Journal: Scientific Reports

Article Title: Utility of nasal swabs for assessing mucosal immune responses towards SARS-CoV-2

doi: 10.1038/s41598-023-44989-5

Figure Lengend Snippet: Methodology for measuring innate and adaptive mucosal immune responses from a single nasal swab. Upon receipt, nasal swabs were thawed and aliquoted. One aliquot was used to assess swab quality by the presence of RNase P. A second aliquot was used to determine total protein concentration using BCA. Nasal swabs were diluted to a standardized concentration of 0.5mg/mL for downstream assays to account for the differences in total protein. Cytokines were measured using a Luminex kit with streptavidin-PE conjugated detection antibody. We reported cytokine values as a fold-change over baseline. We determined total IgA and IgG levels using an ELISA with anti-human IgA or IgG as a capture antibody. A second, HRP-conjugated, anti-human IgA or IgG was used to detect IgA or IgG captured from nasal swab samples. The total peak area under the curve was calculated and used as the variable for total IgA or IgG levels. SARS-CoV-2 specific antibodies were measured using a Luminex based kit with streptavidin-PE conjugated anti-human IgA or IgG secondary antibodies. A “positivity ratio” was calculated by dividing antigen specific IgA/IgG by total IgA/IgG. Neutralizing antibodies were determined using a SARS-CoV-2 Spike-VSV-ΔG- luciferase pseudovirus. Nasal swab material was incubated with the virus for 1 h prior to infecting confluent TMPRSS2 cells. The following day, cells were lysed and luminescence was measured. Percent neutralization was calculated for each swab by comparing the nasal swab + virus luminescence to virus only luminescence.

Article Snippet: To investigate whether mucosal immune responses would trend similarly to systemic responses, we used a similar Luminex Cytokine Human Panel on nasal swab samples diluted to a protein concentration of 0.5 mg/ml.

Techniques: Protein Concentration, Concentration Assay, Luminex, Enzyme-linked Immunosorbent Assay, Luciferase, Incubation, Virus, Neutralization

Journal: Scientific Reports

Article Title: Utility of nasal swabs for assessing mucosal immune responses towards SARS-CoV-2

doi: 10.1038/s41598-023-44989-5

Figure Lengend Snippet: SARS-CoV-2 infection alters cytokine responses differentially in the plasma and nasal cavity over time. Nasal swabs or plasma samples were collected at various times-post testing positive for SARS-CoV-2 and a baseline sample for nasal and plasma pre-infection was used for normalization. Cytokines were assessed by multiplex Luminex assay. ( A , B ) Heat map of the median cytokine fold changes response to each person’s baseline value to account for human variation for nasal swabs ( A ) or plasma samples ( B ). Convalescent stage was split into early (days 21–62) and late (> 62 days) post-infection to study the longitudinal impact of SARS-CoV-2 infection on mucosal cytokine responses ( A ). Ingenuity pathway analyses using predetermined signaling pathways on cytokines that were up or downregulated were assessed for both the nasal and plasma ( A , B ). ( C ) Fold change from baseline in acute, early, or late convalescent for cytokines FGF, VEGF, IL1RA, and IL-8 from nasal swabs. ( D ) Fold change from baseline in acute or convalescent from plasma for TNFα, CCL2, IL1RA, and IL-8. Heat maps and subsequent statistical analyses were conducted in GraphPad Prism version 9. Statistical analyses include a One-way ANOVA with Tukey’s Multiple Comparisons test ( D ). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Plasma, n = 96 for baseline, acute and convalescent; nasal swabs, n = 28 (baseline), n = 12 (acute), and n = 21 (total early + late convalescent). Note, not all individuals had cytokine levels detected in the nasal cavity at baseline or post-infection, which were excluded from this analysis.

Article Snippet: To investigate whether mucosal immune responses would trend similarly to systemic responses, we used a similar Luminex Cytokine Human Panel on nasal swab samples diluted to a protein concentration of 0.5 mg/ml.

Techniques: Infection, Multiplex Assay, Luminex