Assessment of the immunogenicity of biochemically modified hepatitis B virus-like particles and their ability to deliver a medically relevant HIV-1 antigenic sequence

The hepatitis B virus (HBV) is one of the most significant human viruses. Approximately 2 billion people worldwide have been infected with HBV and more than 240 million people are chronically infected (WHO, 2016a). The current preventative HBV vaccine consists of virus-like particles (VLPs) assembled from the small HBV surface antigen (HBsAg-S) embedded in a phospholipid membrane. Approximately 10% of the population are non-responsive to the HBV vaccine, and the development of a HBV vaccine with enhanced immunogenicity for the non-responsive population is needed. Also, for the development of immunogens suitable for therapeutic applications, VLPs with enhanced immunogenicity are required.

The ability of the VLPs to serve as potent immunogens against not only the parent virus but also against inserted foreign antigenic sequences has allowed the development of HBsAg-S VLP platforms capable of inducing immune responses against the malaria parasite, bacterial pathogens, and other viruses. Approximately 37 million people worldwide are infected with HIV-1 (WHO, 2016b), which results in the acquired immunodeficiency syndrome (AIDS) if infection is not treated. Where antiviral drug treatment is available and affordable, infection with HIV-1 is now considered to be a chronic but manageable condition. However, this is no substitute for prevention, and treatment remains inaccessible to many people in developing countries. The use of vaccines remains the most effective strategy to eliminate infectious diseases.

The membrane-proximal external region (MPER) of the HIV-1 envelope protein gp41 is targeted by broadly neutralising antibodies. However, the development of an efficient vaccine to induce neutralising anti-MPER antibodies has not yet been accomplished due in part to the requirement that the MPER epitope be presented in close proximity to a phospholipid membrane. As such, HBsAg-S VLPs may represent a suitable scaffold for the presentation of the MPER in a phospholipid membrane-proximal context for the generation of anti-HIV antibodies.

In order to enhance the immunogenicity of HBV VLPs, the glycan abundance was modulated and enhanced to promote interactions with immune competent cells. Antigenic profiling of hypo- and hyperglycosylated HBsAg-S VLPs revealed that inducing significant differences in the abundance of N-glycans was not detrimental to the formation and structure of HBsAg-S VLPs. Hyperglycosylated VLPs were found to enhance the humoral immune response in an animal model, with an HBsAg-S T116N mutant inducing a significantly earlier and longer lasting antibody response.

To create VLPs capable of stimulating an anti-HIV antibody response, chimeric HBsAg- S VLPs with an inserted HIV-1 MPER sequence were constructed. To enhance VLP immunogenicity, the glycosylation patterns and disulphide bonds within the chimeric HBsAg-S-MPER subunits were modulated. Analysis of HBsAg-S-MPER VLP antigenicity using anti-HBsAg-S and anti-MPER antibodies revealed that inserting the MPER in a membrane-proximal location possibly allowed its presentation in a closer-to-native conformation compared to MPER peptides.

Immunisations in animal models revealed that HBsAg-S-MPER VLPs are capable of inducing MPER-specific antibodies. Changing the disulfide bonding or glycosylation status did not modify MPER exposure, but hyperglycosylation improved the humoral immune response generated by HBsAg-S-MPER VLPs. Importantly, the presence of a glycan at the N3 position adjacent to the inserted MPER sequence was found to both improve the overall immunogenicity of the VLPs and was critical for the stimulation of an effective anti-MPER antibody response. These antibodies were capable of binding to HIV-1 envelope protein complexes expressed on cellular surfaces, but neutralisation of HIV-1 entry was not observed, most likely as a result of an insufficiently raised anti- MPER antibody titre (approximately 1:1,000).

These results demonstrate that HBsAg-S VLPs are a suitable platform for the insertion of the HIV-1 MPER epitope and that biochemical modifications can impact on the immunogenicity of VLPs and their inserted sequences. The finding that hyperglycosylation can both enhance the overall immunogenicity of HBsAg-S VLPs and facilitate the development of humoral immune responses directed toward specific epitopes has significant implications for the development of a more effective HBV vaccine and for the general use of HBsAg-S VLPs as a platform for foreign epitopes.

The chimeric VLPs developed in this study demonstrate that biochemical modifications such as glycosylation alter immunogenicity. This represents an important step toward the design of immunogens with a potentially therapeutic capability. These outcomes demonstrate that HBsAg-S VLPs can be produced with a MPER-specific immunogenicity while presenting the MPER sequence in proximity to a lipid environment. Further assessments of different HBsAg-S-MPER VLP formulations in the presence of adjuvant will be required for the development of a preventative HIV-1 vaccine.