A Rabies Virus Nucleocapsid-like Nanostructure Vaccine Based on Dual-Cationic Lipid Nanoparticles

作者：Zhixiao Zhang, Jingjing Zhang, Changyong Mu, Kaili Ma, Dongxiu Gao, Change Liu, Feng Lin, Xiaowu Peng, Junbo Si, Hongbing Li, Yanrui Su, Fengyuan Zeng, Liping He, An Wang, Chongying Zhou, Zhenxiao Zhang, Y. Wang, Qisheng Li, Qisheng Li, Jiahui Li, Shuiyan Zou, Miaomiao Xing, Huijuan Li, Meng Sun, Weijie Chang, Xiaoxia Yu, Xingguang Liu, Lichun Wang, Yanmei Li, Hongkun Yi, Lichun Zheng, Fuyun He, Qihan Li, Qihan Li · 发表于：Vaccines · 年份：2025 · DOI：10.3390/vaccines13121196 · 研究领域：Rabies epidemiology and control、Virology and Viral Diseases、Bacteriophages and microbial interactions

Background: Rabies virus (RABV) causes approximately 59,000 human deaths annually. Current pre- and post-exposure vaccination relies on inactivated vaccines (INVs) with limited yield and immunogenicity. We engineered a dual-cationic LNP-based nucleocapsid-like nanostructure (NLS) that co-encapsulates RABV G-mRNA and recombinant RABV-N to engage MHC-I/II pathways and enhance protection. Methods: A pVAX-RABV-G plasmid containing 5′/3′UTRs, Kozak, and poly(A) was transcribed in vitro. RABV-N with an N-terminal 6× His tag was expressed in E. coli BL21(DE3) and purified by Ni-Sepharose affinity chromatography. Dual-cationic LNPs (DHA, DOTAP Cl, mPEG-DTA2K, DOPC) were formulated by microfluidics at a 4:1 (G-mRNA:RABV-N) mass ratio. Vaccine quality was assessed by encapsulation efficiency, DLS, PDI, zeta potential, and TEM. Mice received empty LNPs, INV, G-mRNA, or NLS under varied schedules and doses. ELISA measured RABV-G/N-IgG; RFFIT determined neutralizing antibody (nAb) titers; ELISPOT quantified CTL response; qPCR assessed T-cell activation genes. On day 35 after the first immunization of vaccines, mice were challenged intramuscularly with 25 LD50 of CVS-24. Results: G-mRNA purity was >95% and drove strong RABV-G expression in 293T cells. Purified RABV-N was approximately 52 kDa, >90% pure, and reactive to anti-His and anti-N antibodies. NLS achieved >95% encapsulation, a diameter of 136.9 nm, PDI 0.09, and a +18.7 mV zeta potential. A single dose yielded approximatel...