N-2-Hydroxypropyl Trimethyl Ammonium Chloride Chitosan-Aluminum Nano-Adjuvant Elicit Strong Immune Responses in Porcine Epidemic Diarrhea Inactivated Vaccine

Zheng Jin,1 Jiali Liu,1 Sihan Guo,2 Shangen Xu,1 Xiaochen Gong,1,3 Chunjing Zhang,3 Kai Zhao1,2 1Zhejiang Provincial Key Laboratory of Plant Evolutionary Ecology and Conservation, Taizhou Key Laboratory of Biomedicine and Advanced Dosage Forms, School of Life Sciences, Taizhou University, Taizhou, Zhejiang, 318000, People’s Republic of China; 2Engineering Research Center of Agricultural Microbiology Technology, Ministry of Education, Heilongjiang University, Harbin 150080, China; Key Laboratory of Microbiology, College of Heilongjiang Province, School of Life Sciences, Heilongjiang University, Harbin, Heilongjiang, 150080, People’s Republic of China; 3School of Medical Technology, Qiqihar Medical University, Qiqihar, Heilongjiang, 161006, People’s Republic of ChinaCorrespondence: Kai Zhao; Chunjing Zhang, Email [email protected]; [email protected]: Porcine epidemic diarrhea virus (PEDV) inactivated vaccine lacks an effective vaccine adjuvant as an immune activator. The aim of this study was to develop N-2-HACC-Al nano-adjuvant as a high immune-enhancing adjuvant and to make the vaccine suitable for intramuscular and oral administration.Methods: N-2-HACC-Al nano-adjuvant was prepared by ion crosslinking method using the N-2-hydroxypropyl trimethyl ammonium chloride chitosan (N-2-HACC). The N-2-HACC-Al nano-adjuvant was characterised, and its safety was determined by analysing the cytotoxicity and hemolysis. PED inactivated vaccine (N-2-HACC-Al/PEDV) was prepared by electrostatic adsorption method, and mice were inoculated by intramural injection and orally to evaluate the immune enhancement effect and application potential of the N-2-HACC-Al/PEDV.Results: The hemolysis rate was 3.89 ± 0.12% and the activity of PK15 cells was 77.40 ± 1.74%, indicating that the N-2-HACC-Al/PEDV had good biosafety. The levels of PEDV antibodies induced by the N-2-HACC-Al/PEDV were higher than those of commercially available vaccines, both by intramural injection and oral administration. Except for the serum IgG1 levels in the N-2-HACC-Al/PEDV injection group, which were similar to those in the commercial PEDV group, the serum IgG1, IgG2a, IgG2c and sIgA levels in the injection, and the oral groups were significantly higher than those in the commercial group. These results indicated and that N-2-HACC-Al nano-adjuvant significantly enhanced cellular immunity and N-2-HACC-Al nano-adjuvant could deliver PEDV antigen across the mucosal layer of the intestine and induced a strong mucosal immune response.Conclusion: N-2-HACC-Al nano-adjuvant is safe and can efficiently induce humoral, cellular and mucosal immunity efficiently, which provides a new idea for the development of oral mucosal vaccine adjuvant. Keywords: N-2-Hydroxypropyl trimethyl ammonium chloride chitosan, nano-adjuvant, vaccine, porcine epidemic diarrhea, immune effect