Sensitivity to Neutralizing Antibodies and Resistance to Type I Interferons in SARS-CoV-2 R.1 Lineage Variants, Canada

Abstract

Isolating and characterizing emerging SARS-CoV-2 variants is key to understanding virus pathogenesis. In this study, we isolated samples of the SARS-CoV-2 R.1 lineage, categorized as a variant under monitoring by the World Health Organization, and evaluated their sensitivity to neutralizing antibodies and type I interferons. We used convalescent serum samples from persons in Canada infected either with ancestral virus (wave 1) or the B.1.1.7 (Alpha) variant of concern (wave 3) for testing neutralization sensitivity. The R.1 isolates were potently neutralized by both the wave 1 and wave 3 convalescent serum samples, unlike the B.1.351 (Beta) variant of concern. Of note, the R.1 variant was significantly more resistant to type I interferons (IFN-α/β) than was the ancestral isolate. Our study demonstrates that the R.1 variant retained sensitivity to neutralizing antibodies but evolved resistance to type I interferons. This critical driving force will influence the trajectory of the pandemic.

Keywords: COVID-19; Canada; N501Y spike; SARS-CoV-2; VoC; VuM; coronavirus disease; neutralizing antibodies; respiratory infections; severe acute respiratory syndrome coronavirus 2; type I interferons; variant under monitoring; variants of concern; viruses; zoonoses.

Figure 1

Phylogenetic confirmation that SARS-CoV-2 isolates belong to R.1 lineage in study of sensitivity to neutralizing antibodies and resistance to type I interferons in SARS-CoV-2 R.1 lineage variants, Canada. Tree constructed by using maximum-likelihood estimations by executing 1,000 rapid bootstrap inferences and a thorough search with the general time reversible model of nucleotide substitution. Blue indicates R1 isolates (R.1 645, R.1 646) and red SB3 isolate. Variants are highlighted in magenta (Alpha), green (Beta), brown (Delta), mocha (Gamma), and orange (Omicron). The tree was visualized using FigTree version 1.4.2 (http://tree.bio.ed.ac.uk/software/figtree).

Figure 2

Antibody detection in study of sensitivity to neutralizing antibodies and resistance to type I interferons in SARS-CoV-2 R.1 lineage variants, Canada. A, B) S1 (A) and RBD (b) binding IgG determined by using a sandwich ELISA format. Dashed line indicates the limit of detection. C, D) Correlation between S1 and RBD binding IgG for wave 1 (C) and wave 3 (D). E–G) ID₅₀ titers for SB3 (E), R.1 645 (F), and B.1.351 (Beta) VoC (E). Error bars in panels A, B and E–G indicate SD. Statistical significance was calculated by using an unpaired t test for panels A and B and by using 1-way analysis of variance with Tukey multiple comparisons test for panels E–G. ID₅₀, 50% inhibitory dilution; PFU, plaque-forming units; RBD, receptor-binding domain; S1, spike.

Figure 3

Sensitivity of SARS-CoV-2 lineage variants to neutralizing antibodies, Canada. A–C) Sensitivity of SB3, R.1 645, and B.1.351 (Beta) variants to neutralizing antibodies from patients infected with the ancestral virus (wave 1 samples). D–F) Sensitivity of SB3, R.1 645, and B.1.351 (Beta) VoC to neutralizing antibodies from patients infected with the B.1.1.7 (Alpha) VoC (wave 3 samples). For each isolate, we tested 3 different PFU per well: 15,000 (A, D), 1,500 (B, E), and 150 (C, F). Statistical significance was calculated using 1-way analysis of variance with Tukey multiple comparisons test. ID₅₀, 50% inhibitory dilution; PFU, plaque-forming units.

Figure 4

Resistance to type I interferons in SARS-CoV-2 R.1 lineage variants, Canada. A) Sensitivity of SB3, R.1 645, and R.1 646 to IFN-α. B) Sensitivity of SB3, R.1 645, and R.1 646 to IFN-β. C, D) Fold change in IFIT1 transcript levels in response to IFN-α (C) or IFN-β (D) treatment. E, F) Fold change in IRF7 transcript levels in response to IFN-α (E) or IFN-β treatment (F). ISG transcript levels were normalized to GAPDH transcript levels. For testing, Calu-3 cells were either mock-infected or infected with SARS-CoV-2 (50,000 PFU/well) for 1 hour followed by treatment with recombinant IFN (1 or 10 ng/mL). Total RNA was extracted after 72 hours and SARS-CoV-2 RNA levels were determined using quantitative reverse transcription PCR. 1/ΔCT values are represented after normalizing to mock-infected cells. Statistical significance was calculated using 2-way analysis of variance with Tukey multiple comparisons test. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IFIT1, interferon-induced protein with tetratricopeptide repeats; IFN, interferon; IRF7, interferon regulatory factor 7; ISG, IFN-stimulated gene.