Dendritic Cell Activation by Recombinant Hemagglutinin Proteins of H1N1 and H5N1 Influenza A Viruses

Liu, Wen-Chun; Lin, S. H.; Yu, Y. L.; Chu, Chih‐Liang; Wu, Suh-Chin

doi:10.1128/jvi.01316-10

Cited by 36 publications

(38 citation statements)

References 44 publications

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“…Also, compared to complex-type N-glycans from rH1HA, reduced endocytotic strengths were observed in human mDCs induced by complextype N-glycans from rH5HA and rH7HA, suggesting that they may trigger DC activation and/or maturation more efficiently than complex-type N-glycans from rH1HA. Our previous results suggested that HA alone is responsible for this activation via the TLR4/MyD88 pathway (21). However, HA does not exist as a single entity in vivo and it is unclear if this activation has any relevance during virus infection.…”

Section: Discussionmentioning

confidence: 98%

“…mDCs were generated from the bone marrow of C57BL/6 mice as described previously (21). All procedures involving animals were conducted in accordance with guidelines established by the Laboratory Animal Center of National Tsing Hua University (NTHU).…”

Section: Methodsmentioning

confidence: 99%

“…For mouse mDC cytokine production, TNF-␣ produced by mouse mDCs was detected by intracellular staining as previously described (21). Briefly, DCs were treated with phosphate-buffered saline (PBS), 100 ng/ml lipopolysaccharide (LPS; from Escherichia coli O111:B4; Sigma), and 10 or 50 g/ml rHA (H1, H5, or H7) or KIF-rHA (H1, H5, or H7) with or without endo H for 6 h. Brefeldin A (10 g/ml; BioLegend) was added for the final 4 h. Cells were fixed, permeabilized, stained with a mouse anti-TNF-␣ monoclonal antibody (BioLegend), and analyzed with a flow cytometer and Accuri C6 software (BD Biosciences).…”

Section: Methodsmentioning

confidence: 99%

“…The rH1HA, rH5HA, and rH7HA glycoproteins all contained complex-type N-glycans with bi-, tri-, and tetra-antennary structures with one or more terminal sialic acids (Sia [1][2][3][4] Mouse mDC activation and maturation by rH1HA, rH5HA, and rH7HA proteins containing different N-glycans. Purified rHA proteins containing complex-type, high-mannose-type (KIF-treated), or single-GlcNAc-type (KIFϩE-treated) N-glycans were added to cultured, bone marrow-derived mDCs isolated from mice as described previously (21). Flow cytometry was used to measure intracellular TNF-␣ and I Ab (major histocompatibility complex class II), CD40, and CD86 expression levels.…”

Section: Usementioning

confidence: 99%

“…Highly pathogenic avian H5N1 viruses can induce productive infections in human mDCs and mouse primary lung DCs (18), whereas H7N9 viruses only result in impaired IFN production in infected human mDCs (20). We previously reported that recombinant HA proteins from H5N1 and pH1N1 influenza viruses are capable of triggering mouse mDC activation and maturation (21). For this study, we used Chinese hamster ovary (CHO) cell expression to obtain rH1HA, rH5HA, and rH7HA proteins consisting of (i) terminally sialylated complex-type N-glycans, (ii) highmannose-type N-glycans, and (iii) single-N-acetylglucosamine (GlcNAc)-type N-glycans.…”

mentioning

confidence: 99%

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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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Section: Discussionmentioning

confidence: 98%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Usementioning

confidence: 99%

mentioning

confidence: 99%

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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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Glycan‐masking hemagglutinin antigens from stable CHO cell clones for H5N1 avian influenza vaccine development

Chen

Liu

Lin

et al. 2018

Biotech & Bioengineering

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Refocusing of B cell responses can be achieved by preserving the overall fold of the antigen structure but selectively mutating the undesired antigenic sites with additional N-linked glycosylation motifs for glycan-masking the vaccine antigen. We previously reported that glycan-masking recombinant H5 hemagglutin (rH5HA) antigens on residues 83, 127, and 138 (g127+g138 or g83+g127+138 rH5HA) elicited broader neutralizing antibodies and protection against heterologous clades/subclades of high pathogenic avian influenza H5N1 viruses. In this study, we engineered the stably-expressing CHO cell clones for producing the glycan-masking g127+g138 and g83+g127+g138 rH5HA antigens. All of these glycan-masking rH5HA antigens produced in stable CHO cell clones were found to be mostly oligomeric structures. Only the immunization with the glycan-masking g127+g138 but not g83+g127+g138 rH5HA antigens elicited more potent neutralizing antibody titers against four out of five heterologous clades/subclades of H5N1 viral strains. The increased neutralizing antibody titers against these heterologous viral strains were correlated with the increased amounts of stem-binding antibodies, only the glycan-masking g127+g138 rH5HA antigens can translate into more protection against live viral challenges. The stable CHO cell line-produced glycan-masking g127+g138 rH5HA can be used for H5N1 subunit vaccine development. This article is protected by copyright. All rights reserved.

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Influenza Virosomes as Antigen Delivery System

Moser¹,

Amacker²

2012

Novel Immune Potentiators and Delivery Technologies for Next Generation Vaccines

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Dendritic Cell Activation by Recombinant Hemagglutinin Proteins of H1N1 and H5N1 Influenza A Viruses

Cited by 36 publications

References 44 publications

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

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

Glycan‐masking hemagglutinin antigens from stable CHO cell clones for H5N1 avian influenza vaccine development

Influenza Virosomes as Antigen Delivery System

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