[Immunogenicity of mutant and wild HPV16 DNA vaccines].
OBJECTIVE
To evaluate the efficacy of mutation of human papillomavirus 16 (HPV16) E7 in two zinc-binding motifs on HPV16 E7 C terminus on antigen-specific immunity.


METHODS
pcDNA3.1/16E7 and pcDNA3.1/ME7 were successfully constructed by inserting the E7 (ME7) into pcDNA3.1 BamH I, EcoR I cut sites. After intramuscular injection with pcDNA3.1, pcDNA3.1/16E7, and pcDNA3.1/16ME7 on C57BL/6 mice, splenocytes from vaccinated mice was isolated. After have been stimulated with E7-specific peptide, interferon gamma (IFN-gamma), interleukin 2 (IL-2), and cytotoxic T lymphocyte (CTL) were detected by ELISA, Eli-spot, and LDH assay respectively; splenocytes without E7 peptide stimulation were used as control group.


RESULTS
Splenocytes from mice vaccinated with pcDNA/ME7, stimulated with E7 peptide, generated significantly larger number of E7-specific IL-2 compared with pcDNA3.1/16E7, pcDNA3.1, and control group. The E7-specific IL-2 generated in pcDNA-ME7 group was 5-fold of that of pcDNA3.1/16E7, and the difference was statistically significant (P < 0.05). Splenocytes from mice vaccinated with pcDNA/ME7 and stimulated with E7 peptide, generated significantly larger number of E7-specific IFN-gamma compared with other vaccines. pcDNA-ME7 generated a 2-fold increase in the number of E7-specific IFN-gamma compared with wild-type E7 and the difference was statistically significant (P < 0.05). The highest CTL activity in mice vaccinated with pcDNA/ME7 at an E:T ratio of 45:1 was achieved compared with mice vaccinated with other vaccines. The percents of specific lysis generated by pcDNA3.1/ME7, pcDNA3.1/E7, pcDNA3.1, and without vaccination were of (28.7+/-1.2) %, (55+/-2.2) %, (12.5+/-2.0) %, and (11.5+/-1.2) % respectively, and significant difference existed between the former and the latter two groups (P < 0.05). However, no significant difference was found among all the groups without specific E7 peptide stimulation (P > 0.05).


CONCLUSIONS
The mutation of zinc-binding motifs on HPV16 E7 C terminus may greatly enhance the immunogenicity.
Zuo
Ya-gang
YG
Department of Dermatology, PUMC Hospital, CAMS and PUMC, Beijing 100730, China.
Wang
Jia-bi
JB
Jin
Hong-zhong
HZ
Yue-hua
Liu
L
D000818:Animals; D004797:Enzyme-Linked Immunosorbent Assay; D007371:Interferon-gamma / Q000097:blood; D007376:Interleukin-2 / Q000097:blood; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009154:Mutation; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000276:immunology*; D027383:Papillomaviridae / Q000235:genetics / Q000276:immunology*; D050725:Papillomavirus E7 Proteins; D011993:Recombinant Fusion Proteins / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D014611:Vaccination; D019444:Vaccines, DNA / Q000276:immunology*; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics; D014765:Viral Vaccines / Q000276:immunology*
D007376:Interleukin-2; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D011993:Recombinant Fusion Proteins; D019444:Vaccines, DNA; D014764:Viral Proteins; D014765:Viral Vaccines; C059731:oncogene protein E7, Human papillomavirus type 16; D007371:Interferon-gamma