The SARS-CoV-2 Lambda variant exhibits enhanced infectivity and immune resistance

Abstract

SARS-CoV-2 Lambda, a variant of interest, has spread in some South American countries; however, its virological features and evolutionary traits remain unclear. In this study, we use pseudoviruses and reveal that the spike protein of the Lambda variant is more infectious than that of other variants due to the T76I and L452Q mutations. The RSYLTPGD246-253N mutation, a unique 7-amino acid deletion in the N-terminal domain of the Lambda spike protein, is responsible for evasion from neutralizing antibodies and further augments antibody-mediated enhancement of infection. Although this mutation generates a nascent N-linked glycosylation site, the additional N-linked glycan is dispensable for the virological property conferred by this mutation. Since the Lambda variant has dominantly spread according to the increasing frequency of the isolates harboring the RSYLTPGD246-253N mutation, our data suggest that the RSYLTPGD246-253N mutation is closely associated with the substantial spread of the Lambda variant in South America.

Keywords: C.37; COVID-19; Lambda; SARS-CoV-2; spike protein.

Graphical abstract

Figure 1

Epidemic and evolutionary dynamics of the Lambda variant (A) Epidemic dynamics of the Lambda variant in three South American countries. The numbers of the Lambda variant (C.37 lineage) sequences deposited per day from Peru (brown), Chile (dark red), and Argentina (pale blue) are indicated by lines. Gray dots indicate the numbers of SARS-CoV-2 genome sequences deposited in the GISAID database per day from the indicated countries. The raw data are summarized in Tables S1 and S2. (B) Proportion of amino acid replacements in the Lambda variant (C.37 lineage). The top 8 replacements conserved in the S protein of the Lambda variant (C.37 lineage) are summarized. The raw data are summarized in Table S3. (C) An evolutionary timetree of the Lambda variant (C.37 lineage). The coalescent time of the genuine Lambda variants is indicated in the figure. The 3 sequences (GISAID: EPI_ISL_1,532,199 [B.1.1.1 lineage], EPI_ISL_1,093,172 [B.1.1.1 lineage], and EPI_ISL_1,534,656 [C.37 lineage]) and the sister group of Lambda variants are indicated in black. Wuhan-Hu-1 (GISAID: EPI_ISL_1,532,199), the oldest SARS-CoV-2 (isolated on December 26, 2019), is indicated in red. Bars on the internal nodes correspond to the 95% HPD. The tree noted with the GISAID ID and sampling date at each terminal node is shown in Figure S1B. (D) Transition of the effective population size of the Lambda variant and the proportion of the Lambda variant harboring the RSYLTPGD246-253N mutation. The effective population size of the Lambda variant harboring the RSYLTPGD246-253N mutation (left y axis) was analyzed by a Bayesian skyline plot. The initial date was when the first Lambda variant was sampled (November 8, 2020). The 95% HPD is shaded in brown. In the same panel, the proportion of the Lambda variants harboring the RSYLTPGD246-253N mutation for each month (right y axis) is also plotted. The number at each time point indicates the number of Lambda variants harboring the RSYLTPGD246-253N mutation. The number in parentheses indicates the number of Lambda variants deposited in the GISAID database. See also Figure S1 and Tables S1, S2, and S3.

