The Impact of Mutations in SARS-CoV-2 Spike on Viral Infectivity and Antigenicity

Abstract

The spike protein of SARS-CoV-2 has been undergoing mutations and is highly glycosylated. It is critically important to investigate the biological significance of these mutations. Here, we investigated 80 variants and 26 glycosylation site modifications for the infectivity and reactivity to a panel of neutralizing antibodies and sera from convalescent patients. D614G, along with several variants containing both D614G and another amino acid change, were significantly more infectious. Most variants with amino acid change at receptor binding domain were less infectious, but variants including A475V, L452R, V483A, and F490L became resistant to some neutralizing antibodies. Moreover, the majority of glycosylation deletions were less infectious, whereas deletion of both N331 and N343 glycosylation drastically reduced infectivity, revealing the importance of glycosylation for viral infectivity. Interestingly, N234Q was markedly resistant to neutralizing antibodies, whereas N165Q became more sensitive. These findings could be of value in the development of vaccine and therapeutic antibodies.

Keywords: COVID-19; SARS-CoV-2; glycosylation; mutants; pseudotyped virus; spike; variants.

Graphical abstract

Figure 1

Illustration of Amino Acid Changes Selected for This Study (A) Variants and combined variants with D614G across the entire S gene excluding the RBD region. (B) Variants in RBD. (C) Mutants at the putative glycosylation sites (22 sites). This group include mutations we introduced at all 22 putative glycosylation sites (N to Q), a combination of two glycosylation site mutations in RBD, and three naturally occurring variants, N74K, N149H, and T719A, with ablated glycosylation sites. High-frequency amino acid change sites (frequency > 0.1%) are highlighted in red. See also Table S1.

Figure 2

Selection of Susceptible Cell Lines A total of 26 different cell lines were infected with pseudotyped viruses with either SARS-CoV-2 S (A) or VSV G (B). The infected cell lysates were diluted by 10× and analyzed for luminescence activities (RLU). All results were obtained from three independent experiments (mean ± SEM). When the RLU for the tested cell reached 1% of that for Huh-7, it was deemed as a permissive cell line. The dash line indicates the 1% RLU value of Huh-7 cell.

Figure S1

Infectivity Analysis of Variants and Mutants in 293T-ACE2 Cell, Related to Figure 3 RLU values resulting from infection with variant pseudotyped viruses were quantified by luminescence meter and normalized to the reference strain (Wuhan-1) produced in parallel, with the difference by 4-fold being considered as significant; all experiments were conducted three times (mean ± SEM) unless specified. The horizontal dashed lines indicate the threshold of 4-fold difference.

Figure 3

Infectivity Analysis of Variants and Deletions of the Glycosylation Site Mutants Infectivity of natural variants and experimental mutants conducted in 293T-ACE2 (A), Huh-7 (B), Vero (C), and LLC-MK2 (D). RLU values generated with the infection of the variants or mutants, as measured by luminescence meter, were compared with the reference strain (Wuhan-1). A difference by 4-fold is considered significant; all experiments were conducted three times (mean ± SEM) unless specified. See also Figure S1.

Figure 4

Analyses of Antigenicity of the Natural Variants and Experimental Mutants Using a Panel of Neutralizing mAbs Serial dilutions of mAb preparations were pre-incubated with the pseudotyped viruses at 37°C for 1 h before they were added to Huh-7 cells. Luciferase activity was measured 24 h later to calculate EC₅₀ of each antibody. The ratio of EC₅₀ between the variant or mutant strains and the reference strain (Wuhan-1) was calculated and analyzed to generate heatmap using Hem I (Deng et al., 2014). The data were the results from 3–5 replicates. The red and blue boxes indicate the increase or decrease of the neutralization activity as shown in the scale bar. See also Figure S2.

Figure S2

Analyses of Antigenicity of Natural Variants and Experimental Mutants Using a Panel of Neutralizing mAbs, Related to Figure 4 Serial dilutions of mAb preparations were pre-incubated with the virus at 37°C for one hour before they were added to Huh-7 cells. Luciferase activity was measured 24 hours later to calculate EC₅₀ of each antibody. The y axis represents the ratio of EC₅₀ between the variant/mutant strain and the reference strain (Wuhan-1). The data (mean ± SEM) were the results from 3-5 replicates. The vertical dashed lines indicate the threshold of 4-fold difference. The significant changes were marked with colored symbols, blue for decreased, red for increased.

Figure 5

Differential Sensitivity of the Natural Variants and Experimental Mutants to a Panel of Convalescent Serum Samples (A) Serial dilutions of 10 patient serum samples were individually mixed with the pseudotyped viruses at 37°C for 1 h before added to Huh-7 cells for incubation of 24 h to determine the EC₅₀. The experiments were repeated at least 3 times. Hem I software was used to analyze the data and draw the heatmap. The red and blue boxes indicate the increase or decrease of the neutralization activity as shown in the scale bar. (B) Summary of the data from ten serum samples, with the values presented as mean ± SEM. The horizontal dashed lines indicate the threshold of 4-fold difference. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.005, ^∗∗∗∗p < 0.001. See also Figure S3.

Figure S3

Analyses of Antigenicity of the Natural Variants and Experimental Mutants Using Ten Convalescent Serum Samples, Related to Figure 5 The data (mean ± SEM) were the results from 3-5 replicates. The vertical dashed lines indicate the threshold of 4-fold difference. The significant changes were marked with colored symbols, blue for decreased, red for increased.

Figure 6

mAb Epitopes and Antigenicity-Related Sites (A and B) Epitope of the mAb P2B-2F6 on the trimer (A) and RBD (B). (C and D) Epitope of the mAb CB6 on the trimer (C) and RBD (D). (E and F) Epitope of the mAb H014 on the trimer (E) and RBD (F). The monoclonal antibody epitope is indicated in purple. The glycosylation sites are indicated in light blue. The sugar chain is shown in orange. The amino acid sites that coincide with the epitopes of monoclonal antibodies are indicated in green. Amino acid sites that do not coincide with the monoclonal antibody epitope are indicated in dark blue.