Multi-genotype L2Fusion Protein As A Candidate Prophylactic Human Papillomavirus Vaccine

Human papillomavirus (HPV) infection can cause verrucous or papillary lesions on different parts of the body, which is harmful to human health and becomes one of the important pathogens. According to their different carcinogenic, HPV can be divided into low-risk and high-risk kinds. High-risk HPV have15types,including HPV16、18、31、33、39、45、51、52、56、58、59、68、73、82and so on. High-risk HPV infection is related to the occurrence of malignant tumors, for example, esophageal, larynx and tongue cancer, especialy highly associated with the development of cervical cancer, which is the second most frequent malignant female cancer worldwide. Existing cervical mucosa smear screening can be achieved in the early detection of cervical cancer, but false-negative rate of screening and the high cost make it difficult to be popular in the world. In the surgical treatment of advanced cervical cancer has not yielded satisfactory results for high recurrence rate and high cost. Therefore the development of a prophylactic HPV vaccine is an effective way to prevent HPV infection and control the incidence of cervical cancer.HPV vaccine can be divided into prophylactic and therapeutic vaccines. The current success of this study are prophylactic vaccines of the major HPV capsid protein L1, those are, Gardasil, a L1tetravalent vaccine (yeast expression) against HPV6/11/16/18of the Merk Company and Cervarix, GSK’s bivalent vaccine against HPV16/18(insect cell expression), the antibody positive turning rate could be gotten by100%and the protection rate could reach to100%in4.5years, while because of high cost and lacking of cross-protective effects, their using in the world are limited. an ideal HPV vaccine should be inexpensive and has good cross-protection activity, covering as much as possible high-risk types of HPV, and what above has become the aims of the second-generation HPV vaccine research. L2is the minor HPV capsid which is not necessary for capsid formation, but its N-terminal can induce antibody with cross-neutralization activity. The main purpose of this study is to target the L2protein for antigens, and explore the development of a low-cost, broad spectrum HPV prophylactic vaccine.The courses of our work are as follows:at first, according to our preliminary epidemiological survey of HPV in China, we choose HPV16, HPV18and HPV58as target types, and L2N-terminal region could induce cross-neutralising antibody of each type as the target antigens, different length fragment of these regions are expressed in prokaryotic system, then select the best fragment of each type and fuse them, and observe their immunogenicity in the end, hoping for providing experimental basis for the development of a low-cost, broad spectrum of HPV prophylactic vaccines.The main findings are below:Firstly, we constructed11kinds of prokaryotic expression plasmids to express different length fragments of L2protein of HPV16,18and58. The target proteins could be expressed in each plasmid after induced by IPTG and identified by western-blot, after purified,11kinds of proteins with90%purity were gotten. ELISA results showed that different length fragments of L2protein of these three HPV types could induce high titers of antibodies, all were up to1:100,000; we packaged pseudovirus of HPV16,18and58types and established a technology platform and method for HPV neutralizing expriment, according to the experimental results the best L2fragments of each HPV type were selected:HPV16L2(11-150aa), HPV18L2(11-200aa) and HPV58L2(11-150aa), the titers against pseudovirus of the same type were1:640、1:1280and1:1280respectively.Secondly, the best L2fragments of each HPV type selected were fused in two ways:18-16-58and58-16-18, then two pET-9a prokaryotic expression plasmids of HPV16,18and58L2fusion protein had been constructed; each plasmid could express corresponding protein after induced by IPTG, these proteins were identified by western-blot and then purificated, the purity were about90%; Neutralizing experiment in vitro suggested that, the neutralizing titer of antibodies induced by18-16-58trivalent L2fusion protein against pseudovirus of HPV16,18and58were1:3200,1:3200and1:6400respectively, which were better than18-16-58trivalent L2 fusion protein,(that confered that different protein combination may affect exposoure of epitopes), and also could induce cross-neutralizing antibodies against pseudovirus of HPV6,45and52,the titers were1:640,1:1280and1:640respectively. Compared with L2proteins of each HPV type,18-16-58L2fusion protein had better immunogenicity and higher titer of neutralizing antibodies against heterotypic pseudovirus, which mean may have broad spectrum.In conclusion, in this study, three L2fragments inducing antibodies with higher neutralizing activities of HPV16,18and58were selected and then fused, we made preliminary study on their neutralizing activities and meaningful exploration of HPV broad spectrum vaccines, which provided valuable experimental data for in-depth study of this vaccine.