Hepatitis B Surface Antigen-based Vaccines for Human Neoplastic Disease

Abstract

Hepatitis B surface antigen (HBsAg) vaccines elicit strong cytotoxic T-lymphocyte (CTL) responses when delivered as DNA or virus-like particles. When foreign antigens are coexpressed with HBsAg as a fusion protein, powerful antigen-specific CTL responses are achieved. Therefore, HBsAg is an attractive vaccine vector for delivery of disease-related foreign epitopes. The capacity to elicit CTL responses compares favourably when rHBsAg vaccines are delivered by DNA- rather than virus like particle (VLP)-modalities. Human papillomavirus (HPV)-associated carcinoma is the second most frequent cancer causing death in women worldwide. A major feature of the HPV lifecycle is the immortalisation of the squamous epithelium and neoplastic growth, a result of HPV E6 and E7 oncogene expression. E6 and E7 oncoproteins are essential to maintain a transformed cell phenotype and are present in all stages of cervical intra-epithelial neoplasia (CIN), which make them ideal targets for immunotherapeutic therapy. In this study, we have generated an HBsAg-based vaccine platform. Using splice overlap extension (SOE)-PCR the HPV-16 E6 and E7 oncogenes were fused to HBsAg (E7HBE6). In the process, E6 and E7 were mutated to eliminate potential oncogenicity and were codon optimised for enhanced expression. The effector and memory CTL response, as well as tumour protection and therapy afforded by immunisation with E7HBE6 vaccine was evaluated in mice. The E7HBE6 construct was evaluated for the potential to form VLPs. Insect cells expressed E7HBE6 protein at low levels which appeared to form aggregates and did not demonstrate VLP formation. The formation of E7HBE6 VLPs and E7HBE6 protein were undetectable when expressed from two different mammalian cell lines. Observations within these experiments suggested that the E7HBE6 protein maybe unstable. Understanding the CTL response interplay between E6 and E7, within the context of pE7HBE6 is important for its application as a vaccine. Studies were conducted using DNA plasmid HBsAg-HPV vaccine (pE7HBE6). We demonstrated that pE7HBE6 DNA vaccine was immunogenic at a single low dose which was enhanced by boosting. Effector and memory CTL responses to E6 and E7 were maintained for 23 weeks post-immunisation. A maximal CTL response was achieved from a single dose at 30-100μg with a significant response also detected from a single dose of 10μg. When a dominant CTL epitope was eliminated from within pE7HBE6, a subdominant epitope response was detected. The presence of a subdominant epitope enhanced the response to the dominant epitope, at the cost of a decrease in its own effector CTL response. The immunodominance effect was decreased by combining epitope knockout vaccines, allowing processing of E6 and E7 CTL epitopes in separate cells. Furthermore, administration of antigens at separate anatomical sites also interfered with immunodominance. Original antigenic sin was examined by the immunisation of HBsAg-experienced mice with recombinant pE7HBE6 DNA vaccine. Prevention of the growth of HPV-16-associated subcutaneous TC-1 tumour was achieved in 100% of mice by immunisation with pE7HBE6, compared to mice immunised with HBsAg wildtype DNA vaccine, all of which presented with tumour. When pE7HBE6 was used as a therapeutic agent, a significant increase in survival and decreased tumour volume were demonstrated. In a TC-1 tumour metastasis model, where mice were inoculated with TC-1 intravenously, therapy was highly effective. Mice immunised with HBsAg-HPV DNA vaccines (either pE7HBE6, or combined epitope knockouts) remained 100% tumour free compared to mice immunised with HBsAg DNA, of which 80% displayed lung tumours. The results from this study demonstrate that a HBsAg-vectored DNA vaccine encoding both HPV-16 E6 and E7 oncoproteins can be used to provide protection and therapy against a tumour expressing HPV-16 oncogenes. This study supports previously published studies that demonstrates an enhanced HPV-specific CTL response when both E6 and E7 are administered together in the context of a vaccine. These results have generic implications for the design and administration of DNA vaccines encoding chimeric antigens. The results of this study have specific implications for the design of HBsAg-based DNA vaccines delivering HPV antigens for the protection and therapy of HPV-associated squamous carcinomas. An effective therapeutic vaccine designed to treat HPV-associated cancers has the potential to dramatically reduce the burden of cervical carcinoma and associated death rate.