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

Zhang, Zhixiao; Zhang, Jingjing; Mu, Changyong; Ma, Kaili; Gao, Dongxiu; Liu, Chang’e; Feng, Lin; Peng, Xiaowu; Si, Junbo; Li, Hongbing; Su, Yanrui; Zeng, Fengyuan; He, Liping; Wang, An; Zhou, Chongying; Zhang, Zhenxiao; Wang, Yixuan; Li, Qiuqi; Li, Jiahui; Zou, Shuiyan; Xing, Miaomiao; Li, Huijuan; Sun, Meng; Chang, Weijie; Yu, Xiaoxia; Li, Junqing; Wang, Lichun; Li, Yanmei; Yi, Hongkun; Zheng, Lichun; He, Fuyun; Li, Qihan

doi:10.3390/vaccines13121196

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Open AccessArticle

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

by

Zhixiao Zhang

^1,2

,

Jingjing Zhang

^1,2,

Changyong Mu

^1,2,

Kaili Ma

^1,2

,

Dongxiu Gao

¹,

Chang’e Liu

¹,

Lin Feng

¹,

Xiaowu Peng

¹,

Junbo Si

¹,

Hongbing Li

¹,

Yanrui Su

¹,

Fengyuan Zeng

¹,

Liping He

¹,

An Wang

¹,

Chongying Zhou

¹,

Zhenxiao Zhang

¹,

Yixuan Wang

¹,

Qiuqi Li

¹,

Jiahui Li

¹,

Shuiyan Zou

²,

Miaomiao Xing

²,

Huijuan Li

²,

Meng Sun

²,

Weijie Chang

²,

Xiaoxia Yu

²,

Junqing Li

²,

Lichun Wang

¹,

Yanmei Li

¹,

Hongkun Yi

^1,2,

Lichun Zheng

¹,

Fuyun He

¹ and

Qihan Li

^1,* Show full author list Hide full author list

¹

Weirui Biotechnology (Kunming) Co., Ltd., Ciba Biotechnology Innovation Center, Kunming 650000, China

²

Shandong Weigao Litong Biological Products Co., Ltd., Weihai 264200, China

^*

Author to whom correspondence should be addressed.

Vaccines 2025, 13(12), 1196; https://doi.org/10.3390/vaccines13121196

Submission received: 11 October 2025 / Revised: 21 November 2025 / Accepted: 25 November 2025 / Published: 26 November 2025

(This article belongs to the Special Issue Feature Papers of DNA and mRNA Vaccines)

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Abstract

Background: Rabies virus (RABV) causes approximately 59,000 human deaths annually. Current pre and postexposure vaccination relies on inactivated vaccines (INVs) with limited yield and immunogenicity. We engineered a dualcationic LNPbased nucleocapsidlike nanostructure (NLS) that coencapsulates RABV G-mRNA and recombinant RABV-N to engage MHC-I/II pathways and enhance protection. Methods: A pVAXRABVG plasmid containing 5′/3′UTRs, Kozak, and poly(A) was transcribed in vitro. RABVN with an Nterminal 6× His tag was expressed in E. coli BL21(DE3) and purified by NiSepharose affinity chromatography. Dualcationic LNPs (DHA, DOTAP Cl, mPEGDTA2K, DOPC) were formulated by microfluidics at a 4:1 (GmRNA:RABVN) mass ratio. Vaccine quality was assessed by encapsulation efficiency, DLS, PDI, zeta potential, and TEM. Mice received empty LNPs, INV, GmRNA, or NLS under varied schedules and doses. ELISA measured RABV-G/NIgG; RFFIT determined neutralizing antibody (nAb) titers; ELISPOT quantified CTL response; qPCR assessed Tcell activation genes. On day 35 after the first immunization of vaccines, mice were challenged intramuscularly with 25 LD₅₀ of CVS24. Results: GmRNA purity was >95% and drove strong RABVG expression in 293T cells. Purified RABVN was approximately 52 kDa, >90% pure, and reactive to antiHis and antiN 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 approximately 10 IU mL⁻¹ nAb by day 7; two doses peaked at approximately 1000 IU mL⁻¹. Mice showed 100% survival and no viral rebound in brain, spinal cord, and sciatic nerve. NLS induced stronger MHC-I/IIlinked cellular immunity and higher RABV G/Nspecific IFNγ spot frequencies than GmRNA or INV. Conclusions: The dualantigen NLS vaccine codelivering GmRNA and RABVN via dualcationic LNPs robustly activates MHC-I/II, rapidly generates hightiter nAb (≥10 IU mL⁻¹ within 1 week), and sustains potent CD8⁺ CTL and CD4⁺ Th responses. A twodose regimen (days 0 and 21) conferred complete protection, supporting the NLS platform as a nextgeneration rabies vaccine candidate.

Keywords: rabies virus; lipid nanoparticles; nucleocapsid; mRNA vaccine

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MDPI and ACS Style

Zhang, Z.; Zhang, J.; Mu, C.; Ma, K.; Gao, D.; Liu, C.; Feng, L.; Peng, X.; Si, J.; Li, H.; et al. A Rabies Virus Nucleocapsid-like Nanostructure Vaccine Based on Dual-Cationic Lipid Nanoparticles. Vaccines 2025, 13, 1196. https://doi.org/10.3390/vaccines13121196

AMA Style

Zhang Z, Zhang J, Mu C, Ma K, Gao D, Liu C, Feng L, Peng X, Si J, Li H, et al. A Rabies Virus Nucleocapsid-like Nanostructure Vaccine Based on Dual-Cationic Lipid Nanoparticles. Vaccines. 2025; 13(12):1196. https://doi.org/10.3390/vaccines13121196

Chicago/Turabian Style

Zhang, Zhixiao, Jingjing Zhang, Changyong Mu, Kaili Ma, Dongxiu Gao, Chang’e Liu, Lin Feng, Xiaowu Peng, Junbo Si, Hongbing Li, and et al. 2025. "A Rabies Virus Nucleocapsid-like Nanostructure Vaccine Based on Dual-Cationic Lipid Nanoparticles" Vaccines 13, no. 12: 1196. https://doi.org/10.3390/vaccines13121196

APA Style

Zhang, Z., Zhang, J., Mu, C., Ma, K., Gao, D., Liu, C., Feng, L., Peng, X., Si, J., Li, H., Su, Y., Zeng, F., He, L., Wang, A., Zhou, C., Zhang, Z., Wang, Y., Li, Q., Li, J., ... Li, Q. (2025). A Rabies Virus Nucleocapsid-like Nanostructure Vaccine Based on Dual-Cationic Lipid Nanoparticles. Vaccines, 13(12), 1196. https://doi.org/10.3390/vaccines13121196

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

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