The Study Of Human Papillomavirus Virus-like Particle Based Prophylactic Vaccine

Cervical cancer is the second most diagnosed cancer in women worldwide. Infection of high-risk types of human papillomavirus (HPV) has been proved to be the major etiological agent of cervical cancer. There are15high-risk types have been identified, among which HPV16and18are the two most prevalent types all over the world. HPV58is a prevalent high-risk types in Eastern Asia, Central America and South America. HPV major capsid protein L1can self-assemble into virus-like particles (VLPs). Currently licensed HPV L1VLP-based prophylactic vaccines, are demonstrated safe in clinical trials and induce protection against infections and precancerous lesions associated with vaccine included types, but litter cross-protection against HPV45and31infection. There is no report about HPV58longlasting neutralizing antibodies and no commercially available vaccines against HPV58infection. Minor capsid protein L2has cross-neutralizing epitopes, but the immunogenicity of these epitopes are weak. HPV VLPs are potentially highly effective in eliciting strong immune responses reflecting close-packed, regular spaced and high density epitopes on their surface, therefore, chimeric virus-like particles (cVLPs) are formed by display L2cross-neutralizing epitopes on HPV VLPs, and these cVLPs may provide more broad protection than the licensed HPV VLP vaccines.In this study, we expressed HPV16,18,58L1VLPs in Sf9cells using baculovirus expression vector system and purified them. Purities, physicochemical characteristic and immunogenicity of the resultant proteins were indentified, we especially analyzed HPV58longlasting neutralizing antibodies. We also investigated the adjuvant effect of Al(OH)3on HPV L1VLPs and compared the physical characteristic and immunogenicity of chromatography purified HPV L1VLPs with those of CsCl ultracentrifugation purified HPV L1VLPs. Based on the above studies, we cloned, expressed and purified fusion L1-L2proteins by inserting HPV16L2cross-neutralizing epitopes (aa.17-36or aa.56-75) in HPV16L1backbone, and we investigate the insertion site and the tolerated lengths of the inserted L2peptide that still allowed for VLP assembly and inducing neutralizing antibodies. The results are as follows.1. The study of chromatography purified human papillomavirus16/18/58L1virus-like particle vaccine 1.1The purities, physical and epitope characteristic of HPV16,18,58L1VLPsAs demonstrated by the SDS-PAGE with Coomassie blue staining, these HPV16,18,58L1proteins are highly pure. The purities of HPV16,18,58L1proteins were further validated by barely detected Sf9host cell protein (HCP) contaminations by HCP ELISA kit and Western blot by using anti-Sf9-HCP polyclonal antibodies as primary antibody. We next determined the physical characteristic by transmission electron microscopy (TEM) and dynamic light scaling (DLS) assays, the results showed HPV16,18,58L1proteins were properly fold and were highly uniformly assembled into VLPs with the diameters of45-70nm,40-68nm,40-65nm, respectively. By ELISA, we found type-specific neutralizing MAbs H16.V5-, H18J4-and XM58-07-recognized epitopes were confirmed presenting on the surface of HPV16,18,58L1VLPs, respectively. Thus, these data demonstrate that HPV16,18,58L1VLPs purified from Sf9cells are pure, uniform and well-characterized.1.2Comparison of physical characteristic and immunogenicity towards HPV L1VLPs purified by two different methodsDLS results showed in contrast to the homogeneous chromatography purified VLPs, the CsCl ultracentrifugation purified HPV L1VLPs displayed multiple peaks with a relatively wide size range and were heterogeneity. We also compared the immunogenicity of chromatography purified HPV VLPs with that of CsCl ultracentrifugation purified VLPs by immunization HPV16L1VLPs in mice, the results showed the chromatography purified HPV16L1VLPs are highly immunogenic.1.3Immunogenicity study of HPV16,18,58L1VLPs and HPV58long-lasting neutralizing antibodies analysisTo determine the immunogenicity of HPV16,18,58L1VLPs, we calculated the