SARS-CoV-2 variants C.1.2 and B.1.621 (Mu) partially evade neutralization by antibodies elicited upon infection or vaccination

Abstract

Rapid spread of SARS-CoV-2 variants C.1.2 and B.1.621 (Mu variant) in Africa and the Americas, respectively, as well as a high number of mutations in the viral spike proteins raised concerns that these variants might pose an elevated threat to human health. Here, we show that C.1.2 and B.1.621 spike proteins mediate increased entry into certain cell lines but do not exhibit increased ACE2 binding. Further, we demonstrate that C.1.2 and B.1.621 are resistant to neutralization by bamlanivimab but remain sensitive to inhibition by antibody cocktails used for COVID-19 therapy. Finally, we show that C.1.2 and B.1.621 partially escape neutralization by antibodies induced upon infection and vaccination, with escape of vaccine-induced antibodies being as potent as that measured for B.1.351 (Beta variant), which is known to be highly neutralization resistant. Collectively, C.1.2 and B.1.621 partially evade control by vaccine-induced antibodies, suggesting that close monitoring of these variants is warranted.

Keywords: B.1.621; C.1.2; COVID-19; CP: Immunology; CP: Microbiology; Mu; SARS-CoV-2; antibody; neutralization; spike; variants.

Graphical abstract

Figure 1

Spike proteins of SARS-CoV-2 variants C.1.2 and B.1.621 harbor several mutations and enable enhanced entry into certain cell lines (A) Epidemiology of SARS-CoV-2 variants C.1.2 and B.1.621 (as of September 10, 2021; based on data deposited in the GISAID database). Arrowheads indicate the time points when isolates C.1.2 “early” and C.1.2 “late” were detected. (B) Global distribution of SARS-CoV-2 variants C.1.2 and B.1.621 (as of September 10, 2021; based on data deposited in the GISAID database). (C) Schematic overview and S protein domain organization. Mutations compared with SARS-CoV-2 Wuhan-Hu-1 are highlighted in red. Abbreviations: RBD, receptor-binding domain; TD, transmembrane domain. (D) Location of C.1.2 “early,” C.1.2 “late,” and B.1.621-specific mutations in the context of the trimeric spike protein. Color code: light blue, S1 subunit with RBD in dark blue; gray, S2 subunit; orange, S1/S2 and S2′ cleavage sites; red, mutated amino acid residues (compared with SARS-CoV-2 Wuhan-Hu-1; pink labels highlight differences between the two C.1.2 S proteins). (E) Pseudotyped particles harboring the indicated S proteins were inoculated onto the indicated cell lines. Transduction efficiency was quantified at 16–18 h post inoculation and normalized against B.1 S (set as 1). Shown are the average (mean) data from five to nine biological replicates (each performed with technical quadruplicates). Error bars indicate the standard error of the mean (SEM). Statistical significance was analyzed by two-tailed Student’s t test with Welch correction (p > 0.05, not significant [ns]; p ≤ 0.05, ^∗; p ≤ 0.01, ^∗∗; p ≤ 0.001, ^∗∗∗). See also Figure S1.

Figure 2

Spike proteins of SARS-CoV-2 variants C.1.2 and B.1.621 robustly bind ACE2 and efficiently evade antibody-mediated neutralization (A) Protein model of the SARS-CoV-2 RBD in which the ACE2-interacting interface (green) and target regions of therapeutic monoclonal antibodies (circles) are highlighted. (B) Location of C.1.2 “early,” C.1.2 “late,” and B.1.621-specific mutations (red) in the context of the RBD (gray). RBD residues that interact with ACE2 are colored in green. (C) 293T cells expressing the indicated S proteins (or VSV-G) were incubated with soluble ACE2-Fc and secondary antibody and analyzed by flow cytometry (left) and CABA (right). Shown are the average (mean) data from nine (flow cytometry; single samples) or three (CABA, four technical replicates) biological replicates for which ACE2 binding was normalized against B.1 (flow cytometry; set as 1) or the background (CABA; no soluble ACE2-Fc, set as 1). Error bars indicate the SEM. Statistical significance was analyzed by two-tailed Student’s t test with Welch’s correction (flow cytometry) or two-way analysis of variance with Dunnett’s post-hoc test (CABA) (p > 0.05, ns; p < 0.05, ^∗). See also Figure S1. (D) Location of C.1.2 “early,” C.1.2 “late,” and B.1.621-specific mutations (red) in the context of the RBD (gray) epitopes targeted by casirivimab, imdevimab, bamlanivimab, and etesevimab (blue). (E) Pseudotyped particles bearing the indicated S protein were pre-incubated in with the indicated antibodies or antibody cocktails and subsequently inoculated onto Vero cells. At 16–18 h post inoculation, pseudotype entry was quantified and normalized against samples that did not contain antibody (= 0% inhibition). Shown are the average (mean) data from a single biological replicate (performed with technical quadruplicates), and data were confirmed in a separate independent experiment. Error bars indicate the standard deviation. See also Figure S1. (F–H) Pseudotyped particles bearing the indicated S proteins were pre-incubated in the presence of convalescent plasma (F) or serum from individuals either vaccinated with BioNTech/Pfizer’s BNT162b2 vaccine (BNT/BNT (G) or AstraZeneca’s ChAdOx1 nCoV-19 vaccine (AZ/AZ) (H), and subsequently inoculated onto Vero cells. At 16–18 h post inoculation, pseudotype entry was quantified and used for the calculation of the neutralizing titer 50 (NT50). Presented are the data from ten plasma/serum samples per group (black lines and numerical values indicate the median NT50). Further, the median fold reduction in NT50 between SARS-CoV-2 B.1 (set as 1) and the indicated variants was calculated (boxplots indicate the median, quartiles, and range; circles indicate individual samples). Statistical significance was analyzed by two-tailed Mann-Whitney test with 95% confidence level (p > 0.05, ns; p ≤ 0.05, ^∗; p ≤ 0.01, ^∗∗; p ≤ 0.001, ^∗∗∗). See also Figure S2.