Glycan-Dependent Immunogenicity of Recombinant Soluble Trimeric Hemagglutinin

ABSTRACT Recombinant soluble trimeric influenza A virus (IAV) hemagglutinin (sHA3) has proven an effective vaccine antigen against IAV. Here, we investigate to what extent the glycosylation status of the sHA3 glycoprotein affects its immunogenicity. Different glycosylation forms of subtype H5 trimeric HA protein (sH53) 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 sH53 preparations were evaluated: (i) HA proteins carrying complex glycans produced in HEK293T cells; (ii) HA proteins carrying Man9GlcNAc2 moieties, expressed in HEK293T cells treated with kifunensine; (iii) HA proteins containing Man5GlcNAc2 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.

[1]  M. Koopmans,et al.  Profiling of humoral immune responses to influenza viruses by using protein microarray. , 2012, Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases.

[2]  John P. Moore,et al.  Occluding the mannose moieties on human immunodeficiency virus type 1 gp120 with griffithsin improves the antibody responses to both proteins in mice. , 2012, AIDS research and human retroviruses.

[3]  Yan Liu,et al.  A Potent and Broad Neutralizing Antibody Recognizes and Penetrates the HIV Glycan Shield , 2011, Science.

[4]  J. Yewdell,et al.  Fitness costs limit influenza A virus hemagglutinin glycosylation as an immune evasion strategy , 2011, Proceedings of the National Academy of Sciences.

[5]  A. Osterhaus,et al.  Towards universal influenza vaccines? , 2011, Philosophical Transactions of the Royal Society B: Biological Sciences.

[6]  P. Rottier,et al.  Vaccination with a soluble recombinant hemagglutinin trimer protects pigs against a challenge with pandemic (H1N1) 2009 influenza virus. , 2011, Vaccine.

[7]  J. Yewdell,et al.  Glycosylation Focuses Sequence Variation in the Influenza A Virus H1 Hemagglutinin Globular Domain , 2010, PLoS pathogens.

[8]  R. Compans,et al.  Enhanced Immunogenicity of Stabilized Trimeric Soluble Influenza Hemagglutinin , 2010, PloS one.

[9]  Albert D. M. E. Osterhaus,et al.  Recombinant Soluble, Multimeric HA and NA Exhibit Distinctive Types of Protection against Pandemic Swine-Origin 2009 A(H1N1) Influenza Virus Infection in Ferrets , 2010, Journal of Virology.

[10]  B. Bosch,et al.  The influenza A virus hemagglutinin glycosylation state affects receptor-binding specificity. , 2010, Virology.

[11]  E. Steenvoorden,et al.  2‐Picoline‐borane: A non‐toxic reducing agent for oligosaccharide labeling by reductive amination , 2010, Proteomics.

[12]  P. Rottier,et al.  A Single Immunization with Soluble Recombinant Trimeric Hemagglutinin Protects Chickens against Highly Pathogenic Avian Influenza Virus H5N1 , 2010, PloS one.

[13]  Michael Butler,et al.  Expression systems for therapeutic glycoprotein production. , 2009, Current opinion in biotechnology.

[14]  Kay-Hooi Khoo,et al.  Glycans on influenza hemagglutinin affect receptor binding and immune response , 2009, Proceedings of the National Academy of Sciences.

[15]  John P. Moore,et al.  Enzymatic removal of mannose moieties can increase the immune response to HIV-1 gp120 in vivo. , 2009, Virology.

[16]  Chi‐Huey Wong,et al.  The core trisaccharide of an N-linked glycoprotein intrinsically accelerates folding and enhances stability , 2009, Proceedings of the National Academy of Sciences.

[17]  Y. Chau,et al.  DC-SIGN mediates avian H5N1 influenza virus infection in cis and in trans , 2008, Biochemical and Biophysical Research Communications.

[18]  Wei Shi,et al.  Comparative Efficacy of Neutralizing Antibodies Elicited by Recombinant Hemagglutinin Proteins from Avian H5N1 Influenza Virus , 2008, Journal of Virology.

[19]  John P. Moore,et al.  HIV-1 gp120 Mannoses Induce Immunosuppressive Responses from Dendritic Cells , 2007, PLoS pathogens.

[20]  Kimberly B. Ulett,et al.  N-Linked Glycosylation Attenuates H3N2 Influenza Viruses , 2007, Journal of Virology.

[21]  A. Fauci,et al.  HIV-1 gp120 inhibits TLR9-mediated activation and IFN-α secretion in plasmacytoid dendritic cells , 2007, Proceedings of the National Academy of Sciences.

[22]  R. Cummings,et al.  Specificity of DC‐SIGN for mannose‐ and fucose‐containing glycans , 2006, FEBS letters.

[23]  M. Betenbaugh,et al.  Production and N-glycan analysis of secreted human erythropoietin glycoprotein in stably transfected Drosophila S2 cells. , 2005, Biotechnology and bioengineering.

[24]  Claus-Wilhelm von der Lieth,et al.  GlyProt: in silico glycosylation of proteins , 2005, Nucleic Acids Res..

[25]  Roland Contreras,et al.  Structure and function in rhodopsin: High-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N-acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[26]  M. Nussenzweig,et al.  Mannose Receptor-Mediated Regulation of Serum Glycoprotein Homeostasis , 2002, Science.

[27]  M. Ohuchi,et al.  Control of Biological Activities of Influenza Virus Hemagglutinin by Its Carbohydrate Moiety , 1999, Microbiology and immunology.

[28]  H. Klenk,et al.  Regulation of receptor binding affinity of influenza virus hemagglutinin by its carbohydrate moiety , 1997, Journal of virology.

[29]  H. Klenk,et al.  Carbohydrate masking of an antigenic epitope of influenza virus haemagglutinin independent of oligosaccharide size. , 1992, Glycobiology.

[30]  G. Kaushal,et al.  Kifunensine, a potent inhibitor of the glycoprotein processing mannosidase I. , 1990, The Journal of biological chemistry.

[31]  F. Maley,et al.  Characterization of glycoproteins and their associated oligosaccharides through the use of endoglycosidases. , 1989, Analytical biochemistry.

[32]  I. Wilson,et al.  A carbohydrate side chain on hemagglutinins of Hong Kong influenza viruses inhibits recognition by a monoclonal antibody. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[33]  R. J. Webby,et al.  Influenza vaccines. , 2009, Vaccine.

[34]  A. Fauci,et al.  HIV-1 gp120 inhibits TLR9-mediated activation and IFN-{alpha} secretion in plasmacytoid dendritic cells. , 2007, Proceedings of the National Academy of Sciences of the United States of America.

[35]  Arne Skerra,et al.  The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins , 2007, Nature Protocols.

[36]  M. Tate,et al.  Glycosylation as a target for recognition of influenza viruses by the innate immune system. , 2007, Advances in experimental medicine and biology.

[37]  R. Webster,et al.  Glycosylation affects cleavage of an H5N2 influenza virus hemagglutinin and regulates virulence. , 1987, Proceedings of the National Academy of Sciences of the United States of America.