biotinylated spike protein  (R&D Systems)

Journal: iScience

Article Title: HKU1 immune imprinting is associated with post-COVID symptoms after SARS-CoV-2 infection

doi: 10.1016/j.isci.2026.115175

Figure Lengend Snippet: SARS-CoV-2 vaccination boosts pre-existing antibody reactivity to human coronaviruses (HCoVs) (A) Timeline of vaccine administration and peripheral blood sampling in the first (healthcare workers) cohort. (B) IgG responses to the spike protein of SARS-CoV-2 and HCoVs (OC43, HKU1, NL63, and 229E) expressed as arbitrary units per milliliter (AU/mL), shown as box (median, with lower/higher quartiles)-and-whiskers (upper whisker: Q3+1.5xIQR, lower whisker: Q1-1.5xIQR) plots, with all data points. (C) Relative IgG avidity profiles against SARS-CoV-2 and HCoV spike, across time points, with LOESS-smoothed trend lines, showing all data points and 95% confidence intervals (gray shade). (D) Corresponding avidity ranges for SARS-CoV-2 and HCoV spike antigen, shown as box-and-whisker plots (similarly defined as in [B]), with all data points. Paired comparisons were conducted between baseline and 14 days after the first vaccine dose, and between 28 days after the first dose and 14 days after the second dose, using a mixed-effects model (∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001). Sample sizes: baseline ( n = 48), 14 days post-first dose ( n = 46), 28 days post-first dose ( n = 35), 14 days post-second dose ( n = 15), and 28 days post-second dose ( n = 11).

Article Snippet: Ten μL of yellow-green fluorescent NeutrAvidin-labeled microspheres (Thermo Fisher Scientific, Waltham, MA, USA; cat# F8776) were washed with PBS containing 0.1% BSA, resuspended in 10 μL of biotinylated SARS-CoV-2 S1, S2 (ACROBiosystems, cat# S1N-C82E8 and S2N-C52E8), HKU1 (Sino Biological, cat# 40606-V08B-B), and NL63 (Sino Biological, cat# 40604-V08B-B) spike proteins (0.1 μg/μL), and incubated at 36 °C for 2 h. Microspheres were washed twice, resuspended in 1 mL PBS, and aliquoted (10 μL) into a 96-well plate.

Techniques: Sampling, Whisker Assay

Journal: iScience

Article Title: HKU1 immune imprinting is associated with post-COVID symptoms after SARS-CoV-2 infection

doi: 10.1016/j.isci.2026.115175

Figure Lengend Snippet: Recalled HKU1 spike antibody responses display enhanced ADCP function (A) Antibody-dependent cellular phagocytosis (ADCP) responses against HCoV spike and SARS-CoV-2 S1- and S2-domains across vaccine doses and time points. ADCP scores were compared between baseline and 14 days post-first dose, and between 28 days post-first dose and 14 days post-second dose, using a mixed-effects model. Data are shown as box (median, with lower/higher quartiles)-and-whiskers (upper whisker: Q3+1.5xIQR, lower whisker: Q1-1.5xIQR) plots, with all data points. (B) Spearman correlations between HKU1 and NL63 spike, and SARS-CoV-2 S1- and S2-domain-specific ADCP responses, adjusted for multiple comparisons with the Benjamini-Hochberg method. Spearman correlations are shown as a heatmap for each time point. (only significant results are shown; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001). Sample sizes: baseline ( n = 48), 14 days post-first dose ( n = 46), 28 days post-first dose ( n = 35), 14 days post-second dose ( n = 15), and 28 days post-second dose ( n = 11).

Article Snippet: Ten μL of yellow-green fluorescent NeutrAvidin-labeled microspheres (Thermo Fisher Scientific, Waltham, MA, USA; cat# F8776) were washed with PBS containing 0.1% BSA, resuspended in 10 μL of biotinylated SARS-CoV-2 S1, S2 (ACROBiosystems, cat# S1N-C82E8 and S2N-C52E8), HKU1 (Sino Biological, cat# 40606-V08B-B), and NL63 (Sino Biological, cat# 40604-V08B-B) spike proteins (0.1 μg/μL), and incubated at 36 °C for 2 h. Microspheres were washed twice, resuspended in 1 mL PBS, and aliquoted (10 μL) into a 96-well plate.

