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J Virol. 2012 Nov;86(21):11735-44. doi: 10.1128/JVI.01084-12. Epub 2012 Aug 22.

Glycan-dependent immunogenicity of recombinant soluble trimeric hemagglutinin.

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  • 1Virology Division, Department of Infectious Diseases and Immunology, Utrecht University, Utrecht, The Netherlands.

Abstract

Recombinant soluble trimeric influenza A virus (IAV) hemagglutinin (sHA(3)) has proven an effective vaccine antigen against IAV. Here, we investigate to what extent the glycosylation status of the sHA(3) glycoprotein affects its immunogenicity. Different glycosylation forms of subtype H5 trimeric HA protein (sH5(3)) were produced by expression in insect cells and different mammalian cells in the absence and presence of inhibitors of N-glycan-modifying enzymes or by enzymatic removal of the oligosaccharides. The following sH5(3) preparations were evaluated: (i) HA proteins carrying complex glycans produced in HEK293T cells; (ii) HA proteins carrying Man(9)GlcNAc(2) moieties, expressed in HEK293T cells treated with kifunensine; (iii) HA proteins containing Man(5)GlcNAc(2) moieties derived from HEK293S GnTI(-) cells; (iv) insect cell-produced HA proteins carrying paucimannosidic N-glycans; and (v) HEK293S GnTI(-) cell-produced HA proteins treated with endoglycosidase H, thus carrying side chains composed of only a single N-acetylglucosamine each. The different HA glycosylation states were confirmed by comparative electrophoretic analysis and by mass spectrometric analysis of released glycans. The immunogenicity of the HA preparations was studied in chickens and mice. The results demonstrate that HA proteins carrying terminal mannose moieties induce significantly lower hemagglutination inhibition antibody titers than HA proteins carrying complex glycans or single N-acetylglucosamine side chains. However, the glycosylation state of the HA proteins did not affect the breadth of the antibody response as measured by an HA1 antigen microarray. We conclude that the glycosylation state of recombinant antigens is a factor of significant importance when developing glycoprotein-based vaccines, such as recombinant HA proteins.

PMID:
22915811
[PubMed - indexed for MEDLINE]
PMCID:
PMC3486279
Free PMC Article
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