Figure 2

Virological and immunological features of the Lambda variant (A) Pseudovirus assay. HIV-1-based reporter viruses pseudotyped with SARS-CoV-2 S proteins of the parental (B.1), Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Epsilon (B.1.427), and Lambda (C.37) variants and the Lambda + N246-253RSYLTPGD derivative were prepared as described in STAR Methods. The mutations in each variant are listed in Figure S1C. The pseudoviruses were inoculated in HOS-ACE2/TMPRSS2 cells at 1,000 ng HIV-1 p24 antigen, and the percentages of infectivity compared to that of the virus pseudotyped with parental S are shown. (B) Neutralization assay. A neutralization assay was performed using the pseudoviruses with the S proteins of the parental, Lambda, and Lambda + N246-253RSYLTPGD and 18 BNT162b2-vaccinated sera as described in STAR Methods. The raw data are shown in Figure S2B. The number in the panel indicates the fold change in neutralization resistance of Lambda S to the parental S or the Lambda + N246-253RSYLTPGD derivative. (C and D) Structural insights of the mutations in Lambda S. (C) Overlaid overviews of the crystal structure of SARS-CoV-2 S (PDB: 6ZGE, white) (Wrobel et al., 2020) and a homology model of Lambda S (brown) are shown. The mutated residues in the Lambda S and the regions in the NTD and RBD are indicated in red and blue. The squared regions are enlarged in (D). Mutated residues in the NTD (left) and RBD (right) of Lambda S. The residues in parental S and the Lambda S are indicated in red and black. (E) Pseudovirus assay. HIV-1-based reporter viruses pseudotyped with SARS-CoV-2 S proteins bearing respective mutations of the Lambda variant in parental S (G75V, T76I, GT75-76VI, RSYLTPGD246-253N, L452Q, F490S, L452Q/F490S, or T859N) as well as the parental S were prepared. The pseudoviruses were inoculated into HOS-ACE2/TMPRSS2 cells at 1,000 ng HIV-1 p24 antigen, and the percentages of infectivity compared to that of the virus pseudotyped with parental S are shown. (F) Binding affinity of the SARS-CoV-2 S RBD (residues 336-528) to ACE2 by yeast surface display. The K_D values of the binding of the SARS-CoV-2 S RBD expressed on yeast to soluble ACE2 are shown. (G) CTL assay. HLA-A24⁺ CTL lines established from 6 BNT162b2-vaccinated donors were stimulated with 1 nM NF9 peptide or its derivatives, NF9-L452Q (NYNYQYRLF) or NF9-L452R (NYNYRYRLF). The percentage of IFN-γ⁺ cells in CD8⁺ T cells (bottom, n = 6) is shown. Representative fluorescence-activated cell sorting (FACS) plots showing intracellular expression of IFN-γ in the CD8⁺ T cell subset of a vaccinated donor (top, donor GV36-2) are shown in Figure S2H. (H) Neutralization assay. A neutralization assay was performed using the pseudoviruses used in Figure 2B and 18 BNT162b2-vaccinated sera, as described in STAR Methods. The raw data are shown in Figure S2B. The number in the panel indicates the fold change in neutralization resistance to the parental S. (I–L) Effect of monoclonal antibodies. The mean florescence intensity (MFI) of the surface S proteins stained with NTD-targeting NAb clone 4A8 (151 ng/mL–0.21 ng/mL) (Chi et al., 2020) (I) and EAb clone COV2-2490 (1 μg/mL–0.10 μg/mL) (Liu et al., 2021c) (K) antibodies. (J) Antiviral effect of the NTD-targeting NAb clone 4A8 antibody. (L) Enhancing effect of the EAb clone COV2-2490 antibody. The percentages of infectivity compared to that of the virus without antibodies are shown. Assays were performed in triplicate (except for H) or quadruplicate (H), and the mean is shown with the SD. In (A), (E), (F), (I), and (K), statistically significant differences (^∗, p < 0.05) versus parental S were determined by Student’s t test. In (A) and (E), vertical dashed lines indicate 100% (the value of parental S). In (B) and (H), statistically significant differences were determined by the Wilcoxon matched-pairs signed rank test. The p values are indicated in the figure. In (G), statistically significant differences (^∗p < 0.05) versus the NF9 peptide were determined by the Wilcoxon matched-pairs signed rank test. In (I) and (K), vertical dashed lines indicate the MFIs of mock-transfected cells. In (J), statistically significant differences (^∗p < 0.05) versus the value without the antibody were determined by Student’s t test. NS, no statistical significance. In (L), statistically significant differences versus the value without the antibody (^∗p < 0.05) and the parental S (#p < 0.05) were determined by Student’s t test. See also Figure S2 and Tables S4 and S5.

Figure 3

Effect of the nascent NLGS by the RSYLTPGD246-253N mutation on sensitivity to antibodies (A) A scheme showing the generation of the NLGS by the RSYLTPGD246-253N mutation. The NLGS generated in the Lambda S is indicated in red. (B) Pseudovirus assay. HIV-1-based reporter viruses pseudotyped with SARS-CoV-2 S proteins bearing mutations based on the Lambda S (top) and the parental S RSYLTPGD246-253N mutant (bottom) were prepared. The pseudoviruses were inoculated in HOS-ACE2/TMPRSS2 cells at 1,000 ng HIV-1 p24 antigen, and the percentages of infectivity compared to that of the virus pseudotyped with parental S are shown. (C) Neutralization assay. A neutralization assay was performed using pseudoviruses with SARS-CoV-2 S proteins bearing respective mutations based on the Lambda S and the parental S RSYLTPGD246-253N mutant and 18 BNT162b2-vaccinated sera (same as those used in Figure 2B), as described in STAR Methods. The number in the panel indicates the fold change in neutralization resistance of the pseudoviruses, with the mutations indicated to those with respective parental S. (D–G) Effect of monoclonal antibodies. The MFI of the surface S proteins stained with NTD-targeting NAb clone 4A8 (151 ng/mL) (Chi et al., 2020) (D) and EAb clone COV2-2490 (1 μg/mL) (Liu et al., 2021c) (F) antibodies. (E) Antiviral effect of the NTD-targeting NAb clone 4A8 antibody. (G) Enhancing effect of the EAb clone COV2-2490 antibody. The percentages of infectivity compared to that of the virus without antibodies are shown. Assays were performed in triplicate, and the mean is shown with the SD. In (B), statistically significant differences (^∗p < 0.05) versus the Lambda S (top) or the parental S RSYLTPGD246-253N mutant (bottom) were determined by Student’s t test. In (B), (E), and (G), vertical dashed lines indicate 100% (the value of Lambda S [A, top], parental S RSYLTPGD246-253N mutant [A, bottom], or parental S [E and G]). In (C), statistically significant differences were determined by the Wilcoxon matched-pairs signed rank test. The p values are indicated in the figure. In (D)–(G), statistically significant differences (^∗p < 0.05) versus parental S were determined by Student’s t test. In (D) and (F), vertical dashed lines indicate the MFIs of mock-transfected cells. NS, no statistical significance.