Formalin Inactivation of Japanese Encephalitis Virus Vaccine Alters the Antigenicity and Immunogenicity of a Neutralization Epitope in Envelope Protein Domain III

Formalin-inactivated Japanese encephalitis virus (JEV) vaccines are widely available, but the effects of formalin inactivation on the antigenic structure of JEV and the profile of antibodies elicited after vaccination are not well understood. We used a panel of monoclonal antibodies (MAbs) to map the antigenic structure of live JEV virus, untreated control virus (UCV), formalin-inactivated commercial vaccine (FICV), and formalin-inactivated virus (FIV). The binding activity of T16 MAb against Nakayama-derived FICV and several strains of FIV was significantly lower compared to live virus and UCV. T16 MAb, a weakly neutralizing JEV serocomplex antibody, was found to inhibit JEV infection at the post-attachment step. The T16 epitope was mapped to amino acids 329, 331, and 389 within domain III (EDIII) of the envelope (E) glycoprotein. When we explored the effect of formalin inactivation on the immunogenicity of JEV, we found that Nakayama-derived FICV, FIV, and UCV all exhibited similar immunogenicity in a mouse model, inducing anti-JEV and anti-EDII 101/106/107 epitope-specific antibodies. However, the EDIII 329/331/389 epitope-specific IgG antibody and neutralizing antibody titers were significantly lower for FICV-immunized and FIV-immunized mouse serum than for UCV-immunized. Formalin inactivation seems to alter the antigenic structure of the E protein, which may reduce the potency of commercially available JEV vaccines. Virus inactivation by H2O2, but not by UV or by short-duration and higher temperature formalin treatment, is able to maintain the antigenic structure of the JEV E protein. Thus, an alternative inactivation method, such as H2O2, which is able to maintain the integrity of the E protein may be essential to improving the potency of inactivated JEV vaccines.福尔马林灭活的日本脑炎病毒（Japanese encephalitis virus, JEV）疫苗已广泛应用，但福尔马林灭活工艺对JEV抗原结构的影响，以及疫苗接种后诱导的抗体谱特征，目前尚未得到充分阐明。我们使用一组单克隆抗体（monoclonal antibodies, MAbs），对活JEV病毒、未处理对照病毒（untreated control virus, UCV）、福尔马林灭活商用疫苗（formalin-inactivated commercial vaccine, FICV）以及福尔马林灭活病毒（formalin-inactivated virus, FIV）的抗原结构进行了图谱解析。相较于活病毒与未处理对照病毒，T16单克隆抗体对中山株来源的FICV以及数株FIV的结合活性显著降低。T16单克隆抗体属于弱中和性JEV血清型复合抗体，可在病毒吸附后的阶段抑制JEV感染。T16表位被定位至包膜糖蛋白（envelope glycoprotein, E）结构域III（domain III, EDIII）内的329、331及389位氨基酸残基。在探究福尔马林灭活对JEV免疫原性的影响时，我们发现中山株来源的FICV、FIV及UCV在小鼠模型中均表现出相似的免疫原性，可诱导抗JEV以及抗EDII 101/106/107表位特异性抗体。然而，相较于UCV免疫组小鼠血清，FICV免疫组与FIV免疫组小鼠血清中针对EDIII 329/331/389表位的IgG抗体及中和抗体滴度均显著更低。福尔马林灭活工艺似乎会改变E蛋白的抗原结构，这可能会降低商用JEV疫苗的效价。采用过氧化氢（H₂O₂）而非紫外线（UV）、短时高温福尔马林处理进行病毒灭活，可维持JEV E蛋白的抗原结构。因此，诸如过氧化氢这类可维持E蛋白完整性的替代灭活工艺，对于提升灭活JEV疫苗的效价或至关重要。