Strain-Specific Variability in Viral Kinetics, Cytokine Response, and Cellular Damage in Air-Liquid Cultures of Human Nasal Organoids After Infection with SARS-CoV-2

Abstract

SARS-CoV-2 variants have demonstrated distinct epidemiological patterns and clinical presentations throughout the COVID-19 pandemic. Understanding variant-specific differences at the respiratory epithelium is crucial for understanding their pathogenesis. Here, we utilized human nasal organoid air-liquid interface (HNO-ALI) cell cultures to compare the viral replication kinetics, innate immune response, and epithelial damage of six different strains of SARS-CoV-2 (B.1.2, WA, Alpha, Beta, Delta, and Omicron). All variants replicated efficiently in HNO-ALIs, but with distinct replication kinetic patterns. The Delta variant exhibited delayed replication kinetics, achieving a steady state at 6 days post-infection compared to 3 days for other variants. Cytokine analysis revealed robust pro-inflammatory and chemoattractant responses (IL-6, IL-8, IP-10, CXCL9, and CXCL11) in WA1, Alpha, Beta, and Omicron infections, while Delta significantly dampened the innate immune response, with no significant induction of IL-6, IP-10, CXCL9, or CXCL11. Immunofluorescence and H&E analysis showed that all variants caused significant ciliary damage, though WA1 and Delta demonstrated less destruction at early time points (3 days post-infection). Together, these data show that, in our HNO-ALI model, the Delta variant employs a distinct "stealth" strategy characterized by delayed replication kinetics and epithelial cell innate immune evasion when compared to other variants of SARS-CoV-2, potentially explaining a mechanism that the Delta variant can use for its enhanced transmissibility and virulence observed clinically. Our findings demonstrate that variant-specific differences at the respiratory epithelium could explain some of the distinct clinical presentations and highlight the utility of the HNO-ALI system for the rapid assessment of emerging variants.

Keywords: SARS-CoV-2; airway organoids; host responses; variants.

Figure 1

Replication kinetics and morphologic analysis of SARS-CoV-2 infected HNO-ALIs. (A) Apical log₁₀ viral gene copy numbers for two HNO-ALIs for strains Washington, B.1.2, Alpha, Beta, Delta, and Omicron, (B) corresponding to basolateral log₁₀ viral gene copy numbers. Error bars (standard deviation) represent two technical replicates of two donor HNO-ALIs. (C). Forest plot demonstrating the adjusted beta coefficient estimate with 95% confidence intervals, represented by dots and T-bars, respectively, between strains of SARS-CoV-2 by days post infection (dpi). (D,E) H&E images of single adult HNO-ALI line at 3 and 6 dpi with six strains of SARS-CoV-2. Scale bar is 100 µm. Black inverted triangles indicate cilia, arrows indicate goblet cells, and asterisks indicate cell damage. (F) Comparison of epithelial area of HNO-ALIs between strains of SARS-CoV-2 or mock infection. (G) Area of epithelium of six strains of SARS-CoV-2 at 3 and 6 dpi. Error bars (standard deviation) represent two technical replicates of two donor HNO-ALIs.

Figure 2

Epithelial cytokine and chemokine response during infection with different strains of SARS-CoV-2. Cytokine/chemokine secretion was modeled on the following factors: infection condition, cell line, cell surface, and dpi. Adjusted odds ratio estimates and their associated 95% confidence intervals are represented by dots and T-bars, respectively. (A) Forest plot showing the adjusted odds ratio with 95% confidence interval for overall cytokine secretion between strains of SARS-CoV-2. (B) Forest plot showing the adjusted odds ratio with 95% confidence interval cytokine expression of IL-6, (C) IL-8, (D) IP-10, (E) CXCL9, and (F) CXCL11.

Figure 3

Epithelial cytokine and chemokine groups during infection with different strains of SARS-CoV-2. Forest plot showing the adjusted odds ratio with 95% confidence interval for grouped cytokine secretion between strains of SARS-CoV-2. Groups include (A) chemoattractant, (B) anti-viral, (C) maturational, (D) metalloproteinases, (E) pro-inflammatory, and (F) regulatory cytokines.

Figure 4

Location of cytokine and chemokine release over time. Cytokine/chemokine secretion was modeled on the following factors: infection condition, cell line, cell surface, and dpi. Forest plot with adjusted odds ratio with 95% confidence intervals of apical or basolateral expression of cytokines. Individual cytokines are listed below their respective groups. Adjusted odds ratio estimates and their associated 95% confidence intervals are represented by dots and T-bars, respectively. Time points are as follows: (A) 1 dpi. (B) 3 dpi. (C) 6 dpi.

Figure 5

Ciliary damage and percentage of basal cells in HNO-ALIs infected with strains of SARS-CoV-2. (A,B) Representative IF imaging of a single adult HNO at 3 and 6 days post-infection (dpi) with six strains of SARS-CoV-2. Basal cells are stained in red by Krt5, ciliated cells are stained in green by acetylated alpha tubulin, SARS-CoV-2 viral particles are stained in gray by anti-spike protein antibody, and cellular nuclei are stained in blue by DAPI. Scale bar is 100 µm. (C) Area of ciliated epithelium in HNO-ALIs infected with strains of SARS-CoV-2 at 3 or 6 dpi. Error bars (standard deviation) represent two technical replicates from two donor HNO-ALIs. (D) Percentage of basal cells (number of Krt5 positive cells/ total cells) in HNO-ALIs infected with strains of SARS-CoV-2 at 3 or 6 dpi. Error bars (standard deviation) represent two technical replicates from two donor HNO-ALIs. (E) Forest plot of mock versus cilia area by virus strain modeled on the following factors: cell line, infection condition, and dpi. Each strain was compared against mock. Adjusted risk ratio estimates and their associated 95% confidence intervals are represented by dots and T-bars, respectively. (F) Forest plot of mock versus percentage of basal cells by viral strain was modeled on the following factors: cell line, infection condition, and dpi. Each strain was compared against mock. Adjusted risk ratio estimates and their associated 95% confidence intervals are represented by dots and T-bars, respectively.

Figure 6

Mucous secretion in HNO-ALIs infected with six strains of SARS-CoV-2. (A,B) Representative IF imaging of a single adult HNO at 3 and 6 days post-infection (dpi) with six strains of SARS-CoV-2. Basal cells are stained in red by Krt5, goblet cells are stained in green by Muc5AC, SARS-CoV-2 viral particles are stained in gray by anti-spike protein antibody, and cellular nuclei are stained in blue by DAPI. Scale bar is 100 µm. (C) The area of mucus (Muc5AC+ area) in HNO-ALIs infected with strains of SARS-CoV-2 at 3 or 6 dpi. Error bars (standard deviation) represent two technical replicates from two donor HNO-ALIs. (D) Percentage of goblet cells (number of Muc5AC positive cells/ total cells) in HNO-ALIs infected with strains of SARS-CoV-2 at 3 or 6 dpi. Error bars (standard deviation) represent two technical replicates from two donor HNO-ALIs. (E) Forest plot of mock versus mucous area by virus strain modeled on the following factors: cell line, infection condition, and dpi. Each strain was compared against mock. Adjusted risk ratio estimates and their associated 95% confidence intervals are represented by dots and T-bars, respectively. (F) Forest plot of mock versus percentage of goblet cells (Muc5AC+ cells with DAPI+ nuclei over total number of DAPI+ cells) by viral strain was modeled on the following factors: cell line, infection condition, and dpi. Each strain was compared against mock. Adjusted risk ratio estimates and their associated 95% confidence intervals are represented by dots and T-bars, respectively.