope for our panel of strongly neutralizing MAbs. Therapeutic studies in mice demonstrated protection against lethality caused by genotype I and III strains when MAbs were administered as a single dose even 5 days after infection. This information may inform the development of vaccines and therapeutic antibodies as emerging strains and genotypic shifts become more prevalent.
Highlights
Japanese encephalitis virus (JEV) remains a leading cause of viral encephalitis worldwide
We screened ~3,800 hybridoma supernatants from five independent fusions for binding to JEV-infected cells by flow cytometry and direct virus binding by enzyme-linked immunosorbent assay (ELISA) and cloned 13 JEV monoclonal antibodies (MAbs) by limiting dilution for further characterization
We found that alanine substitution of certain amino acids (e.g., T321, D332, and I383), which correspond to sites in E-DIII-lateral ridge (LR), caused loss of binding of most of the neutralizing murine and human MAbs tested, especially JEV-31, JEV-131, JEV-143, and hJEV-69 (Fig. 4A and B)
Summary
Japanese encephalitis virus (JEV) remains a leading cause of viral encephalitis worldwide. We describe the development of a panel of mouse and human neutralizing monoclonal antibodies (MAbs) that inhibit infection in cell culture of four different JEV genotypes tested. IMPORTANCE Japanese encephalitis virus (JEV) is a vaccine-preventable cause of viral encephalitis, the inactivated and live attenuated platforms available are derived from strains belonging to a single genotype (GIII) due to its historical prevalence in areas of JEV epidemics. Despite the existence of inactivated and live attenuated vaccine platforms, Japanese encephalitis virus (JEV) remains a primary cause of viral encephalitis. It is prevalent in Asia, with approximately 68,000 clinical cases [1, 2] and an estimated 10,000 to 15,000 deaths per year [1]. Structural analysis of the JEV E protein shows a smaller dimer interface with increased contacts at the E-DI-DIII pocket compared to those of related flaviviruses [15]
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