Vaccination of monoglycosylated hemagglutinin induces cross-strain protection against influenza virus infections

Chen, Juine-Ruey; Yu, Yueh‐Hsiang; Tseng, Yeu-Yang; Chiang, Wan-Ling; Chiang, Ming-Feng; Ko, Young-Chai; Chiu, Yi-Kai; Ma, Hsiu-Hua; Wu, Chung‐Yi; Jan, Jia-Tsrong; Lin, Kuo‐I; Ma, Che; Wong, Chi‐Huey

doi:10.1073/pnas.1323954111

Cited by 64 publications

(64 citation statements)

References 25 publications

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“…Thus, our findings have also important practical impact, since they provide basic information that explains the improved ability of HA with simple N -linked oligosaccharides to elicit StRAbs and provide broader immunity to drifted and shifted HAs [56], [57], what is consistent with a temperature-dependent glycan steric hindrance of StRAb binding to mature and viral HA.…”

Section: Discussionsupporting

confidence: 70%

Biogenesis of Influenza A Virus Hemagglutinin Cross-Protective Stem Epitopes

et al. 2014

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Antigenic variation in the globular domain of influenza A virus (IAV) hemagglutinin (HA) precludes effective immunity to this major human pathogen. Although the HA stem is highly conserved between influenza virus strains, HA stem-reactive antibodies (StRAbs) were long considered biologically inert. It is now clear, however, that StRAbs reduce viral replication in animal models and protect against pathogenicity and death, supporting the potential of HA stem-based immunogens as drift-resistant vaccines. Optimally designing StRAb-inducing immunogens and understanding StRAb effector functions require thorough comprehension of HA stem structure and antigenicity. Here, we study the biogenesis of HA stem epitopes recognized in cells infected with various drifted IAV H1N1 strains using mouse and human StRAbs. Using a novel immunofluorescence (IF)-based assay, we find that human StRAbs bind monomeric HA in the endoplasmic reticulum (ER) and trimerized HA in the Golgi complex (GC) with similar high avidity, potentially good news for producing effective monomeric HA stem immunogens. Though HA stem epitopes are nestled among several N-linked oligosaccharides, glycosylation is not required for full antigenicity. Rather, as N-linked glycans increase in size during intracellular transport of HA through the GC, StRAb binding becomes temperature-sensitive, binding poorly to HA at 4°C and well at 37°C. A de novo designed, 65-residue protein binds the mature HA stem independently of temperature, consistent with a lack of N-linked oligosaccharide steric hindrance due to its small size. Likewise, StRAbs bind recombinant HA carrying simple N-linked glycans in a temperature-independent manner. Chemical cross-linking experiments show that N-linked oligosaccharides likely influence StRAb binding by direct local effects rather than by globally modifying the conformational flexibility of HA. Our findings indicate that StRAb binding to HA is precarious, raising the possibility that sufficient immune pressure on the HA stem region could select for viral escape mutants with increased steric hindrance from N-linked glycans.

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Section: Discussionsupporting

confidence: 70%

Biogenesis of Influenza A Virus Hemagglutinin Cross-Protective Stem Epitopes

et al. 2014

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“…In addition, a number of groups have attempted to trigger a broadly neutralizing antibody response toward influenza hemagglutinins, by artificially modulating HA glycosylation (125, 126). Therefore, HA glycosylation is a clear indicator of virus fitness and evolution (11, 13, 21, 122).…”

Section: Resultsmentioning

confidence: 99%

Integrated Omics and Computational Glycobiology Reveal Structural Basis for Influenza A Virus Glycan Microheterogeneity and Host Interactions

Khatri

Klein

White

et al. 2016

Molecular & Cellular Proteomics

114

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Despite sustained biomedical research effort, influenza A virus remains an imminent threat to the world population and a major healthcare burden. The challenge in developing vaccines against influenza is the ability of the virus to mutate rapidly in response to selective immune pressure. Hemagglutinin is the predominant surface glycoprotein and the primary determinant of antigenicity, virulence and zoonotic potential. Mutations leading to changes in the number of HA glycosylation sites are often reported. Such genetic sequencing studies predict at best the disruption or creation of sequons for N-linked glycosylation; they do not reflect actual phenotypic changes in HA structure. Therefore, combined analysis of glycan micro and macro-heterogeneity and bioassays will better define the relationships among glycosylation, viral bioactivity and evolution. We present a study that integrates proteomics, glycomics and glycoproteomics of HA before and after adaptation to innate immune system pressure. We combined this information with glycan array and immune lectin binding data to correlate the phenotypic changes with biological activity. Underprocessed glycoforms predominated at the glycosylation sites found to be involved in viral evolution in response to selection pressures and interactions with innate immune-lectins. To understand the structural basis for site-specific glycan microheterogeneity at these sites, we performed structural modeling and molecular dynamics simulations. We observed that the presence of immature, high-mannose type glycans at a particular site correlated with reduced accessibility to glycan remodeling enzymes. Further, the high mannose glycans at sites implicated in immune lectin recognition were predicted to be capable of forming trimeric interactions with the immune-lectin surfactant protein-D.

