HPV6L2E7E6Fusion Protein As A Candidate Therapeutic HPV Vaccine

Human papillomaviruses (HPVs), a group of very important infectious agents, play a key role in induction of proliferative or even malignant change of human skin and mucosa in different parts. According to their pathogenicity, HPVs can be divided into low-risk and high-risk groups, the former is associated with skin and genital wart, the latter has close relationship with cervical cancer. Genital wart (GW) is one of the most popular sexually transmitted diseases in the world, although not normally fatal, GW brings patients psychological stress and high cost of treatments. The close relationship between GW and HPVs infection has already been clear, in which HPV-6,11are dominantly involved. Currently, common treatments for GW are physical therapy (such as freezing, laser, etc.) and drug therapy, although effective and can clear the warts within a short time, can not remove the tumor cells or virus-infected cells completely, so it is easy to relapse. A more effective method of prevention and treatment of GW is need badly.HPV vaccines could be divided into two groups:prophylactic and therapeutic vaccines. Prophylactic vaccines have now made significant progress, two vaccines produced by Merck and GSK respectively has come into market, the main strategy of which is focused on inducing high neutralizing antibody titers of relative conformation-depended B cell epitopes in HPV capsid protein L1to prevent infections. Therapeutic vaccines have acquired relatively slow advancements, their mechanism is that virus antigens induce T cell immune response in vivo to eliminate tumor cells and virus-infected cells. As the two early proteins E6and E7of HPV are expressed in the whole viral life cycle, they have become the majority choice of target antigens in therapeutic vaccines research. Many high-risk HPV16/18therapeutic vaccines have carried through II clinical research and some effective results have been proved, when it comes to low-risk HPV, there is only one HPV6L2E7fusion protein therapeutic vaccine developed by GSK, which has obtained a certain GW treatment effect on HPV6-only-infected patients in a phase Ⅱ clinical experiment.The design of this work is:based on HPV6L2E7fusion protein, we add in the E6protein which has been found with stronger immunogenicity in recent clinical research to build a L2E7E6fusion protein. Considering that the full length of L2gene fused with E7and E6genes may affect the protein expression level, we choose3different lengths of L2gene fragments, thus1-360bp,1-600bp and full length, fuse with E7and E6genes. IFN-y ELISPOT, ELISA, HPV6pseudovirus neutralizing experiments are used to compare the cellular and humoral immune responses of different fusion proteins, then select one fusion protein to refine the immune evaluation, including screening and sub-type determination of relevant epitopes. The main research results are as follows:1. Three prokaryotic expression plasmids for HPV6L2, E7and E6genes respectively were constructed, His-6L2, GST-6E7and GST-6E6proteins were expressed and purified, which could be used for testing corresponding antibodies.2. Three prokaryotic expression plasmids for HPV6L2gene fragments with3different lengths fused with E7and E6genes were constructed,6L236oE7E6and6L2600E7E6proteins were expressed, identified and purified. Animal experiment results showed that there were no significant differences in immune responses between these two proteins plus CpG adjuvant, they both could induce strong cellular immune response in C57BL/6mice, and the same neutralizing antibody titer against HPV6pseudovirus were1:2560.3. We selected6L236oE7E6protein which had higher expression level in E.coli for further evaluation of immune responses. In cellular immune response,6L236oE7E6protein plus CpG adjuvant was the best, secondly was6L236oE7E6protein without adjuvant, and6L2360E7E6plus Al (OH)3adjuvant was the weakest; ELISA result showed that there were no significant differences in IgG antibody titers among these groups; In pseudovirus neutralization experiment,6L236oE7E6plus CpG adjuvant could induce neutralizing and cross-neutralizing antibody against HPV6,11,16,18,58,45with1:3200,1:1600,1:800,1:800,1:200,1:800titers respectively; result of growth inhibition of cancer in mice showed that6L236oE7E6plus CpG adjuvant could inhibit tumor growth and delay tumor formation, and even some mice had been completely protected from tumor.4. Two T cell epitopes of HPV6E7and E6proteins with the strongest reaction had been screened out, which were located in E71-15and E637-51peptides, both epitopes were8-amino-acid, the detail sequences were N’-VTLKDIVL-C and N’-YSYAYKHL-C respectively, intracellular cytokine staining experiment confirmed that both are the CD8+subtype T cell epitopes, more importantly, in-vivo CTL experiment in mice proved that E637.51had a strong CTL epitope.In conclusion, in this study, HPV6L236oE7E6fusion protein with high expression level in E.coli as a candidate therapeutic vaccine for HPV6associated diseases was made and we had preliminarily evaluated its cellular and humoral immune responses, particularly neutralizing and cross-neutralizing antibody levels, which could provide valuable experimental data for in-depth study on this kind of vaccine. The fusion protein could elicit strong CD8+T cellular and humoral immune responses in C57BL/6mice, and have the potential as a candidate therapeutic HPV vaccine for HPV6relative diseases.