Multi-genotype HPV L2 Fusion Protein Vaccine Protecting Against Cervical Cancer

Objective According to preliminary epidemiological survey of HPV in China,the top five HPV infection in are 16,18,31,52 and 58.So we will take the five types as research objects,to develop an five L2 fusion protein and detect its immunogenicity.It provides laboratory basis for research and development of economic broad-spectrum HPV L2 prophylactic vaccine which suitable for the characteristics of cervical cancer in China.Methods We have completed preliminary study for HPV16;,18,58 L2 fusion protein,so this article first take HPV31and 52 as research objects to construct an bivalent HPV L2 fusion protein.According to the amino acid sequences of HPV31 and 52 L211-200aa published in the GenBank database,we artificially synthesized the HPV31 and 52 L2 fusion gene which was optimized according to Escherichia coli codon usage and encodes 11-200 amino acid of HPV31 and HPV52 L2,then cloned it into pET-9a vector.The HPV31-52 L2 fusion protein was expressed in Prokaryotic expression system and the mice were immunized with the fusion protein after purification.The immunogenicity was detected in immunized mice by serum antibody detection,in vitro and vivo neutralization assay.The mice were immuned by the bivalent HPV31-52 L2 fusion protein and trivalent HPV 18/16/58 L2 fusion protein mixed aluminum adjuvants,immunogenicity was compared with produced by five valence fusion protein.ResultsFirst,we packed and prepared six different types HPV pseudovirus contain HPV1618,31,45,52,58 in vivo and in vitro,the virus titer was in 108TCID50/ml-109/TCID50/ml.Second,we designed and artificially synthesized a prokaryotic expression plasmid including optimized HPV31 and 52 L2 11-200aa according to Escherichia coli codon usage,which could expressed HPV31-52 L2N 11-200aa fusion protein.The recombinant plasmid could expressed corresponding target protein when it was induced by IPTG.Purify the fusion protein after it was identified by western-blot,a purity of more than 90%of the fusion protein was obtained.Third,mice were immunized with the purified HPV31-52 L2N 11-200aa fusion protein combined with aluminum adjuvant,ELISA results showed that the fusion protein could induce high titers of total antibody.Theimmunogenicity of plus adjuvant group was stronger than protein group.They induced total antibody titers against HPV31 and 52 L2 were respectively 1:102400,1:51200 and 1:51200,1:12800.In vitro neutralization assay,the neutralizing titer of antibodies induced by the fusion protein combined with aluminum adjuvant group against pseudovirus of HPV31,52 were 1:1280,were much stronger than without adjuvant group.The cross protection antibody levels of plus adjuvant group was also stronger than protein group.The cross neutralizing titer of antibodies induced by the fusion protein combined with aluminum adjuvant group against pseudovirus of HPV16,18,45,58 were respectively 1:160,1:80,1:1280,1:640,and without aluminum adjuvant group were 1:80,1:40,1:640,1:160.In vivo neutralization assay,when mice were challenged by HPV pseudovirus,mice were all detected by fluorescence signal in the control groupwhile no mice were detected in the experimental group.It indicating that the fusion protein provided significant protection for mice against infecting five HPV pseudovirus.Fourth,mice were immuned by the bivalent HPV31-52 L2 fusion protein and trivalent HPV18/16/58 L2 fusion protein mixed aluminum adjuvants.In vitro neutralization assay,the neutralizing titer of antibodies against pseudovirus of HPV31,52 were 1:1600,the neutralizing titer of antibodies against pseudovirus of HPV 16,18,5 8 were 1:3200,the cross neutralizing titer of antibodies against pseudovirus of HPV45 was 1:3200.Compared with neutralizing antibodies and cross neutralization antibody induced by single bivalent HPV31-52 L2 fusion proteins were significantly enhanced.Compared with single trivalent HPV18/16/58 L2 fusion protein,cross neutralization antibody induced were significantly enhanced,while neutralization antibody levels against pseudovirus of the same type HPV 16,18 were no significant enhanced,against pseudovirus of HPV58 was reduced.In vitro neutralization assay,when mice were challenged by five types HPV pseudovirus,mice were all detected by fluorescence signal in the control group,while no mice were detected in the experimental group.It indicated that the fusion protein provided protection for mice against HPV pseudovirus.ConclusionFirst,HPV31-52 L2 N 11-200aa fusion protein was highly expressed in E.coli,the amount of fusion protein is nearly 20%of the total bacterial protein.The purified fusion protein with aluminum adjuvant could induce specific high titer of IgG antibodies,also induce the neutralizing antibodies and cross-neutralizing antibodies.Animal infection model also illustrated the fusion protein had certain protective effect on preventing HPV infection.Second,mice were immuned by the bivalent HPV31-52 L2 fusion protein and trivalent HPV18/16/58 L2 fusion protein mixed aluminum adjuvants.Compared with neutralizing antibodies and cross neutralization antibody induced by single bivalent HPV31-52 L2 fusion proteins were all significantly enhanced,compared with cross neutralization antibody induced by single trivalent HPV 18/16/5 8 L2 fusion protein was enhanced while neutralization antibody were not.The above research provided laboratory basis for research and development of HPV L2 protein vaccine.