[Enhancement of human papillomavirus type 16E6E7 vaccine-induced specific immune response by coimmunization with B7-1 co-stimulatory gene].

OBJECTIVE: To develop a therapeutic vaccine against human tumors associated with human papillomavirus type 16E6E7 (HPV16E6E7) which is modified from a Chinese patient of the cervical cancer which possessing the antigenicity and no transforming activity, and explore more active vaccine for inducing cellular immunity with mouse co-stimulatory molecular B7-1 gene. METHODS: The modified E6E7 gene expression plasmid pVR1012-fmE6E7 was constructed and transfected Cos-7 cells, and the E7 protein specific expression was testified by immunofluorescence assay. C57BL/6 mice were immunized intramuscularly with pVR1012-fmE6E7 alone or in combination with B7-1 gene expression plasmid (pcDNA3.1-B7-1). The activity of cytotoxic T lymphocytes (CTLs) was analyzed with 51Cr specific release assay and the specific antibody in sera was analyzed by indirect ELISA. HPV16 positive C57BL/6 tumor cells C3 were inoculated subcutaneously in the vaccinated mice to assay the growth of transplanted tumors. RESULTS: The specific CTLs and antibody from immunized mice were induced efficaciously by the E6E7 gene immunization, and co-administration of B7-1 gene could significantly enhanced the CTLs immune responses of fmE6E7, and protected 33% immunized mice against C3 tumor cells challenge. In contrast, all the mice immunized only with fmE6E7 gene developed transplanted tumors after C3 cells challenge. There was no difference in E7 specific antibody responses between mice immunized with the E6E7 gene only and co-administration with B7-1 gene. CONCLUSIONS: The modified E6E7 gene can be used as target gene for developing DNA vaccine, and B7-1 gene may represent an attractive adjuvant for enhancement of the specific cellular immune responses.

Expression of human papillomavirus type 6 L1 and L2 isolated in China and self assembly of virus-like particles by the products.

To study variations of genome late region of human papillomavirus type 6 (HPV-6) isolated in China and assembling capabilities of the encoded capsid proteins, HPV-6 L1 and L2 sequences were cloned and used for expression in Bac-to-Bac baculovirus expression systems (Gibco BRL). Based upon L1 and L2 overlapping sequence two sequences (GenBank accession number AY015006, AY015008) of HPV-6 late region (2869 bp long) were assembled and classified into HPV-6b by phylogenetic analysis. Compared with prototype sequence, nine point mutations were found, including four missense mutations. L1, instead of L2, could self-assemble into virus-like particles (VLPs) in Sf9 nucleus. VLPs self-assembled by L1 alone (L1-VLPs) and by L1 plus L2 (L1+L2-VLPs) were purified and further characterized. Both types of VLPs were spherical particles with a diameter of approximately 50 nm. L1+L2 VLPs comprising L1 and L2 in the molar ratio of about 4:1 possessed the HPV-6 L1 VLP surface and conformational epitopes. In co-expression assay with a series of MOI combination of L1 and L2 recombinant baculoviruses (total MOI=10), existence of L2 of certain level enhanced L1 production by 0.8 fold and VLP production by 3 - 4 folds under experimental conditions. In conclusion, variation rate of HPV-6 genome late region is less than 0.28% and the substitutions A to G at position 7081 and G to A at 7099 may represent region characteristics. The cloned HPV-6 L1 and L2 sequences can be expressed efficiently in Sf9 cells, and the expressed products (L1 or L1+L2) can self-assemble into VLPs that resemble naturally occurring virions.

[Identification and assessment of multiple human papillomavirus types in condyloma acuminata lesions from patients with genital warts in Beijing area].

OBJECTIVE: To identify and assess multiple human papillomavirus types in condyloma acuminatum lesions from patients with genital warts in Beijing area, and compare different features between otherwise healthy and immunosuppressed patients. METHODS: PCR, RFLP and nucleotide sequencing analysis were used to determine HPV types from individual lesions. RESULTS: The predominant type from other healthy patients was HPV6, secondly HPV11. The mean age of patients infected by HPV6 was lower than that of HPV11 and HPV6 + 11. While lesions from immunosuppressed patients were often contained HPV11 or mixed with HPV6. Besides, HPV types 16 and 53 were detected from infected lesions than other HPV types. CONCLUSIONS: HPV6 was the major pathogen of condyloma acuminatum, but infected patients were at lower ages. While HPV11 was most often detected from immunosuppressed patients. As a low risk virus in normal genital tract, HPV53 also could be a pathogen in genital warts.