ED50value of each VLPs. The ED50values of HPV16,18,58L1VLPs were0.0275μg,0.0172μg,0.1140μg, respectively. Next, by pseudovirus neutralization assays, monovalent vaccines as well as trivalent vaccine were able to induce high levels of neutralizing antibodies against corresponding immunogen type(s). There were no significant differences in vaccine component type-specific neutralizing antibody titers between trivalent and corresponding monovalent vaccines (P>0.05). High levels of longlasting neutralizing antibodies are crucial for long-term protection against HPV infection. Results of pseudovirus neutralization assay showed anti-HPV58neutralizing antibody titers in both trivalent and mono58immunized mice were maintained at high levels (above103) for at least48weeks. These results indicated that HPV16,18,58L1VLPs purified by chromatography are highly immunogenic to elicit effective neutralizing antibody responses.1.4Adjuvant effect of Al(OH)3on HPV L1VLPs.To investigate the adjuvant effect of Al(OH)3on HPV L1VLPs, we immunized low doses or high doses of mono16, mono18, mono58, trivalent vaccines with or without Al(OH)3in mice, the results showed only in low doses groups, Al(OH)3significantly increased anti-HPV16, anti-HPV18neutralizing antibodies in mono and trivalent vaccines, while didn’t significantly increase anti-HPV58neutralizing antibodies. To investigate the dose-response of HPV VLPs to Al(OH)3, we immunized different amount of Al(OH)3with HPV18L1VLPs, the results showed the anti-HPV18neutralizing antibodies increased by increasing Al(OH)3amount.2. The study of human papillomavirus16chimeric virus-like particle vaccine2.1Construction and identification of recombinant baculovirus transfer vectorsUsing overlap PCR, we inserted HPV16L2aa.56-75(sE) or two copies of HPV16L2aa.17-36or tandem HPV16L2aa.17-36(dE) into the HPV16L1DE loop (aa.136/137) or H4helix (aa.430/433), respectively and we called these HPV16L1-L2genes DE/sE, H4/sE, DE/ddE, H4/ddE, DE/sdE, respectively. These HPV16L1-L2genes which verified by DNA sequencing were cloned to pFastBacl vector. The proteins were expressed in insect cells by using Bac-to-Bac system. These proteins were purified by chromatography methods.2.2Analysis of purified HPV16L1-L2proteinsThe results of TEM showed that H4/sE assembled into VLPs, DE/ddE only formed pentamers, H4/ddE and DE/sE apparently formed VLPs and amorphous aggregates, DE/sdE existed in less-ordered conformations, indicating pentamers or protein aggregates. We analysis HCP contaminations by Western blot using anti-Sf9-HCP polyclonal antibodies. The results showed the amount of Sf9HCPs in purified H4/sE, DE/sE, DE/ddE were less than10%, while the amount in purified DE/sdE were 10%-20%.2.3Immunogenicity analysis of HPV16L1-L2proteinsWe subcutaeous immunized with HPV16L1-L2proteins or HPV16LI VLPs (contrast group) with freund’s adjuvant in BALB/c mice. The results showed HPV16L1VLPs, H4/sE, DE/sE, H4/ddE, DE/ddE, DE/sdE induced anti-HPV16neutralizing antibodies of3.2×104,1.8×104,2.7×103,1.2×102,5R101,<50respectively. H4/sE induced the highest neutralizing antibodies in immunized HPV16L1-L2proteins groups and they were comparable with the neutralizing antibodies induced by HPV16L1VLPs. Both H4helix and DE loop is more tolerated for insertion of sE than ddE. Insertion epitope (sE or ddE) in H4helix induced higher antibodies than those of DE loop.Taken together, we obtained highly pure, uniform and well-characterized HPV16,18,58L1VLPs from Sf9cells. Low-dosage immunization with HPV58L1VLPs alone or co-administrated with HPV16and HPV18L1VLPs is sufficient to induce high levels of long-lasting neutralizing antibodies in mice. Our results suggest that the highly immunogenic HPV16,18,58L1VLPs are good candidates for developing trivalent vaccine against HPV16,18,58infections. The investigation about insertion site and the tolerated lengths of the inserted L2peptide that still allowed for VLP assembly and inducing neutralizing antibodies will lad foundation for further research on HPV cVLPs which may provide more broad protection than the licensed HPV VLP vaccines.