Techniques: Whisker Assay

Journal: iScience

Article Title: HKU1 immune imprinting is associated with post-COVID symptoms after SARS-CoV-2 infection

doi: 10.1016/j.isci.2026.115175

Figure Lengend Snippet: HKU1 antibody reactivity was protective against SARS-CoV-2 infection but increased the risk of post-COVID symptoms in vaccinated, infection-naive individuals (A) Comparison of HCoV spike IgG levels (HKU1, OC43, NL63, and 229E; log-transformed data) at baseline, between individuals infected and those who were not at the one-year follow-up. Comparisons were performed using a Wilcoxon rank-sum test ( n = 700, infected = 509, uninfected = 191). (B) Effects of HCoV spike IgG levels, time interval between the first and second vaccine dose (days), use of mRNA vaccines only, sex, and age at baseline, on the risk of SARS-CoV-2 infection at the one-year follow-up. (C) Comparison of HCoV spike IgG levels at baseline, between individuals who developed post-COVID symptoms versus those who did not, by the one-year follow-up visit. Comparisons were performed using a Wilcoxon rank-sum test ( n = 509, post-COVID symptoms = 165, no post-COVID symptoms = 335; missing data = 9). (D) Effects of HCoV spike IgG levels, time interval between the first and second dose (days), use of mRNA vaccines only, sex, and age at baseline, on the risk of post-COVID symptoms at the one-year follow-up. (B and D) Show adjusted odds ratios with 95% confidence intervals on a logarithmic scale (in blue) where the dashed horizontal line indicates no effect (OR = 1), with corresponding p -values (in red). (E–G) Antibody-dependent complement deposition (ADCD) between individuals with the highest (78 th centile, n = 88) and lowest (22 nd centile, n = 88) HKU1 spike (E), SARS-CoV-2 S1 (F) and S2 (G) IgG levels. Comparisons were performed using a Wilcoxon rank-sum test. For (A, C, E–G) data are shown as box (median, with lower/higher quartiles)-and-whiskers (upper whisker: Q3+1.5xIQR, lower whisker: Q1-1.5xIQR) plots, with all data points. For (B, D): Odds Ratios (OR) with 95% confidence intervals (blue line) and p value (in red), on a log scale. (∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001).

Article Snippet: Ten μL of yellow-green fluorescent NeutrAvidin-labeled microspheres (Thermo Fisher Scientific, Waltham, MA, USA; cat# F8776) were washed with PBS containing 0.1% BSA, resuspended in 10 μL of biotinylated SARS-CoV-2 S1, S2 (ACROBiosystems, cat# S1N-C82E8 and S2N-C52E8), HKU1 (Sino Biological, cat# 40606-V08B-B), and NL63 (Sino Biological, cat# 40604-V08B-B) spike proteins (0.1 μg/μL), and incubated at 36 °C for 2 h. Microspheres were washed twice, resuspended in 1 mL PBS, and aliquoted (10 μL) into a 96-well plate.

Techniques: Infection, Comparison, Transformation Assay, Vaccines, Whisker Assay

Journal: iScience

Article Title: HKU1 immune imprinting is associated with post-COVID symptoms after SARS-CoV-2 infection

doi: 10.1016/j.isci.2026.115175

Figure Lengend Snippet: SARS-CoV-2 vaccination boosts pre-existing antibody reactivity to human coronaviruses (HCoVs) (A) Timeline of vaccine administration and peripheral blood sampling in the first (healthcare workers) cohort. (B) IgG responses to the spike protein of SARS-CoV-2 and HCoVs (OC43, HKU1, NL63, and 229E) expressed as arbitrary units per milliliter (AU/mL), shown as box (median, with lower/higher quartiles)-and-whiskers (upper whisker: Q3+1.5xIQR, lower whisker: Q1-1.5xIQR) plots, with all data points. (C) Relative IgG avidity profiles against SARS-CoV-2 and HCoV spike, across time points, with LOESS-smoothed trend lines, showing all data points and 95% confidence intervals (gray shade). (D) Corresponding avidity ranges for SARS-CoV-2 and HCoV spike antigen, shown as box-and-whisker plots (similarly defined as in [B]), with all data points. Paired comparisons were conducted between baseline and 14 days after the first vaccine dose, and between 28 days after the first dose and 14 days after the second dose, using a mixed-effects model (∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001). Sample sizes: baseline ( n = 48), 14 days post-first dose ( n = 46), 28 days post-first dose ( n = 35), 14 days post-second dose ( n = 15), and 28 days post-second dose ( n = 11).

Article Snippet: For HKU1-specific B cells, biotinylated HKU1 spike proteins were purchased from Sino Biological, Beijing, China (cat# 40606-V08B-B).

Techniques: Sampling, Whisker Assay

Journal: iScience

Article Title: HKU1 immune imprinting is associated with post-COVID symptoms after SARS-CoV-2 infection

doi: 10.1016/j.isci.2026.115175

Figure Lengend Snippet: HKU1 immune imprinting predominantly targets the SARS-CoV-2 S2 domain Percentages of antigen-specific memory B cells, IgG + B cells, or plasmablasts calculated as the number of antigen-specific cells within each subset divided by total B cells, IgG + B cells, or plasmablasts, respectively, across time points (flow cytometry). Log10-transformed data are shown as box (median, with lower/higher quartiles)-and-whiskers (upper whisker: Q3+1.5xIQR, lower whisker: Q1-1.5xIQR) plots, with all data points, and LOESS-smoothed trend lines with 95% confidence intervals (gray shade). (A–C) Percentage of S1 and S2-specific cells within memory and IgG+ memory B cells, and plasmablasts. Comparisons were performed between S1 and S2 groups using a Wilcoxon signed-rank test. (D–F) Percentages of HKU1-specific B cells within total and IgG+ memory B cells, and plasmablasts. Comparisons assessed between sequential vaccine time point pairs, using mixed-effects models (only significant results are shown; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001). Sample sizes: baseline ( n = 20); 14 days ( n = 30) and 28 days post-first dose ( n = 19); and 14 days ( n = 22) and 28 days post-second dose ( n = 18).

