Preliminary Study On The Expression, Purification And Immunological Activity Of HPV16 L2 Multi - Epitope And Modified IgG1 Fc Fusion Protein

Human papillomavirus (HPV) can be divided into over150types with distinct tropisms. The mucosal HPV are subdivided into two groups:high-risk HPVs and low-risk HPVs. Persistent infections of high-risk HPVs have been proved to be the major etiological agents of cervical cancer. There are15high risk types have been identified, including HPV16,-18,-45,-58,-52,-31,-33and so on. Low-risk HPVs are mainly associated with benign genital warts, and about90%of verruca acuminates were caused by infections with HPV types6and11. The cutaneous HPVs cause benign cutaneous warts, such as HPV5can cause epidermodysplasia verruciformis. Currently, two HPV vaccines have been commercially developed based on HPV major capsid protein L1, but their protection are mainly for HPV16and HPV18infection and associated lesions, and can only prevent70%of Cervical cancer worldwide. A plurality of neutralizing epitopes are mainly located in N terminal region of HPV L2. HPV16L2amino acid (aa.)17-36is a major neutralizing epitope, which can induce cross-protective antibodies and broadly protective immunity, which is the focus in studying of HPV L2prophylactic vaccine.Antigen capture mediated by FcyR-immune complexes performs important functions on DC maturation, antigen internalization, antigen presentation, B maturation, antigen-specific humoral responses and antigen-specific T cell responses. In this study, two modified Fc-fusion proteins (E3R4and E12R4) consisting of three or twelve repeats of HPV16L2aa.17-36epitope (E3or E12) and a modified human IgG1Fc scaffold (R4) were expressed in baculovirus expression system as secreted proteins. The recombinant proteins were maintained high levels of expression, with approximately3mg of E3R4or2.5mgof E12R4obtained in100ml culture medium, and purified by rProtein A affinity chromatography. E3R4was stable, but E12R4was instable, which had partial degradation. ELISA analysis showed that E3R4and E12R4both can bind with HPV16L2epitope specific monoclonal antibodies (RG-1) effectively. BALB/c mice were respectively immunized with E3R4and E12R4formulated in Freunds’adjuvant, and the antisera were tested with pseudovirus-based ELISA. E3R4and E12R4induced significantly higher titers of HPV16,-18,-45,-52,-58,-6,-11and-5PsV cross-reactive antibodies than E3peptide and R4scaffold. E3R4induced significantly higher titers of antibodies than E12R4, which was instable. Our results demonstrate that delivering antigen by using R4scaffold can enhance the immunogenicity of antigen. E3R4fusion protein expressed in insect cell can induce broad-spectrum antibodies which can bind to multiple HPV types. This vaccine produced in this system is high level to expression and easy to purification. Thus, this study laid a basis for the research of a novel HPV L2prophylactic vaccine.