Chitosan Nanovaccines as Efficient Carrier Adjuvant System for IL-12 with Enhanced Protection Against HBV

Huajun Zhao,1 Haigang Wang,1 Yifei Hu,1 Dongqing Xu,1 Chunlai Yin,2 Qiuju Han,1 Jian Zhang1 1Institute of Immunopharmaceutical Sciences, School of Pharmaceutical Sciences, Shandong University, Jinan, People’s Republic of China; 2Department of Immunology, Dalian Medical University, Dalian, People’s Republic of ChinaCorrespondence: Jian ZhangInstitute of Immunopharmaceutical Sciences, School of Pharmaceutical Sciences, Shandong University, Jinan, People’s Republic of ChinaTel +86-531-8838-3781Fax +86-531-8838-3782Email zhangj65@sdu.edu.cnPurpose: Alum adjuvant in HBV prophylactic vaccines is poor in inducing cellular immunity with the inhibition of IL-12 secretion, and approximately 5– 10% of immunised individuals fail to clear HBV upon infection. IL-12 plasmids (pIL-12) as adjuvants enhance significant humoral and cellular immune response in vaccines. However, finding a novel delivery system to protect pIL-12 from enzymatic degradation and achieve efficient delivery remains a major challenge.Methods: We prepared the chitosan nanovaccine-loaded IL-12 expression plasmid (termed as “Ng(-)pIL-12”) and analysed the physicochemical properties, encapsulation efficiency and safety. Then, we evaluated the efficiency of Ng(-)pIL-12 for prophylactic HBV vaccine. Serum samples were collected and analysed for IL-12, HBsAg, anti-HBs IgG, IgG1 and IgG2b. Liver tissues were collected and analysed for HBV DNA and RNA. BMDCs and lymphocytes were collected and analysed for HBV-specific immune responses. To further confirm the long-term protective immune response against HBV, these immunised mice were challenged with hydrodynamic injection of pAAV/HBV 1.2 plasmid on day 56 after the initiation of immunisation.Results: Chitosan nanovaccine prepared with CS and γ-PGA could load pIL-12 effectively and safely, and IL-12 was efficiently produced in vivo. Interestingly, Ng(-)pIL-12 adjuvant combined with HBsAg induced higher levels of anti-HBs IgG, IgG1 and IgG2b, promoted maturation and presentation capacity of DCs, especially CD8α+/CD103+ DCs. Meanwhile, Ng(-)pIL-12 adjuvant generated robust HBV-specific CD8+ T and CD4+ T cell responses. More importantly, Ng(-)pIL-12 adjuvant triggered terminally differentiated effector memory responses with strong anti-HBV effects.Conclusion: Chitosan nanovaccines as an efficient carrier adjuvant system for pIL-12 combined with HBsAg induced protective anti-HBs IgG and enhanced HBV-specific CD8+ T and CD4+ T cell responses, and achieved long-term memory response against HBV, making it a promising candidate for prophylactic HBV vaccines.Keywords: nanovaccines, hepatitis B, prophylactic vaccine, CD8+ T cells, long-term memor