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

Abstract

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.

N-Linked Glycan Sites on the Influenza A Virus Neuraminidase Head Domain Are Required for Efficient Viral Incorporation and Replication

“…However, three of the conserved N2 head domain sites contain the N-X-T sequence (Asn146, Asn200, and Asn234), and the gel shifts that were observed indicate the different sites are generally recognized. These observations suggest that the individual sites could provide a subtle increase in the replication efficiency of H1N1 IAVs that was not detected by our assays, but it is equally plausible that the conserved sites provide a growth or immunogenicity advantage in vivo, which has been reported for several other viral glycoproteins (50)(51)(52).…”

Antibody Response to the Furin Cleavable Twenty-Seven Amino Acid Peptide (p27) of the Fusion Protein in Respiratory Syncytial Virus (RSV) Infected Adult Hematopoietic Cell Transplant (HCT) Recipients

“…Therefore, investigators usually assume that RSV p27 is not present on the mature RSV F protein. However, immunization of mice with the F N116Q DNA containing a mutation of the N-glycosylation sequon located within the p27 domain of the RSV F protein elicited enhanced antibody responses and viral protection upon viral challenge [30]. This finding suggests that the removal of N116 glycan unmasked an epitope possibly within the p27 domain that augmented protective antibodies against RSV infection in mice.…”

Isolation and Characterization of Clinical RSV Isolates in Belgium during the Winters of 2016–2018

“…The F protein is indispensable for virus infection [39] and its activity can be linked to the formation of syncytia [36,40]. Mean syncytium size and mean syncytium frequency were determined after 48h of infection with a 0.6% Avicel ®® overlay to minimize free particle movement.…”

“…The plaque reduction assay was performed as described by Leemans A. et al [36]. Briefly, HEp-2 cells were seeded at a concentration of 17,500 cells/well in a clear 96-well plate (Falcon) 24 h prior to inoculation.…”