Presentation Of Receptor-Contacting Loop Of Human Ige On The Hbcag Particles

Immunoglobulin of class E(IgE) plays a central role in allergies and allergic asthma. Therapeutic anti-IgE vaccine is expected to elicit autologous antibodies targeting the endogenous IgE molecules and neutralize allergic mechanism. Virus-like particles (VLPs) are a form of immunogens capable of breaking self-tolerance and represent an attractive platform for designing the therapeutic vaccines against self-antigens, such as the IgE. We designed VLPsfrom the core protein of hepatitis B virus (HBcAg). Arrays of the receptor-contacting epitopes of the human IgE were engineered on the VLP surfaces.Objectives: Primary objective of this study was to develop IgE-epitope antigen that induces antibodies against the receptor-contacting epitope on the human IgE molecule.Methods: Genes encoding the empty carrier and HBcAg with insertion of IgE epitope were constructed de novo and cloned into pET expression vectors. Ultracentrifugation in sucrose gradients and size exclusion chromatography (SEC) were used to purify VLPs.Electron microscopy and dynamic light scattering were used for proof of the VLPs appearance. Mice were immunized, andwestern blotswere conducted with immune sera to reveal the presence of the induced anti-(human)IgE antibodies. Titers of the anti-IgE were measured in ELISA.Results: Two types ofplasmid constructs for expression of the VLPs carrying IgEepitope arrays were produced. One typerepresents aninsertion of an immunogenic peptide into HBcAg by construction of a contiguous fusion protein in which the peptide is flanked with sequences of the carrier. The other type is different in that the carrier protein is “split” into two polypeptide chains which upon expression remain associated in a stable VLP-forming subunit. The splitting was achieved by construction of a bicistronic RNA (s.c. “SplitCore” technology). The obtained IgE-epitope antigens with contiguous sequences appeared to be incapable of formation of VLPs. On the contrary, the split core protein efficiently formed VLPs. Immunization of mice with the VLPs presenting the IgE Cε3 FG loop resulted in the development of high titers of antibodies-recognizing human IgE in ELISA.Conclusion: Recombinantly expressed VLPs presenting the IgE Cε3 FG loop elicitthe anti-IgE antibodies upon immunization. Application of the SplitCore technology to construct IgE-epitope antigens can result in a pan-allergy anti-IgE vaccine.