Interferon resistance of emerging SARS-CoV-2 variants

Abstract

The emergence of SARS-CoV-2 variants with enhanced transmissibility, pathogenesis, and resistance to vaccines presents urgent challenges for curbing the COVID-19 pandemic. While Spike mutations that enhance virus infectivity or neutralizing antibody evasion may drive the emergence of these novel variants, studies documenting a critical role for interferon responses in the early control of SARS-CoV-2 infection, combined with the presence of viral genes that limit these responses, suggest that interferons may also influence SARS-CoV-2 evolution. Here, we compared the potency of 17 different human interferons against multiple viral lineages sampled during the course of the global outbreak, including ancestral and five major variants of concern that include the B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma), B.1.617.2 (delta), and B.1.1.529 (omicron) lineages. Our data reveal that relative to ancestral isolates, SARS-CoV-2 variants of concern exhibited increased interferon resistance, suggesting that evasion of innate immunity may be a significant, ongoing driving force for SARS-CoV-2 evolution. These findings have implications for the increased transmissibility and/or lethality of emerging variants and highlight the interferon subtypes that may be most successful in the treatment of early infections.

Keywords: COVID-19; SARS-CoV-2; innate immunity; interferons; variants of concern.

Fig. 1.

Selection of SARS-CoV-2 strains for IFN sensitivity studies. (A) Global distribution of SARS-CoV-2 clades. GISAID.org plotted the proportion of deposited sequences in designated clades against collection dates. The 10 isolates chosen are noted by colored dots. (B) SARS-CoV-2 strains selected for this study included representatives of lineages A, B, B.1, B.1.351, and B.1.1.7 (SI Appendix, Table S1). Lineage P.1 (which branched off from lineage B.1.1.28), B.1.617.2, and B.1.1.529 were added after the initial preprint submission and were evaluated for IFNβ and IFNλ1 sensitivity. Lineage B isolates encode the D614G mutation associated with increased transmissibility. *Amino acid mutations were relative to the reference hCOV-19/Wuhan/WIV04/2019 sequence.

Fig. 2.

Sensitivity of SARS-CoV-2 strains to IFN-I and IFN-III. (A) Antiviral assay using recombinant IFNs (2 pM) in A549-ACE2 cells. The red line corresponds to the qPCR detection limit (90 copies/reaction, or 1.8 × 10⁴ copies/mL). Viral copy numbers in two D614G+ isolates, showing a similar rank order of IFNs from least to most potent. (B) The average fold inhibition relative to mock for lineage B, B.1, B.1.351, and B.1.1.7 isolates are shown. The most potent IFNs are shown top to bottom. Bars and error bars correspond to means and SDs. (C) Log-transformed IFN-inhibition values relative to mock for the five different SARS-CoV-2 strains were compared to previously published HIV-1 inhibition values, based on percentage of inhibition of HIV-1 p24⁺ gut lymphocytes relative to mock as measured by flow cytometry (3). Each dot corresponds to an IFNα subtype. Linear regression was performed using GraphPad Prism. Significant correlations (P < 0.05) were highlighted with a red best-fit line. (D) Heatmap of fold inhibition of representative strains from the lineages noted. Colors were graded on a log-scale from highest inhibition (yellow) to no inhibition (black). (E) Comparison of IFN-I sensitivities between lineage B versus B.1, B.1.351, and B.1.1.7. The mean fold inhibition values relative to mock were compared in a pairwise fashion for the 14 IFN-Is. The average fold inhibition values were noted. Differences were evaluated using a nonparametric, two-tailed Wilcoxon matched-pairs signed rank test. NS, not significant; ****P < 0.0001.

Fig. 3.

Dose titration of ancestral lineage B versus five variants of concern against IFNβ and IFNλ1. IFNβ and IFNλ1 dose titrations in (A)–(F) correspond to separate infections and used the lineage B isolate Germany/BavPat1/2020 isolate as a control. A549-ACE2 cells were pretreated with serial 10-fold dilutions of IFNs for 18 h in triplicate and then infected with SARS-CoV-2. Supernatants were collected after 24 h, SARS-CoV-2 N1 copy numbers were determined by qPCR in triplicate, and then the mean copy numbers were normalized against mock as 100%. Error bars correspond to SDs. Nonlinear best-fit regression curves of mean normalized infection levels were used to interpolate IC₅₀s (green dotted lines). IFN sensitivity of the ancestral lineage B isolate was compared to (A) ancestral lineage A (USA-WA1/2020); (B) B.1.1.7 (Alpha) isolates USA/CA_CDC_5574/2020 (CA) and England/204820464/2020 (UK); (C) B.1.351 (Beta) (South Africa/KRISP-EC-K005321/2020); (D) P.1 (Gamma) (Japan/TY7-503/2021); (E) B.1.617.2 (Delta) isolates USA/PHC658/2021 (ORF7a⁻) and USA/MD-HP05647/2021 (ORF7a⁺); and (F) B.1.1.529 (Omicron) (USA/MD-HP20874/2021). *In (B), the datapoint at 200 pM IFNλ1 for lineage B (0.54) precluded efforts for IC₅₀ determination and was excluded in the curve fitting. In (A) and (C), the datapoint for lineage B at 0.002 pM IFNβ was excluded as the inhibition curve was significantly skewed to the left relative to four other lineage B titrations. (F) IC₅₀ and (G) residual infection levels at the highest IFN doses were compared between lineage B (five independent titrations) and VOCs. Datapoint shapes and colors correspond to those in (A)–(F). Differences in the mean values were evaluated using a two-tailed Student’s t test or Mann-Whitney U test, depending on whether the data distribution was normal. *P < 0.05; ***P < 0.001. Fold differences between lineage B and VOCs are indicated.

Fig. 4.

Evidence for increasing IFN resistance of SARS-CoV-2 in primary HBECs during the course of the COVID-19 pandemic. (A) Basal stem cells from healthy donor airway tissue were used to generate HBEC cultures. (Upper Left) Basal cells were isolated from human donor lungs, expanded to passage 1, and then differentiated at ALI on Transwell filters. (Upper Right) En face view of whole-mount-labeled ALI at day 21 (ALI+21d). Mature HBECs contain a large number of ciliated (red) and mucous (green) cells. (Lower Panel) Schematic of ALI culture and SARS-CoV-2 infection timeline. At ALI+21d, untreated or IFN-treated HBECs were infected in quadruplicate with 10⁸ vRNA N1 copies of SARS-CoV-2 strains representing lineage A (USA-WA1/2020), lineage B (Germany/BavPat1/2020), Alpha (England/204820464/2020), Delta (USA/MD/HP05647/2021), and Omicron (USA/MD-HP20874/2021). (B) Apical viral shedding up to 4 dpi was determined by qPCR quantification of viral N1 RNA copies. Mean copy numbers per microliter input are shown ±SEMs for 2 h and 2, 3, and 4 dpi, showing that differences between mock- and IFNβ-treated cells became more apparent at 4 dpi. (C) Relative sensitivity of globally dominant SARS-CoV-2 isolates in HBECs against (Left) IFNβ, (Center) IFNα2, and (Right) IFNλ1, plotted with time since the first COVID-19 case reported on December 10, 2019 (day 0) (38). Error bars correspond to SEMs in quadruplicate experiments. Correlations were evaluated by linear regression.