[Linkage of modified human papillomavirus type 16 E7 to CD40 ligand enhances specific CD8+ T-lymphocyte induction and anti-tumour activity of DNA vaccine].
OBJECTIVE
To develop human papillomavirus (HPV) 16 DNA vaccine for the treatment of HPV16 infection and its related tumors.


METHODS
HPV16 oncogene E7 was modified by combined approaches including insertion and replication of specific region of E7 gene, murine codon optimization, and point-mutation at transforming regions of the E7 protein. The resulting artificial gene, named as mE7, was obtained by gene synthesis. The mE7 gene was then genetically fused to murine CD40 ligand (CD40L) by overlapping PCR to form the mE7/CD40L fusion gene. The mE7/CD40L gene was inserted into pVR1012 plasmid and then immunized C57/BL6 mice intramuscularly. The E7-specific IFN-gamma-secreting CD8+ T cells were analyzed with EIISPOT, and E7-specific antibody was measured by indirect ELISA. FACS assays were performed to analyze the activation of E7-specific Th cells. Mice were vaccinated, followed by tumor challenged or challenged before immunization. Tumor growth was observed.


RESULTS
The mE7 DNA vaccine elicited an increased E7-specific antibody level (P < 0.01), E7-specific IFN-gamma-secreting CD8+ T (P < 0.01), and CD4+ T cells number (P < 0.05), compared with those of mice immunized with wE7 gene. Furthermore, the mE7/CD40L DNA vaccine elicited an increased number of E7-specific IFN-gamma secreting CD8+ T cell compared with that of mice immunized with mE7 gene (P < 0.01); however, no significant differences were found between mice immunized with the mE7 gene and mE7/CD40L fusion gene in the E7-specific antibody production and Th cell activation. In the preventive experiment, all mice received the mE7 or mE7/CD40L remained tumor-free 7 weeks after challenges with TC-1 tumor cells, while the wE7 group exhibited tumor growth within 2 weeks. In the therapeutic experiment, all the mice in the wE7 group exhibited tumor growth within 8 days, while among mice receiving the mE7 and mE7/CD40L, 30% and 45% of mice remained tumor-free after TC-1 challenge, respectively. HE staining of tumor tissues showed copious lymphocytes infiltration around tumor cells in mE7 and mE7/CD40L mice with regression of tumor growth.


CONCLUSIONS
The mE7 DNA vaccine increases the E7-specific humoral and cellular immune responses, and the fusion of CD40L to mE7 gene enhances the specific immune responses and anti-tumor effects against HPV16 E7-expressing murine tumors. mE7/CD40L may therefore be a suitable and promising target for HPV16 therapeutic vaccine.
Wang
Qing-yong
QY
Department of Biophysics and Structural Biology, Institute of Basic Medical Sciences, CAMS and PUMC, Beijing 100005, China.
Xu
Yu-fei
YF
Fan
Dong-sheng
DS
Peng
Qing-lin
QL
Zhang
Ting
T
Xu
Xue-mei
XM
D000818:Animals; D019013:CD40 Antigens / Q000235:genetics* / Q000276:immunology; D019496:Cancer Vaccines / Q000235:genetics / Q000276:immunology* / Q000627:therapeutic use; D045744:Cell Line, Tumor; D050939:Gene Fusion; D052162:Human papillomavirus 16 / Q000276:immunology*; D007111:Immunity, Cellular; D056724:Immunity, Humoral; D051379:Mice; D008810:Mice, Inbred C57BL; D009368:Neoplasm Transplantation; D050725:Papillomavirus E7 Proteins / Q000235:genetics* / Q000276:immunology; D053918:Papillomavirus Vaccines / Q000235:genetics / Q000276:immunology* / Q000627:therapeutic use; D019444:Vaccines, DNA / Q000235:genetics / Q000276:immunology* / Q000627:therapeutic use
D019013:CD40 Antigens; D019496:Cancer Vaccines; D050725:Papillomavirus E7 Proteins; D053918:Papillomavirus Vaccines; D019444:Vaccines, DNA