Preparation of human papillomavirus 16 E7 peptide vaccine and its effectiveness in vitro and in vivo
LIAO Shu-jie, HU Xiao-ji, HAN Ling-fei, JIANG Xue-feng, XIA Xi, WANG Wei, LU Yun-ping, WANG Shi-xuan, MA Ding
Published 2009-12-25
Cite as Chin J Obstet Gynecol, 2009,44(12): 903-908. DOI: 10.3760/cma.j.issn.0529-567x.2009.12.006
Abstract
Objective To prepare the human papillomavirus (HPV) 16 peptide vaccine and explore the effect in vitro and in vivo. Methods (1) Prediction of the major histocompatibility complex (MHC) class I restricted T cell epitopes by bioinformatics target at transporter associated with antigen processing (TAP) and named by E7Pa, E7Pb, E7Pc separately. (2)In vivo, the C57BL/6 mice were divided into five groups with same amounts randomly after loading with TC-1 cells (HPV 16 positive tumor cells from C57BL/6 mouse), named as E7Pa + CpG,E7Pb + CpG,E7Pc + CpG (as experiment groups, and added 50 μg/ml E7Pa, E7Pb, E7Pc, respectively), CpG(as positive control group and added Con A with 12 mg/L final concentration) and blank control group (without any treatment). The T cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) assay at different time points;the lactate dehydrogenase (LDH) delivery method was used to test the cytolytie T lymphocyte (CTL) activity of mouse splenic lymphocyte in different ratio of effector cells and target cells (E:T);the related cytokines in tumor tissue and mouse peripheral blood were evaluated by real-time PCR and enzyme-linked immunosorbent assay (ELISA), respectively. The tumor volumes were measured to contrast the therapeutic effect in different groups. Results (1) Three peptide named E7Pa, E7Pb, E7Pc were successfully preparated which had high affinity and specificity. (2) After vaccination of 24, 48, 72,96 hours, MTT results shown that the proliferation rate in E7Pa + CpG group were(131±32)%, (302±15)%, (552±28)%, (731±24)% individually, which were much higher than those in blank control [(72± 15) %, (120 ± 57) %, (176 ±41)%, (288±29)% ;P<0.01], and the other groups i. e. E7Pb + CpG,E7Pc +CpG and CpG groups all proliferated much higher than those in blank control group with statistic signification (P<0. 05), but there was no significant difference between groups(P>0.05);the LDH delivery assay showed that when the ratio of E:T was 100:1, the activity of CTL in the E7Pa + CpG group was most powerful than the other groups with statistic signification (P<0. 01). Meanwhile, the ratio of E:T was concentration-dependent. Compared E7Pb + CpG, E7Pc + CpG or CpG groups with blank control group, there were significantly difference(P<0. 05) ,while there was no significant difference between groups(P >0. 05). The mRNA levels of interferon γ (IFN-γ), interleukin-2 (IL-2) in tumor tissue and peripheral blood in E7Pa + CpG group were significantly higher than those in blank control group (P<0. 01), which was the similar results when compared E7Pb + CpG, E7Pc + CpG or CpG groups with control group (P < 0. 05), and without significant difference between groups(P > 0. 05). The tumor volumes were suppressed obviously in all the experiment groups, especially at the 60th days, the volumes in ETPa + CpG group were much smaller than that in blank control group with statistic signification (P < 0. 01),which was the similar results that E7Pb + CpG, E7Pc + CpG or CpG groups had difference than blank control group with statistic signification (P < 0. 05), and without significant difference between groups(P >0. 05). Conclusion The HPV16 E7 peptide target at TAP combination with CpG as a vaccine could treat effectively the HPV16 E7 positive tumor in experiment.
Key words:
Papillomavirus; human; Papillomavirus vaccines; Vaccines; subunit; Oncogene proteins; viral; Cytosine nuctcotides; Cell line; tumor
Contributor Information
LIAO Shu-jie
Department of Gynecologic and Obstetric, Tongji Hospital, Tongji Medical College,Huazhong University of Science and Technology, Wuhan 430030, China
HU Xiao-ji
Department of Gynecologic and Obstetric, Tongji Hospital, Tongji Medical College,Huazhong University of Science and Technology, Wuhan 430030, China
HAN Ling-fei
Department of Gynecologic and Obstetric, Tongji Hospital, Tongji Medical College,Huazhong University of Science and Technology, Wuhan 430030, China
JIANG Xue-feng
Department of Gynecologic and Obstetric, Tongji Hospital, Tongji Medical College,Huazhong University of Science and Technology, Wuhan 430030, China
XIA Xi
Department of Gynecologic and Obstetric, Tongji Hospital, Tongji Medical College,Huazhong University of Science and Technology, Wuhan 430030, China
WANG Wei
Department of Gynecologic and Obstetric, Tongji Hospital, Tongji Medical College,Huazhong University of Science and Technology, Wuhan 430030, China
LU Yun-ping
Department of Gynecologic and Obstetric, Tongji Hospital, Tongji Medical College,Huazhong University of Science and Technology, Wuhan 430030, China
WANG Shi-xuan
Department of Gynecologic and Obstetric, Tongji Hospital, Tongji Medical College,Huazhong University of Science and Technology, Wuhan 430030, China
MA Ding
Department of Gynecologic and Obstetric, Tongji Hospital, Tongji Medical College,Huazhong University of Science and Technology, Wuhan 430030, China