Enhancing the immunogenicity and functionality of hepatitis B surface antigen virus-like particles
Creators: name: JOE, Carina Citra Dewi
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 310607
subject: Nanobiotechnology
classificationCode: 401807
subject: Nanomaterials

description: Hepatitis B virus (HBV) infection is the most common liver infection in the world, and can lead to cirrhosis, Iiver cancer or liver failure. There were an estimated 887,000 deaths worldwide in 2015 due to complications from HBV infection. Current treatment for chronic HBV infection is generally ineffective and inefficient, thus vaccination remains the most successful and cost-effective prophylactic approach in controlling HBV infection. The biologically active components of the commercial HBV vaccines (Engerix®B, Recombivax HB®, and Heplisav-B®) are recombinant hepatitis B virus-like particles (VLPs) expressed in yeast, composed of the hepatitis B surface antigen and host-derived lipids that can self-assemble into a highly organised, multimeric structure. Hepatitis B VLPs, also referred to as hepatitis B small surface antigen (HBsAgS) efficiently stimulate both humoral and cell-mediated immunity, affording protection from infection in ≥90% immunised recipients. However, commercial vaccines fail to elicit protective antibodies in 5-10% immunocompetent subjects. Therefore, there is a need to improve the efficacy of HBV vaccine in this particular group of people. My research aimed to develop approaches to improve the immunogenicity of the HBV-based VLPs that form the basis of a number of successful commercial vaccines.<br><br>
A number of approaches for VLPs production were undertaken in my study, in the first instance, the suspension adapted HEK293 mammalian cells were transiently transfected to express HBsAgS VLPs, allowing for a rapid and cost-effective HBsAgS VLP production. Furthermore, an immunoaffinity capture-based purification method using anti-Flag monoclonal antibody column was developed for a rapid, scalable, reproducible, and efficient downstream processing that provides increased recovery of highly pure VLPs (&gt;90%) as an alternative to the ultracentrifugation-based method.<br><br>
A number of strategies to enhance protective immune responses to HBsAgS VLPs were employed in my study, such as engineering an additional glycosylation site, T116N to increase the amount of N-linked glycans in the HBsAgS VLP which promote antigen uptake by dendritic cells (DCs) and will subsequently enhance humoral immune responses. Another strategy involved mutating three cysteine residues, C121A, C124A, and C147A to disrupt the disulphide bonds in the structure of HBsAgS VLP to facilitate the access of proteolytic enzymes for the generation of precursor epitopes which will subsequently promote antigen presentation in the cross-presentation pathway and enhance cell-mediated immune responses.<br><br> Mice immunised with T116N-HBsAgS VLPs in combination with adjuvant showed a sustained, high antibody titre compared to Engerix®B and the other test VLP groups. One week following four immunisations, the mice immunised with T116N-HBsAgS VLPs in combination with adjuvant had an endpoint anti-HBsAgS titre of 1: 1966000 whilst wild-type (WT)-HBsAgS in the absence of adjuvant, WT-HBsAgS in combination with adjuvant, T116N-HBsAgS in the absence of adjuvant, and Engerix®B immunised mice had endpoint anti-HBsAgS titres of 1: 30700, 1: 232100, 1: 225200, and 1: 129700, respectively. In contrast, the mice immunised with triple cysteine mutant (TCM) HBsAgS VLP in the absence of adjuvant did not elicit significantly higher cytotoxic T lymphocyte (CTL)-mediated immune response compared to WT-HBsAgS VLP-immunised mice which was confirmed with in vivo CTL assays.<br><br>
Immunisation with self-antigens arrayed on VLPs has been reported to successfully induce autoantibody responses, for example, mice immunised with a self-antigen displayed on the surface of papillomavirus VLPs demonstrated 1000-fold higher autoantibody titres compared to immunisation with a fusion protein alone. In my study, a novel tumour-associated epitope identified on the epidermal growth factor receptor (EGFR) amino acids 287-302 that is recognised by the monoclonal antibody 806 (mAb806), was used for display on the surface of HBsAgS VLP. Interestingly, mAb806 only binds to either tumour-associated EGFR deletion mutants, observed in 40-50% of glioblastomas or following overexpression of the WT EGFR, and is currently in Phase III clinical trials as an antibody-drug conjugate for the treatment of glioblastoma.<br><br>
Chimeric HBsAgS VLPs displaying human EGFR-derived epitope successfully elicited epitope-specific antibody responses. Surprisingly, the specific humoral antibody responses were also elicited by HBsAgS VLPs presenting the corresponding mouse EGFR sequence i.e. a self-antigen. The most encouraging observation for the VLP-based human and mouse 806 immunogen was that the pooled polyclonal antisera was shown to bind to the cell-surface EGFR carrying the C271A mutation, highlighting the capability of the elicited anti-806 antibodies to recognise the cell-surface EGFR on living cells. Interestingly, cross-reactivity was observed between mAb806 with the mouse EGFR which was confirmed using both recombinant mouse protein corresponding to EGFR vIII and the flow cytometry following transient transfection of HEK293 cells with vector expressing EGFR carrying the C271A mutation and to my knowledge, this is the first known study to report this finding.<br><br>
The hyperglycosylated (T116N) HBsAgS VLPs were shown to enhance humoral immune responses due to the increase in glycan density, hence, glycoengineered VLP represents an attractive strategy for the development of superior vaccine candidates and optimised VLP delivery platforms. An increase in high mannose-type glycans was observed in the majority of VLPs expressed in my study which was correlated with improved uptake by professional antigen-presenting cell (APCs) and DCs in particular, due to the expression of membrane bound mannose receptors on these cells. Finally, the results of immunogenicity studies using chimeric HBsAgS VLPs displaying EGFR-derived epitope provide encouraging prospects for future development of vaccine-based cancer immunotherapies that are capable of eliciting autoantibodies and actively target cancers associated with EGFR/EGFR vIII overexpression.