Evaluating the Impact of N-Glycan Sequon Removal in the p27 Peptide on RSV F Protein Immunogenicity and Functionality

Abstract

Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infections in young children, elderly and immunocompromised patients worldwide. The RSV fusion (F) protein, which has 5-6 N-glycosylation sites depending on the strain, is a major target for vaccine development. Two to three of these sites are located in the p27 peptide, which is considered absent in virions. Prior research from our group showed that removing the N-glycan at position 116 (N116) in p27 led to higher neutralizing antibody responses and better protection against RSV. In this study, the effect of single, double and triple N-glycan deletion mutations in F p27 was evaluated. Surprisingly, all mutants exhibited similar expressions and functionality to the wild-type F protein. All F p27 glycomutants induced neutralizing antibodies and lowered lung viral loads after an RSV challenge in a mouse model. Although N-glycans in p27 influence immune responses, their exact role in RSV biology remains unclear. Possibly, these glycans, which are mostly conserved, play a role in other aspects of virus replication and biology.

Keywords: DNA immunization; N-glycosylation; RSV; fusion protein; p27 peptide.

Figure 1

Immunofluorescence staining of cell surface expression of F p27 glycomutants. BSR T7/5 cells were transfected with pCAXL plasmid DNA encoding RSV F p27 glycosylation mutants. The nuclei were stained with DAPI (blue). F proteins are visualized with polyclonal goat anti-RSV antibodies and secondary donkey anti-goat IgG (AF488) (green). Images were acquired through confocal fluorescence microscopy (scale bar = 12 µM).

Figure 2

Fusion assay assessing the effect of loss of RSV F p27 N-glycans on syncytium formation. (A) Fluorescent images of the respective F p27 glycomutants. Nuclei were stained with DAPI (blue) and RSV F proteins were stained with palivizumab and secondary goat anti-human IgG AF488 (green) (scale bar = 50 µm). (B) Syncytium frequency. (C) Mean syncytium size. Data represent the mean ± SD of three independent repeats. * p < 0.05. (one-way ANOVA).

Figure 3

Total and neutralizing antibody responses. Two subsequent immunizations of the indicated F DNA constructs were intramuscularly administered to BALB/c mice. Serum was collected 3 weeks after prime immunization (A) and 2 weeks after boost immunization but before the challenge (B). Total (left) and neutralizing (right) antibody titers were determined. The dotted line represents the detection limit. ** p < 0.01. **** p < 0.0001 (one-way ANOVA).

Figure 4

Total (A) and neutralizing (B) antibody responses and corresponding lung viral loads (C) after challenging BALB/c mice previously immunized with DNA. Mice were intramuscularly immunized twice with the indicated plasmids (prime immunization on day 0 and boost immunization on day 21). Five days post RSV challenge (day 61), sera were collected, and the total (A) and neutralizing (B) antibody titers were determined. Five days post challenge with 1 × 10⁶ PFU of RSV A2-K-line19F, the lungs were collected and homogenized. The relative RNA levels (C) in the infected lungs were determined via RT-qPCR. The dotted line represents the detection limit (A,B). * p < 0.05. **** p < 0.0001 (one-way ANOVA).

Figure 5

Cross-neutralization of post-challenge serum. Mice were intramuscularly immunized twice with the indicated plasmids (prime immunization on day 0 and boost immunization on day 21). Five days post RSV challenge (day 61), serum was collected, and the cross-neutralization capacities were determined with PRNTs. Dotted lines represent limit of detection (one-way ANOVA).