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. 2023 Apr 12;14:2081. doi: 10.1038/s41467-023-37697-1

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — A Illustration of construction of DelNS1-RBD4N-DAF. B Immunofluorescence (IF) assay of RBD expression with or without DAF in MDCK cells. MDCK cells were mock infected or infected with DelNS1, DelNS1-RBD or DelNS1-RBD-DAF viruses in the backbone of A/California/4/2009 (H1N1)¹⁶ at an MOI of 1 for 10 h and cells processed for IFA using antibodies specific for RBD (red). Nuclei were stained with DAPI (blue). Images are representative of three independent experiments. C Confirmation of N-glycosylation in RBD of DelNS1-RBD4N-DAF LAIV. MDCK cells were mock infected or infected with DelNS1-RBD (WT, lineage A virus), DelNS1-RBD with individual N-glycosylation mutations (A372T, G413N, D428N, or P521N), or DelNS1-RBD4N (4N) LAIV virus at an MOI of 1 for 10 h. Cell lysates were analyzed by western blot using anti-RBD antibody and anti-NP antibody. DelNS1-RBD, DelNS1-RBD4N, and DelNS1 LAIVs were also used to infect A549, BHK21 and MDCK cells to detect the expression of RBD. Images are representative of three independent experiments. D Schedule of immunization and blood collection for BALB/c mice. E Estimation of anti-RBD antibodies in mice prime-boost immunized intranasally with 2 × 10⁶ pfu of DelNS1-RBD-DAF with wild-type (WT) SARS-CoV-2 strain (lineage A) RBD (RBD_WT-DAF, n = 4) or DelNS1-RBD with WT-RBD (RBD_WT, n = 5) or DelNS1 vector (n = 5). At weeks 4 and week 6, blood was collected from mice and tested for anti-S1 RBD-specific IgG titers by ELISA assay and neutralization activity against pseudovirus expressing wild-type SARS-CoV-2 spike protein. F Estimation of anti-RBD antibodies in mice prime-boost immunized intranasally with 2 × 10⁶ pfu of DelNS1-RBD-DAF with Delta (B.1.617.2) RBD (Delta-DAF, n = 8) or Beta (B.1.351) RBD (Beta-DAF, n = 8), or DelNS1-RBD4N-DAF with Delta RBD4N (Delta4N-DAF, n = 8) or Beta RBD4N (Beta4N-DAF, n = 8), or DelNS1 vector (n = 8). At week 6, blood was collected from mice and tested for anti-S1 RBD-specific IgG titers and neutralization activity against pseudoviruses expressing spike proteins of SARS-CoV-2 variants. LOD: lower limit of detection. Error bars represent mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Dunn’s multiple comparisons test: ****p < 0.0001, **p < 0.01, *p < 0.05, ns not significant. Numerical labels indicate fold difference. Mouse cartoon created with BioRender.com.