Influence of bovine respiratory syncytial virus F glycoprotein N-linked glycans on in vitro expression and on antibody responses in BALB/c mice☆

Klink, Holly A; Brady, Ryan P; Topliff, Christina L.; Eskridge, Kent M.; Srikumaran, Subramaniam; Kelling, Clayton L.

doi:10.1016/j.vaccine.2005.12.067

Cited by 3 publications

(5 citation statements)

References 26 publications

(19 reference statements)

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“…(Figure 1D). F2 contains two N-linked glycans at 27 N and 70 N (numbering based on full length wild type RSV-F sequence), 11 and SDS-PAGE suggested only one glycan was lost (Figure 1A, right panel and Figure 1B). A mass spectrometric analysis was used to pinpoint exact site of glycan loss.…”

Section: ■ Resultsmentioning

confidence: 99%

“…The site of observed glycan shedding was immediately adjacent to one of the amino acid sequences that constitute site Ø . Klink et al showed that deletion of 70 N glycan in bovine RSV resulted in higher immune response compared to fully glycosylated wild-type . Zimmer et al found that loss of 70 N glycosylation led to significantly increased fusion activity .…”

Section: Discussionmentioning

confidence: 99%

“…13 Klink et al showed that deletion of 70 N glycan in bovine RSV resulted in higher immune response compared to fully glycosylated wild-type. 11 Zimmer et al found that loss of 70 N glycosylation led to significantly increased fusion activity. 18 Glycans are generally considered as low in immunogenicity.…”

Section: ■ Discussionmentioning

confidence: 99%

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Non-Enzymatic and Site-Specific Glycan Shedding: A Novel Protein Degradation Pathway Observed in a Stabilized Form of RSV Prefusion F Protein

Qian

Yearley

Tian³

et al. 2018

Anal. Chem.

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Stability is one of the critical attributes of a protein-based therapeutic or vaccine product, which is directly linked to product quality and efficacy. Elucidating protein degradation pathways is required to obtain thorough understanding of the product and ensure degradation products are properly monitored. We observed a unique protein degradation involving nonenzyme catalyzed loss of a complete N-linked glycan under stress condition from an engineered respiratory syncytial virus (RSV) prefusion F protein (RSVPreF3). Investigations involving mass spectrometry, molecular modeling, and mutagenesis revealed that the glycan shedding was site-specific, dependent on structural elements, and required a glycine residue immediately following the site of glycosylation. The glycan loss did not negatively affect the binding between the main immunogenic epitope Site Ø and the neutralizing antibody D25. Further study indicated that the glycan shedding followed a similar but different mechanism than that of conventional deamidation. Since glycosylation is an important attribute for many recombinant therapeutic proteins or vaccine antigens, the finding from this study suggests the need to monitor this new type of degradation, especially when glycosylation has an impact on efficacy or safety.

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Section: ■ Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Non-Enzymatic and Site-Specific Glycan Shedding: A Novel Protein Degradation Pathway Observed in a Stabilized Form of RSV Prefusion F Protein

Qian

Yearley

Tian³

et al. 2018

Anal. Chem.

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“…Additionally, the immunogenicity of viral glycoproteins is often determined by their glycosylation profile. The attachment of N-linked glycans can affect the recognition of antibodies by shielding antigenic sites on the protein [ 24 , 25 , 26 , 27 , 28 , 29 ]. For example, N-linked glycans might shield up to 52% of the RSV F protein surface for antibody recognition [ 30 ].…”

Section: Introductionmentioning

confidence: 99%

“…In the context of DNA vaccines or live-attenuated vaccines (LAVs), this approach was already broadly investigated for multiple viruses [ 25 , 27 , 29 , 31 , 32 ]. Interestingly, mice immunized with a specific bRSV F glycomutant showed higher antibody responses compared with the wild type (WT) bRSV F protein [ 24 ]. However, it is not clear how the individual N-linked glycans impact the immunogenicity of the hRSV fusion protein and to which extent the observations of the glycomutant bRSV F protein immunizations can be extrapolated to hRSV F.…”

Section: Introductionmentioning

confidence: 99%

Removal of the N-Glycosylation Sequon at Position N116 Located in p27 of the Respiratory Syncytial Virus Fusion Protein Elicits Enhanced Antibody Responses after DNA Immunization

Leemans

Boeren

Gucht

et al. 2018

Viruses

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Prevention of severe lower respiratory tract infections in infants caused by the human respiratory syncytial virus (hRSV) remains a major public health priority. Currently, the major focus of vaccine development relies on the RSV fusion (F) protein since it is the main target protein for neutralizing antibodies induced by natural infection. The protein conserves 5 N-glycosylation sites, two of which are located in the F2 subunit (N27 and N70), one in the F1 subunit (N500) and two in the p27 peptide (N116 and N126). To study the influence of the loss of one or more N-glycosylation sites on RSV F immunogenicity, BALB/c mice were immunized with plasmids encoding RSV F glycomutants. In comparison with F WT DNA immunized mice, higher neutralizing titres were observed following immunization with F N116Q. Moreover, RSV A2-K-line19F challenge of mice that had been immunized with mutant F N116Q DNA was associated with lower RSV RNA levels compared with those in challenged WT F DNA immunized animals. Since p27 is assumed to be post-translationally released after cleavage and thus not present on the mature RSV F protein, it remains to be elucidated how deletion of this glycan can contribute to enhanced antibody responses and protection upon challenge. These findings provide new insights to improve the immunogenicity of RSV F in potential vaccine candidates.

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The RSV F p27 peptide: current knowledge, important questions

et al. 2023

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Respiratory syncytial virus (RSV) remains a leading cause of hospitalizations and death for young children and adults over 65. The worldwide impact of RSV has prioritized the search for an RSV vaccine, with most targeting the critical fusion (F) protein. However, questions remain about the mechanism of RSV entry and RSV F triggering and fusion promotion. This review highlights these questions, specifically those surrounding a cleaved 27 amino acids long peptide within F, p27.

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Influence of bovine respiratory syncytial virus F glycoprotein N-linked glycans on in vitro expression and on antibody responses in BALB/c mice☆

Cited by 3 publications

References 26 publications

Non-Enzymatic and Site-Specific Glycan Shedding: A Novel Protein Degradation Pathway Observed in a Stabilized Form of RSV Prefusion F Protein

Non-Enzymatic and Site-Specific Glycan Shedding: A Novel Protein Degradation Pathway Observed in a Stabilized Form of RSV Prefusion F Protein

Removal of the N-Glycosylation Sequon at Position N116 Located in p27 of the Respiratory Syncytial Virus Fusion Protein Elicits Enhanced Antibody Responses after DNA Immunization

The RSV F p27 peptide: current knowledge, important questions

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