A Monoclonal Antibody Specific for Japanese Encephalitis Virus with High Neutralizing Capability for Inclusion as a Positive Control in Diagnostic Neutralization Tests.

Japanese encephalitis virus (JEV) is the most common cause of viral encephalitis in Asia, and it is increasingly a global public health concern because of its recent geographic expansion. Although commercial vaccines are available and used in some endemic countries, JEV continues to cause illness, with more than 60,000 cases reported annually. To develop a reproducible positive control antibody useable in diagnosis of JEV infections, murine hybridomas were developed from mice inoculated with a combination of IXIARO JEV vaccine and JEV domain III of the envelope protein (E-DIII). Monoclonal antibodies (MAbs) were characterized for their ability to neutralize virus in vitro. Monoclonal antibody 17BD3-2 was found to be JEV specific and highly neutralizing, with a plaque reduction neutralization test (PRNT)90 endpoint titer of 1.25 µg/mL. The functional epitopes were mapped using virus neutralization escape variants to amino acid residues S309, K312, and G333 in E-DIII. This MAb may be substituted for human immune sera used as a positive control in PRNT for distribution to public health laboratories worldwide in potential future outbreaks of JEV.

A humanized monoclonal antibody neutralizes yellow fever virus strain 17D-204 in vitro but does not protect a mouse model from disease.

The yellow fever virus (YFV) vaccine 17D-204 is considered safe and effective, yet rare severe adverse events (SAEs), some resulting in death, have been documented following vaccination. Individuals exhibiting post-vaccinal SAEs are ideal candidates for antiviral monoclonal antibody (MAb) therapy; the time until appearance of clinical signs post-exposure is usually short and patients are quickly hospitalized. We previously developed a murine-human chimeric monoclonal antibody (cMAb), 2C9-cIgG, reactive with both virulent YFV and 17D-204, and demonstrated its ability to prevent and treat YF disease in both AG129 mouse and hamster models of infection. To counteract possible selection of 17D-204 variants that escape neutralization by treatment with a single MAb (2C9-cIgG), we developed a second cMAb, 864-cIgG, for use in combination with 2C9-cIgG in post-vaccinal therapy. MAb 864-cIgG recognizes/neutralizes only YFV 17D-204 vaccine substrain and binds to domain III (DIII) of the viral envelope protein, which is different from the YFV type-specific binding site of 2C9-cIgG in DII. Although it neutralized 17D-204 in vitro, administration of 864-cIgG had no protective capacity in the interferon receptor-deficient AG129 mouse model of 17D-204 infection. The data presented here show that although DIII-specific 864-cIgG neutralizes virus infectivity in vitro, it does not have the ability to abrogate disease in vivo. Therefore, combination of 864-cIgG with 2C9-cIgG for treatment of YF vaccination SAEs does not appear to provide an improvement on 2C9-cIgG therapy alone.

Development of a small animal peripheral challenge model of Japanese encephalitis virus using interferon deficient AG129 mice and the SA14-14-2 vaccine virus strain.

Japanese encephalitis virus (JEV) is the most common cause of viral encephalitis in Asia, and it is increasingly a global public health concern due to its recent geographic expansion. While commercial vaccines are available and used in some endemic countries, JEV continues to be a public health problem, with 50,000 cases reported annually. Research with virulent JEV in mouse models to develop new methods of prevention and treatment is restricted to BSL-3 containment facilities, confining these studies to investigators with access to these facilities. We have developed an adult small animal peripheral challenge model using interferon-deficient AG129 mice and the JEV live-attenuated vaccine SA14-14-2, thus requiring only BSL-2 containment. A low dose of virus (10PFU/0.1ml) induced 100% morbidity in infected mice. Increased body temperatures measured by implantable temperature transponders correlated with an increase in infectious virus and viral RNA in serum, spleen and brain as well as an increase in pro-inflammatory markers measured by a 58-biomarker multi-analyte profile (MAP) constructed during the course of infection. In the future, the MAP measurements can be used as a baseline for comparison in order to better assess the inhibition of disease progression by other prophylactic and therapeutic agents. The use of the AG129/JEV SA14-14-2 animal model makes vaccine and therapeutic studies feasible for laboratories with limited biocontainment facilities.