Supplementary Material
Comparison of PLA Microparticles and Alum as Adjuvants for H5N1 Influenza Split Vaccine: Adjuvanticity Evaluation and Preliminary Action Mode Analysis
Weifeng Zhang,†,‡,§ Lianyan Wang,†,§ Yuan Liu,†,‡ Xiaoming Chen,†,‡ Jiahui Li,† Tingyuan Yang,† Wenqi An,# Xiaowei Ma,# Ruowen Pan,# Guanghui Ma*,†
†National Key Laboratory of Biochemical Engineering, PLA Key Laboratory of Biopharmaceutical Production & Formulation Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China
‡University of Chinese Academy of Sciences, Beijing 100049, PR China
#Hualan Biological Engineering, Inc., Xinxiang 453003, Henan Province, PR China
Corresponding Author
*Chinese Academy of Sciences, Institute of Process Engineering, National Key Laboratory of Biochemical Engineering, Bei-Er-Jie No.1, Zhong-Guan-Cun, Haidian District, Beijing, 100190, PR China. Tel/Fax: +86 10 82627072; E-mail: .
Author Contributions
§These authors contributed equally.
Supplementary Methods
Determination of antigen trafficking into draining lymph nodes
Balb/c mice (n=3) were intramuscularly vaccinated with different vaccine formulations containing FITC-Ag conjugate. At pre-determined time points post injection, mice were euthanized, and single cell suspension was prepared from draining lymph nodes (popliteal lymph nodes). The cells were stained with anti-CD11c antibody (eBioscience, USA) to specifically identify DCs. The percentage of FITC-Ag+ cells was determined by flow cytometry (CyanTM ADP flow cytometer, Beckman Coulter, USA) and analyzed using Summit software (version 4.3.02).
Supplementary Figures
Fig. S1 The effect of PLA microparticles and alum on lymphocytes activation and effector memory T cell response. Balb/c mice (n=6) were intramuscularly vaccinated twice at a 2-week interval. On day 28 post primary immunization, mice were euthanized, and splenocytes were harvested and stimulated with HA (HA, 2.5 μg/ml; splenocytes, 5.0×106 cells/ml) in a 37℃ humidified incubator with 5% CO2 for 72 h. Flow cytometry assay was performed to determine the percentages of activated lymphocytes (CD69＋) and effector memory (CD44hiCD62Llow) T cells. (A) CD4+ T cell activation; (B) CD8+ T cell activation; (C) B cell activation; (D) effector memory CD4+ T cells; (E) effector memory CD8+ T cells. Data are shown as mean ± SEM (n=6). *p＜0.05, **p＜0.01, and ***p＜0.001.
Fig. S2 The effect of PLA microparticles and alum on antigen trafficking into draining lymph nodes. Balb/c mice (n=3) were intramuscularly vaccinated with different vaccine formulations containing FITC-Ag conjugate. At pre-determined time points, mice were euthanized, and single cell suspension was prepared from draining lymph nodes (popliteal lymph nodes). The cells were stained with anti-CD11c antibody (eBioscience, USA) to specifically identify DCs. The percentage of FITC-Ag+ cells was determined by flow cytometry (CyanTM ADP flow cytometer, Beckman Coulter, USA). (A) Representative flow cytometry plot of percentage of FITC-Ag+ DCs in draining lymph node (24 h); (B) Percentage of FITC-Ag+ DCs at different time points post vaccine injection. Data are shown as mean ± SEM (n=3).