Influenza HA and the importance of glycosylation

After the 2009 2010 H1N1 influenza pandemic, US public health experts concluded that non-egg based, recombinant DNA technologies using genetic sequences should be given priority for influenza vaccine development. Influenza virus-like particles (VLPs) produced in insect cells are considered a promising new vaccine approach to prevent influenza virus infections. However, it has been shown that carbohydrate structures found on the surface of influenza A viruses differ significantly between viral subtypes. Since hemagglutinin (HA) is the most relevant antigenic glycoprotein, specific attention must be paid when it comes to the production of influenza vaccines. On the one hand, efficacy of a vaccine might by impaired by the fact that certain glycans shield important antigenic sites. On the other hand carbohydrate structures might serve as adjuvants and increase the desired immune response, or cause undesired immune response, bearing the risk of allergic reactions. In order to evaluate the impact of specific N-linked glycan structures present on viral hemagglutinin on immunogenicity, we will produce a set of either non-glycosylated VLPs or VLPs carrying carbohydrate structures ranging from oligomannosidic to complex biantennary type. This will be accomplished using the hence developed SweetBac system, which is based on the introduction of various specific glycozymes in a modifyied baculovirus genome (MultiBac). The resulting SweetBac backbones will then be used for the expression of VLPs in different insect cell lines. Purification of VLPs will be performed using polymethacrylate monoliths, so-called CIM-disks and CIM tubes. The structure and position of carbohydrate structures present on HA will be elucidated using different mass spectrometry methods. In order to get detailed information about the impact of different carbohydrate structures on immunogenicity we will vaccinate BALB/c mice with purified VLPs and evaluate the breath of immune response as well as the allergenic potential. VLPs showing significantly increased immunogenic properties and a high degree of cross-reactivity will finally be tested in a challenge study.

After the 2009 2010 H1N1 influenza pandemic, US public health experts concluded that non-egg based, recombinant DNA technologies using genetic sequences should be given priority for vaccine development. Virus-like particles (VLPs) produced in insect cells are considered a promising new vaccine approach to prevent e.g. influenza virus infections. Nevertheless, these insect cell expressed proteins sometimes suffer from reduced biological activity and unwanted side effects. Several studies have demonstrated that glycosylation might be an explanation for these effects. We therefore investigated approaches for improving immunogenicity of insect cell expressed Influenza A Virus-Like particles and at the same time for reducing their allergenic potential. Dendritic cells (DC) have been shown to play a key role in the initiation of immune responses by binding and up taking antigen. This uptake is often accomplished using mannose specific receptors. In order to investigate the impact of mannose residues on the immunogenic behavior of insect cell expressed VLPs we chemically blocked an enzyme in the cells that allows for the production of VLPs carrying oligomannosidic N-glycans. These showed significantly enhanced binding and uptake characteristics to na?ve DCs. The presence of another carbohydrate structure, a specifically linked fucose residue, bears the risk of causing immediate hypersensitivity reactions in patients with allergy. To diminish the fucose de novo synthesis pathway of different insect cell lines, we integrated a bacterial enzyme in the baculovirus genome. This setup gives the possibility to produce VLPs almost free from fucose. A binding assay of non-fucosylated VLPs with sera from patients with allergy showed a 10-fold decrease in IgE antibody binding levels compared to wildtype variants. These results might help to improve the baculovirus insect cell system for the production of biopharmaceuticals by fine-tuning the immunogenic behaviour of the product and on the other hand greatly reducing the risk of unwanted allergic reactions to the product.