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Abstract

The link between the human papillomavirus (HPV) and cervical cancer has been well established since the 1980s and today it is known that >95 % of cervical cancer cases arise from persistent HPV infection of the cervix. Moreover, there has been accumulating evidence addressing HPV as an etiological agent in other anogenital cancers, oropharyngeal cancers and skin cancers. The recognition of HPV as an oncogenic agent initiated the HPV vaccine studies to reduce HPV infection and cervical cancer incidence. Current prophylactic HPV vaccines on the market are all based on L1 virus-like particle (VLP) mixture from different HPV types. Despite their high efficacy and safety profile, the current vaccines have a high degree of type-specific response. Moreover, the high production and transportation costs of the current vaccines drove efforts to seek broadly protective and economically feasible alternative HPV antigens. The L2 capsid protein has been identified as an alternative as a prophylactic vaccine antigen capable of cross-protection via the conserved epitope residing at the N-terminus between 20-38 residues. On the other hand, lower immunogenicity compared to L1 antigens is the main obstacle of L2-based antigens. Previously in our group, L2(20-38) epitopes from eight different HPV types were fused by covalently linking and insertion into thioredoxin from hyperthermophilic archaea Pyrococcus furiosus as scaffold (Trx-8mer) and the immunogenicity of Trx-8mer was increased when it was heptamerized through the OVX313 domain (Trx-8mer-OVX313). The rationale behind the multimerization is that B cell receptor cross-linking due to the presentation of repetitive epitopes triggers signaling and induces potent B cell responses. I aimed to multimerize Trx-8mer (i) by mtDod platform, which is a dodecin from Mycobacterium tuberculosis assembling into dodecameric nanoparticles, and (ii) by HPV 16 L1 capsomers that are pentameric subunits of VLPs. For the former, Trx-8mer was connected to mdDod either directly by producing a fusion protein or by coupling via DogTag/DogCatcher molecular glue after the purification of the two partners. Multimerization via capsomers was performed only via DogTag/DogCatcher. The mtDod antigens and capsomer antigens were produced in E.coli as an economically-feasible production process. I demonstrated that the mtDod platform displays L2 epitopes when it is fused with the Trx-8mer at the gene level (Trx-8mer-mtDod) and vaccination with Trx-8mer-mtDod adjuvanted with either SWE or AddaVax induces neutralizing antibody response against vaccine and cross-type HPV. Furthermore, heterologous immunization by priming with Trx-8mer-OVX313 followed by boosting with Trx-8mer-mtDod significantly enhanced the neutralizing antibody titers induced by L2 epitopes. However, the enhanced titers were not higher than those induced by homologous immunization with Trx-8mer-OVX313. In contrast, the most remarkable result was obtained when mtDod nanoparticles (DogTag-mtDod) were decorated with Trx-8mer (DogCatcher-Trx-8mer) molecules after being purified separately via DogTag/DogCatcher. These nanoparticles are more immunogenic than both Trx-8mer-mtDod and Trx-8mer-OVX. The second multimerization strategy was to decorate capsomers with either Trx-8mer and 8mer epitopes with the aid of DogTag/DogCatcher. Insertion of DogTag into the DE loop of the L1 protein did not disrupt the capsomer structure and the tag was accessible to react with DogCatcher partners. (DogCatcher-Trx-8mer, DogCatcher-8mer). I showed that the decoration of capsomers shields the L1 epitopes so that the vaccination of mice with decorated capsomers induced only L2-specific antibodies with titers that were significantly higher than those induced by monomeric Trx-8mer. As an attempt to use the capsomers as a platform for a therapeutic antigen, DogCatcher-Trx-HPV 16 E7(49-57)x3 was designed to decorate capsomers with HPV 16 E7 cytotoxic T cell epitope (E7(49-57)). Mice immunized with the mentioned antigen exhibited E7(49-57)-specific T cell response. These findings demonstrate that Trx-8mer multimerized with mtDod particles and HPV 16 L1 capsomers via DogTag/DogCatcher is a promising candidate antigen for prophylactic HPV vaccines. Besides, the modular DogTag-capsomers can be further investigated as a platform to produce therapeutic antigens.