Article Snippet: For HKU1-specific B cells, biotinylated HKU1 spike proteins were purchased from Sino Biological, Beijing, China (cat# 40606-V08B-B).

Techniques: Flow Cytometry, Transformation Assay, Whisker Assay

Journal: iScience

Article Title: HKU1 immune imprinting is associated with post-COVID symptoms after SARS-CoV-2 infection

doi: 10.1016/j.isci.2026.115175

Figure Lengend Snippet: Recalled HKU1 spike antibody responses display enhanced ADCP function (A) Antibody-dependent cellular phagocytosis (ADCP) responses against HCoV spike and SARS-CoV-2 S1- and S2-domains across vaccine doses and time points. ADCP scores were compared between baseline and 14 days post-first dose, and between 28 days post-first dose and 14 days post-second dose, using a mixed-effects model. Data are shown as box (median, with lower/higher quartiles)-and-whiskers (upper whisker: Q3+1.5xIQR, lower whisker: Q1-1.5xIQR) plots, with all data points. (B) Spearman correlations between HKU1 and NL63 spike, and SARS-CoV-2 S1- and S2-domain-specific ADCP responses, adjusted for multiple comparisons with the Benjamini-Hochberg method. Spearman correlations are shown as a heatmap for each time point. (only significant results are shown; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001). Sample sizes: baseline ( n = 48), 14 days post-first dose ( n = 46), 28 days post-first dose ( n = 35), 14 days post-second dose ( n = 15), and 28 days post-second dose ( n = 11).

Article Snippet: For HKU1-specific B cells, biotinylated HKU1 spike proteins were purchased from Sino Biological, Beijing, China (cat# 40606-V08B-B).

Techniques: Whisker Assay

Journal: iScience

Article Title: HKU1 immune imprinting is associated with post-COVID symptoms after SARS-CoV-2 infection

doi: 10.1016/j.isci.2026.115175

Figure Lengend Snippet: HKU1 antibody reactivity was protective against SARS-CoV-2 infection but increased the risk of post-COVID symptoms in vaccinated, infection-naive individuals (A) Comparison of HCoV spike IgG levels (HKU1, OC43, NL63, and 229E; log-transformed data) at baseline, between individuals infected and those who were not at the one-year follow-up. Comparisons were performed using a Wilcoxon rank-sum test ( n = 700, infected = 509, uninfected = 191). (B) Effects of HCoV spike IgG levels, time interval between the first and second vaccine dose (days), use of mRNA vaccines only, sex, and age at baseline, on the risk of SARS-CoV-2 infection at the one-year follow-up. (C) Comparison of HCoV spike IgG levels at baseline, between individuals who developed post-COVID symptoms versus those who did not, by the one-year follow-up visit. Comparisons were performed using a Wilcoxon rank-sum test ( n = 509, post-COVID symptoms = 165, no post-COVID symptoms = 335; missing data = 9). (D) Effects of HCoV spike IgG levels, time interval between the first and second dose (days), use of mRNA vaccines only, sex, and age at baseline, on the risk of post-COVID symptoms at the one-year follow-up. (B and D) Show adjusted odds ratios with 95% confidence intervals on a logarithmic scale (in blue) where the dashed horizontal line indicates no effect (OR = 1), with corresponding p -values (in red). (E–G) Antibody-dependent complement deposition (ADCD) between individuals with the highest (78 th centile, n = 88) and lowest (22 nd centile, n = 88) HKU1 spike (E), SARS-CoV-2 S1 (F) and S2 (G) IgG levels. Comparisons were performed using a Wilcoxon rank-sum test. For (A, C, E–G) data are shown as box (median, with lower/higher quartiles)-and-whiskers (upper whisker: Q3+1.5xIQR, lower whisker: Q1-1.5xIQR) plots, with all data points. For (B, D): Odds Ratios (OR) with 95% confidence intervals (blue line) and p value (in red), on a log scale. (∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001).

Article Snippet: For HKU1-specific B cells, biotinylated HKU1 spike proteins were purchased from Sino Biological, Beijing, China (cat# 40606-V08B-B).

Techniques: Infection, Comparison, Transformation Assay, Vaccines, Whisker Assay