TY - JOUR A1 - Radke, Lars A1 - Sandig, Grit A1 - Lubitz, Annika A1 - Schließer, Ulrike A1 - von Horsten, Hans Henning A1 - Blanchard, Véronique A1 - Keil, Karolin A1 - Sandig, Volker A1 - Giese, Christoph A1 - Hummel, Michael A1 - Hinderlich, Stephan A1 - Frohme, Marcus T1 - In Vitro Evaluation of Glycoengineered RSV-F in the Human Artificial Lymph Node Reactor JF - Bioengineering N2 - Subunit vaccines often require adjuvants to elicit sustained immune activity. Here, a method is described to evaluate the efficacy of single vaccine candidates in the preclinical stage based on cytokine and gene expression analysis. As a model, the recombinant human respiratory syncytial virus (RSV) fusion protein (RSV-F) was produced in CHO cells. For comparison, wild-type and glycoengineered, afucosylated RSV-F were established. Both glycoprotein vaccines were tested in a commercial Human Artificial Lymph Node in vitro model (HuALN®). The analysis of six key cytokines in cell culture supernatants showed well-balanced immune responses for the afucosylated RSV-F, while immune response of wild-type RSV-F was more Th1 accentuated. In particular, stronger and specific secretion of interleukin-4 after each round of re-stimulation underlined higher potency and efficacy of the afucosylated vaccine candidate. Comprehensive gene expression analysis by nCounter gene expression assay confirmed the stronger onset of the immunologic reaction in stimulation experiments with the afucosylated vaccine in comparison to wild-type RSV-F and particularly revealed prominent activation of Th17 related genes, innate immunity, and comprehensive activation of humoral immunity. We, therefore, show that our method is suited to distinguish the potency of two vaccine candidates with minor structural differences. KW - Glycoengineering KW - fucosylation KW - RSV KW - F-Protein KW - NanoString Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-9831 SN - 2306-5354 VL - 4 IS - 3 ER - TY - JOUR A1 - Sandig, Grit A1 - von Horsten, Hans Henning A1 - Radke, Lars A1 - Blanchard, Véronique A1 - Frohme, Marcus A1 - Giese, Christoph A1 - Sandig, Volker A1 - Hinderlich, Stephan T1 - Engineering of CHO Cells for the Production of Recombinant Glycoprotein Vaccines with Xylosylated N-glycans JF - Bioengineering N2 - Xylose is a general component of O-glycans in mammals. Core-xylosylation of N-glycans is only found in plants and helminth. Consequently, xylosylated N-glycans cause immunological response in humans. We have used the F-protein of the human respiratory syncytial virus (RSV), one of the main causes of respiratory tract infection in infants and elderly, as a model protein for vaccination. The RSV-F protein was expressed in CHO-DG44 cells, which were further modified by co-expression of β1,2-xylosyltransferase from Nicotiana tabacum. Xylosylation of RSV-F N-glycans was shown by monosaccharide analysis and MALDI-TOF mass spectrometry. In immunogenic studies with a human artificial lymph node model, the engineered RSV-F protein revealed improved vaccination efficacy. KW - CHO KW - glycoengineering KW - respiratory syncytial virus KW - vaccine KW - xylose Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-9620 SN - 2306-5354 VL - 4 IS - 2 ER -