Abstract：Objective: To construct dengue virus envelope protein EDIII tetravalent recombinant vaccines and to analyze its immunogenicity. Methods: Firstly, the gene sequences encoding dengue virus type 1-4 envelope protein EDIII were analyzed by bioinformatics methods, and the representative DENV1-4 EDIII was selected and connected by GS linker to obtain chimeric EDIII; Then, the chimeric gene coding EDIII was inserted into the multiple cloning site of the expression vector pColdIII to constructe the recombinant vector pColdIII-EDIII. The constructed pColdIII-EDIII plasmid was transformed into E.coli BL21 cells and induced by IPTG. The recombinant protein EDIII was purified by Ni-NTA affinity chromatography and confirmed by SDS-PAGE and Western blot analysis. The purified protein was used to immune BABL/c mice. After boosting twice, to detect the antibody titers in serum and the secretion of cytokines by ELISA to evaluate the immune response. Results: The mice immunized with EDIII could induce specific antibodies and the antibody titers was 1:40 000. The mouse spleen lymphocytes stimulated by antigen could produce cytokine including IFN-r, IL-4, IL-10, which were significantly higher in the experimental group. Conclusion: Our studies indicates that dengue virus envelope protein EDIII tetravalent vaccine can induce specific humoral and cellular immune response, and give priority to with the humoral immune response, which laid the foundation for the research of a new type of chimeric tetravalent vaccine in the future.
[1] KHALIL S M, TONKIN D R, MATTOCKS M D, et al. A tetravalent alphavirus-vector based Dengue vaccine provides effective immunity in an early life mouse model[J]. Vaccine, 2014, 32(32): 4068-4074.
[2] DE PAULA S O, LIMA D M, DE OLIVEIRA FRANÇA R F, et al. A DNA vaccine candidate expressing engue-3 virus prM and E proteins elicits neutralizing antibodies and protects mice against lethal challenge[J]. Arch Virol, 2008,153(12): 2215-2223.
[3] APT D, RAVIPRAKASH K, BRINKMAN A, et al. Tetravalent neutralizing antibody response against four dengue serotypes by a single chimeric dengue envelope antigen[J]. Vaccine, 2006, 24(3): 335-344.
[4] WEI J, CHEN H, AN J. Recent progress in dengue vaccine development[J]. Virol Sin, 2014, 29(6): 353-363.
[5] COLLER B A, CLEMENTS D E. Dengue vaccines: progress and challenges[J]. Curr Opin Immunol, 2011, 23(3):391-398.
[6] 陈文静, 黄崇安, 陈静, 等. 潜伏相关抗原UL138蛋白的制备及其用于人巨细胞病毒感染血清学检测的评价[J]. 温州医科大学学报, 2015, 45(10): 703-708.
[7] 刘巧琼, 涂建新, 林晓云, 等. 沙眼衣原体主要外膜蛋白21-387的原核表达及其免疫原性[J]. 温州医科大学学报, 2015, 45(2): 95-100.
[8] 张志珊, 严延生, 翁育伟. 登革病毒重组亚单位疫苗的构建及免疫原性分析[J]. 中华实验和临床病毒学杂志, 2010,24(6): 430-433.
[9] HUGHES H R, CRILL W D, CHANG G J. Manipulation of immunodominant dengue virus E protein epitopes reduces potententtial antibody-dependent enhancement[J]. Virol J,2012, 9: 115.
[10] ZHANG Z S, YAN Y S, WENG Y W, et al. High-level expression of recombinant dengue virus type 2 envelope domain III protein and induction of neutralizing antibodies in BALB/C mice[J]. J Virol Methods, 2007, 143(2): 125-131.
[11] WAHALA W M, KRAUS A A, HAYMORE L B, et al. Dengue virus neutralization by human immune sera: role of envelope protein domain III-reactive antibody[J]. Virology, 2009, 392(1): 103-113.
[12] LAMBRECHTS L. Predicting Wolbachia potential to knock down dengue virus transmission[J]. Ann Transl Med, 2015, 3(19): 288-292.
[13] FAHIMI H, ALLAHYARI H, HASSAN Z M, et al. Dengue virus type-3 envelope protein domain III; expression and immunogenicity[J]. Iran J Basic Med Sci, 2014, 17(11): 836-843.
[14] ZHAO H, JIANG T, ZHOU X Z, et al. Induction of neutralizing antibodies against four serotypes of dengue viruses by MixBiEDIII, a tetravalent dengue vaccine[J]. PLoS One, 2014, 9 (1): e86573.
[15] POGGIANELLA M, SLON CAMPOS J L, CHAN KR, et al. Dengue E protein domain III-based DNA immunisation induces strong antibody responses to all four viral serotypes[J]. PLoS Negl Trop Dis, 2015, 9(7): e0003947.
[16] SONG Y C, LIU H H, CHEN I H, et al. A purified recombinant lipopeptide as adjuvant for cancer immunotherapy[J]. Biomed Res Int, 2014, 2014: 349783.
[17] CHEN Y C, CHUN H H, MEI Y C, et al. Recombinant lipidated dengue-4 envelope protein domain III elicits protective immunity[J]. Vaccine, 2014, 32(12): 1346-1353.
[18] QUINAN B R, FLESCH I E, PINHO T M, et al. An intact signal peptide on dengue virus E protein enhances immunogenicity for CD8(+) T cells and antibody when expressed from modified vaccinia Ankara[J]. Vaccine, 2014, 32(25):2972-2979.
[19] ARORA U, TYAGI P, KHANNA N, et al. Chimeric Hepatitis B core antigen virus-like particles displaying the envelope domain III of dengue virus type 2[J]. J Nanobiotechnology, 2012, 10: 30. DOI: 10.1186/1477-3155-10-30.
[20] REDDY P B, PATTNAIK P, TRIPATHI N K, et al. Expression, purification and evaluation of diagnostic potential and immunogenicity of dengue virus type 3 domain III protein [J]. Protein Pept Lett, 2012, 19(5): 509-519.
[21] GUZMAN M G, HERMIDA L, BERNARDO L, et al. Domain III of the envelope protein as a dengue vaccine target [J]. Expert Rev Vaccines, 2010, 9(2): 137-147.
[22] MATHEW A, TOWNSLEY E, ENNIS F A. Elucidating the role of T cells in protection against and pathogenesis of dengue virus infections[J]. Future Microbiol, 2014, 9(3): 411-425.
[23] TSAI T T, CHUANG Y J, LIN Y S, et al. An emerging role for the anti-inflammatory cytokine interleukin-10 in dengue virus infection[J]. J Biomed Sci, 2013, 20: 40. DOI: 10.1186/1423-0127-20-40.
[24] BABU J P, PATTNAIK P, GUPTA N, et al. Immunogenicity of a recombinant envelope domain III protein of dengue virus type-4 with various adjuvants in mice[J]. Vaccine 2008, 26(36): 4655-4663.
[25] BATRA G, RAUT R, DAHIYA S, et al. Pichia pastoris expressed dengue virus type 2 envelope domain III elicits virusneutralizing antibodies[J]. J Virol Methods, 2010, 167(1):10-16.
