Encapsidating artificial human papillomavirus-16 mE7 protein in human papillomavirus-6b L1/L2 virus like particles..
BACKGROUND
Human papillomaviruses (HPVs) can infect squamous or mucosal epithelia and cause cervical cancer or genital warts. Coinfection with multiple HPV types is a common finding of many epidemiological studies. Therefore, it is necessary to develop a vaccine, which can eradicate established HPV infections and prevent other HPV infections. In this study, we generated chimeric virus like particles (cVLPs) composed of HPV-6b L1, HPV-6b L2 and one artificial HPV-16 mE7 proteins.


METHODS
The artificial HPV-16 mE7 gene was designed by codon modification, point mutation and gene shuffling then chemically synthesized and subcloned behind HPV-6b L2. HPV-6b L1 and L2-mE7 were expressed in insect cells by using Bac-to-Bac system. The generated cVLPs were purified by CsCl gradient ultracentrifuge and analyzed by immunoblot, electron microscope and haemagglutination assay.


RESULTS
The HPV-6b L1 and L2-mE7 proteins were well expressed in insect cells and could selfassemble into cVLPs, whose diameter was about 55 nm and similar to that of HPV-6b L1/L2 VLPs. Intact cVLPs could be recognized by H6.M48 neutralizing monoclonal antibody and HPV-6b L2 polyclonal antibody, while the denatured cVLPs, but not the intact cVLPs, were reactive to HPV-16 E7 polyclonal antibody. HPV-6b L1/L2-mE7 cVLPs haemagglutinated mouse erythrocytes as efficiently as HPV-6b L1/L2 VLPs did.


CONCLUSIONS
The insertion of the 158 amino acid HPV-16 mE7 protein behind L2 did not disrupt the correct assembling of cVLPs. The morphological characteristics and haemagglutinating activity of cVLPs were similar to those of HPV-6b L1/L2 VLPs. The cVLPs retained conformational B cell epitopes of HPV-6 VLPs and HPV-16 mE7 protein had an internal location in the cVLPs. Therefore, large modified E7 protein with higher immunogenicity could be incorporated into cVLPs by fusing to the C-terminus of L2, which would help to improve the therapeutic effects of L1/L2-E7 cVLPs.
Xu
Yu-fei
YF
Department of Biophysics and Structural Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China.
Wang
Qing-yong
QY
Zhang
Hong-tao
HT
Han
Ye-hua
YH
Song
Guo-xing
GX
Xu
Xue-mei
XM
D000818:Animals; D001483:Base Sequence; D036022:Capsid Proteins / Q000276:immunology*; D006386:Hemagglutination Tests; D051379:Mice; D008810:Mice, Inbred C57BL; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000276:immunology*; D050725:Papillomavirus E7 Proteins; D053918:Papillomavirus Vaccines / Q000276:immunology*; D014764:Viral Proteins / Q000276:immunology*; D014771:Virion / Q000276:immunology* / Q000648:ultrastructure
D036022:Capsid Proteins; C082746:L2 oncogene viral capsid protein, Human papillomavirus type 6; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D053918:Papillomavirus Vaccines; D014764:Viral Proteins; C059731:oncogene protein E7, Human papillomavirus type 16; C091560:L1 protein, Human papillomavirus type 6