Abstract
Hemagglutinin glycoprotein (HA) is a principle influenza vaccine antigen. Recombinant HA-based vaccines become a potential alternative for traditional approach. Complexity and variation of HA N-glycosylation are considered as the important factors for the vaccine design. The number and location of glycan moieties in the HA molecule are also crucial. Therefore, we decided to study the effect of N-glycosylation pattern on the H5 antigen structure and its ability to induce immunological response. We also decided to change neither the number nor the position of the HA glycosylation sites but only the glycan length. Two variants of the H5 antigen with high mannose glycosylation (H5hm) and with low-mannose glycosylation (H5Man5) were prepared utilizing different Pichia strains. Our structural studies demonstrated that only the highly glycosylated H5 antigen formed high molecular weight oligomers similar to viral particles. Further, the H5hm was much more immunogenic for mice than H5Man5. In summary, our results suggest that high mannose glycosylation of vaccine antigen is superior to the low glycosylation pattern. Our findings have strong implications for the recombinant HA-based influenza vaccine design.
Highlights
Traditional manufacturing of influenza vaccines involves using living viruses and presents unique technical and biosafety challenges
The DNA encoding the extracellular domain of HA with deletion of the cleavage site (EpiFluDatabase Accession No EPI15789) from H5N1 avian influenza virus (A/swan/Poland/305-135V08/ 2006 clade 2.2.2) was cloned into pJAZs1 vector using BsaI (Thermo Scientific, USA) and pPICZαC using ClaI and NotI restriction sites. pJAZs1/H5Man5 and pPICZαC/H5 antigen with high mannose glycosylation (H5hm) plasmids were linearized with PmeI (Thermo Scientific, USA) and used for the electroporation of the SuperMan5 (GlycoSwitch, Biogramma tics, USA) and KM 71 (Invitrogen, USA) P. pastoris strains
We decided to study the effect of N-glycosylation pattern without changing the number or the position of glycosylation sites
Summary
Traditional manufacturing of influenza vaccines involves using living viruses and presents unique technical and biosafety challenges. Serious limitations of the egg-based method forced researchers for a new solution in the area of influenza vaccine development. A relevant characteristic of the influenza virus is its capacity to change constantly, which is caused by mutations in the genes encoding two proteins: hemagglutinin (HA) and neuraminidase [1]. Those changes are usually activated by two mechanisms: antigenic shift and antigenic drift.
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