N-glycosylation converts non-glycoproteins into mannose receptor ligands and reveals antigen-specific T cell responses in vivo
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Christoph Kreer1, Janina M. Kuepper2, Matthias Zehner1, Thomas Quast1, Waldemar Kolanus1, Beatrix Schumak2,*, Sven Burgdorf1,*
1Life and Medical Sciences (LIMES) Institute, University of Bonn, 53115 Bonn, Germany
2Institute for Medical Microbiology, Immunology and Parasitology (IMMIP), University Hospital Bonn, 53105 Bonn, Germany
*These authors have contributed equally to this work
Sven Burgdorf, email: firstname.lastname@example.org
Keywords: antigen presentation, Komagataella phaffii, receptor-mediated endocytosis, T cell activation, cross-presentation
Received: May 03, 2016 Accepted: December 05, 2016 Published: December 28, 2016
N-glycosylation is generally accepted to enhance the immunogenicity of antigens because of two main reasons. First, the attachment of glycans enables recognition by endocytic receptors like the mannose receptor (MR) and hence increased uptake by dendritic cells (DCs). Second, foreign glycans are postulated to be immunostimulatory and their recognition could induce DC activation. However, a direct comparison between the immunogenicity of N-glycosylated vs. de-glycosylated proteins in vivo and a direct effect of N-glycosylated antigens on the intrinsic capacity of DCs to activate T cells have not been assessed so far.
To analyze whether enforced N-glycosylation is a suited strategy to enhance the immunogenicity of non-glycosylated antigens for vaccination studies, we targeted non-glycoproteins towards the MR by introduction of artificial N-glycosylation using the methylotrophic yeast Komagataella phaffii (previously termed Pichia pastoris). We could demonstrate that the introduction of a single N-X-S/T motif was sufficient for efficient MR-binding and internalization. However, addition of N-glycosylated proteins neither influenced DC maturation nor their general capacity to activate T cells, pointing out that enforced N-glycosylation does not increase the immunogenicity of the antigen per se. Additionally, increased antigen-specific cytotoxic T cell responses in vivo after injection of N-glycosylated compared to de-glycosylated proteins were observed but this effect strongly depended on the epitope tested. A beneficial effect of N-glycosylation on antibody production could not be detected, which might be due to MR-cross-linking on DCs and to concomitant differences in IL-6 production by CD4+ T cells.
These observations point out that the effect of N-glycosylation on antigen immunogenicity can vary between different antigens and therefore might have important implications for the development of vaccines using K. phaffii.
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