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“…However, the complex-type N-glycans of rH5HA proteins produced in CHO cells induced better immunogenicity than insect cell-expressed rH5HA proteins in terms of serum hemagglutination inhibition and neutralization titers, thereby enhancing the protection of mice against lethal H5N1 viral challenges (22). The single-GlcNAc N-glycan forms of rH1HA and rH5HA proteins have also been reported to induce more potent T cells and a more diverse B-cell repertoire, as well as to provide better protective immunity in mice and ferrets (30,31).…”

Section: Discussionmentioning

confidence: 99%

Influenza Virus Hemagglutinin Glycoproteins with Different N-Glycan Patterns Activate Dendritic Cells In Vitro

Liu

Lin

Spearman

et al. 2016

J Virol

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Influenza virus hemagglutinin (HA) N-glycans play important regulatory roles in the control of virus virulence, antigenicity, receptor-binding specificity, and viral escape from the immune response. Considered essential for controlling innate and adaptive immune responses against influenza virus infections, dendritic cells (DCs) trigger proinflammatory and adaptive immune responses in hosts. In this study, we engineered Chinese hamster ovary (CHO) cell lines expressing recombinant HA from pandemic H1, H5, and H7 influenza viruses. rH1HA, rH5HA, and rH7HA were obtained as wild-type proteins or in the presence of kifunensine (KIF) or further with endo-␤-N-acetylglucosaminidase-treated KIF (KIF؉E) to generate single-N-acetylglucosamine (GlcNAc) N-glycans consisting of (i) terminally sialylated complex-type N-glycans, (ii) high-mannose-type N-glycans, and (iii) single-GlcNAc-type N-glycans. Our results show that high-mannose-type and single-GlcNAc-type N-glycans, but not complex-type N-glycans, are capable of inducing more active hIL12 p40, hIL12 p70, and hIL-10 production in human DCs. Significantly higher HLA-DR, CD40, CD83, and CD86 expression levels, as well reduced endocytotic capacity in human DCs, were noted in the high-mannose-type rH1HA and single-GlcNAc-type rH1HA groups than in the complex-type N-glycan rH1HA group. Our data indicate that native avian rHA proteins (H5N1 and H7N9) are more immunostimulatory than human rHA protein (pH1N1). The high-mannose-type or single-GlcNAc-type N-glycans of both avian and human HA types are more stimulatory than the complex-type N-glycans. HA-stimulated DC activation was accomplished partially through a mannose receptor(s). These results provide more understanding of the contribution of glycosylation of viral proteins to the immune responses and may have implications for vaccine development. IMPORTANCEInfluenza viruses trigger seasonal epidemics or pandemics with mild-to-severe consequences for human and poultry populations. DCs are the most potent professional antigen-presenting cells, which play a crucial role in the link between innate and adaptive immunity. In this study, we obtained stable-expression CHO cells to produce rH1HA, rH5HA, and rH7HA proteins containing distinct N-glycan patterns. These rHA proteins, each with a distinct N-glycan pattern, were used to investigate interactions with mouse and human DCs. Our data indicate that native avian rHA proteins (H5N1 and H7N9) are more immunostimulatory than human rHA protein (pH1N1). High-mannose-type and single-GlcNAc-type N-glycans were more effective than complex-type N-glycans in triggering mouse and human DC activation and maturation. We believe these results provide some useful information for influenza vaccine development regarding how influenza virus HA proteins with different types of N-glycans activate DCs.

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Vaccination of monoglycosylated hemagglutinin induces cross-strain protection against influenza virus infections

Cited by 64 publications

References 25 publications

Biogenesis of Influenza A Virus Hemagglutinin Cross-Protective Stem Epitopes

Biogenesis of Influenza A Virus Hemagglutinin Cross-Protective Stem Epitopes

Integrated Omics and Computational Glycobiology Reveal Structural Basis for Influenza A Virus Glycan Microheterogeneity and Host Interactions

Influenza Virus Hemagglutinin Glycoproteins with Different N-Glycan Patterns Activate Dendritic Cells In Vitro

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