Glycan-dependent immunogenicity of recombinant soluble trimeric hemagglutinin.
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.
Virology Division, Department of Infectious Diseases and Immunology, Utrecht University, Utrecht, The Netherlands.
Robert P
RP
de Vries
Cornelis H
CH
Smit
Erwin
E
de Bruin
Alan
A
Rigter
Erik
E
de Vries
Lisette A H M
LA
Cornelissen
Dirk
D
Eggink
Nancy P Y
NP
Chung
John P
JP
Moore
Rogier W
RW
Sanders
Cornelis H
CH
Hokke
Marion
M
Koopmans
Peter J M
PJ
Rottier
Cornelis A M
CA
de Haan
D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D002460:Cell Line; D002645:Chickens; D004586:Electrophoresis; D005260:Female; D006385:Hemagglutination Inhibition Tests; D006388:Hemagglutinins / Q000737:chemistry* / Q000276:immunology*; D006801:Humans; D009980:Influenza A virus / Q000276:immunology*; D007313:Insecta; D013058:Mass Spectrometry; D051379:Mice; D008810:Mice, Inbred C57BL; D011134:Polysaccharides / Q000032:analysis*; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology
D000914:Antibodies, Viral; D006388:Hemagglutinins; D011134:Polysaccharides; D011994:Recombinant Proteins