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. 2024 Nov 20;9(12):3254–3267. doi: 10.1038/s41564-024-01860-1

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, 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 licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

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Fig. 4 — a, Analytical SEC analysis of RSV-A and RSV-B F protein with or without foldon, as determined in cell supernatant. RSV-A F carries substitutions N67I, S215P and D486N. RSV-B F carries substitutions P101Q, I152M, L203I, S215P, D486N and D489Y. Trimeric (T) or monomeric (M) F is indicated. b, PreF expression and stability depicted as trimer peaks in analytical SEC at day 0 versus the CR9501 binding ratio at day 0 and 6. Stabilizing substitutions were screened in a semi-stable consensus backbone (‘BB’; RSV-A F with foldon and D486N). Substitutions in blue were selected for follow-up. c, Analytical SEC on RSV-A and RSV-B F without a foldon (Δfoldon) in cell supernatant. RSV-A Δfoldon contains P101Q, S215P, Q354L, D486N, E487L and D489Y. RSV-B Δfoldon contains P101Q, I152M, L203I, S215P, Q354L, D486N, E487L and D489Y. T is indicated. d, Stability of indicated variants as determined by analytical SEC on supernatants after 15 min of incubation at indicated temperatures. The backbone is RSV-A Δfoldon described in c. e, Stability of indicated variants as determined by analytical SEC on supernatants stored for 0 or 16 days at 4 °C. The backbone is RSV-B Δfoldon described in c. f, SEC-MALS (~162 kDa) of RSV-A F Δfoldon trimers with stabilizing substitutions P101Q, S215P, Q354L, D486N, E487L, D489Y, F505W and S509F. Variants contain either a short HR2 (residues 1–513) or a long HR2 (residues 1–524). g, Melting temperature (Tm₅₀) of RSV-A F Δfoldon variants with a short or long HR2 sequence, as described in f, determined by differential scanning fluorimetry (DSF). Data reported as the average (blue line) of n = 3 technical replicates (grey lines). h, Antigenicity profile of RSV-A F Δfoldon variants with a short or long HR2, as described in f, measured by BLI. RSV postF protein is included as a control⁵². Antibody specificity is indicated. The binding rate is reported as the average of three technical replicates, and individual data points are represented by open circles.

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