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

Epidemic dynamics of the Lambda variant in South American countries

As of June 29, 2021, 1,908 genome sequences of the Lambda variant belonging to the PANGO C.37 lineage have been isolated from 26 countries and deposited in GISAID. Although the Lambda variant was first reported in Peru in December 2020 (WHO, 2021a), our in-depth analysis revealed that this variant was first detected in Argentina on November 8, 2020 (GISAID: EPI_ISL_2158693) (Figure 1A; seeSTAR Methods for details). The prevalence of the Lambda variant is increasing in South American countries, including Peru, Chile, and Argentina (Figure 1A andTable S1), suggesting that this variant is spreading predominantly in these countries (Table S2). We then generated a maximum likelihood-based phylogenetic tree of the Lambda variant (Figure S1A). Although there were some isolates that were misclassified as Lambda, which could be the “sister group of Lambda variants,” our phylogenetic tree indicated the monophyly of the “genuine Lambda variants” isolates (Figure S1A).

Association of the Lambda variant spread with the increasing frequency of isolates harboring the RSYLTPGD246-253N mutation

In the 1,908 sequences of the Lambda variant (C.37 lineage), the majority of the S protein sequence of the genuine Lambda variants contains 6 substitution mutations (G75V, T76I, L452Q, F490S, D614G, and T859N) and a 7-amino acid deletion in the NTD (RSYLTPGD246-253N) (Figure S1A). The Lambda variant first isolated in Argentina contains the 6 amino acid mutations as well as the deletion in the S protein. However, the sister group of Lambda variants does not contain any of the 7 mutations, excluding D614G in their S proteins (Figure S1A).

We then analyzed the mutations in the S protein using the 1,908 sequences of the Lambda variant (C.37 lineage) and showed that the 6 amino acid mutations are highly (>90%) conserved (Figure 1B). Although a large deletion in the NTD, the RSYLTPGD246-253N mutation, is also highly conserved, and only 287 of the 1,908 sequences (15.0%) of the Lambda variant (C.37 lineage) genomes do not harbor this mutation (Figures 1B andS1A). To determine whether the epidemic dynamic of the Lambda variant is associated with the emergence of the RSYLTPGD246-253N mutation, we examined all of the amino acid changes in the S protein of the SARS-CoV-2 genomes deposited in the GISAID database (2,084,604 sequences; as of June 29, 2021). Our analysis showed that the RSYLTPGD246-253N mutation was first found in Argentina on November 8, 2020, which is the first isolate of the Lambda variant (Figure 1A), suggesting that this deletion event uniquely occurred in the ancestral lineage of the genuine Lambda variants. We then analyzed the molecular evolutionary dynamics of the Lambda variants by performing Bayesian tip-dating analysis. We showed that the estimated time to the most recent common ancestor of the genuine Lambda variants was approximately July 12, 2020 (95% highest posterior density [HPD], January 5, 2020–October 22, 2020) (Figures 1C andS1B). To infer the population dynamics of the lineage, we performed a Bayesian skyline plot analysis. This analysis showed that the effective population size of the Lambda variant increased at the beginning of 2021 (Figure 1D). Intriguingly, when we plotted the proportion of the Lambda variant with the RSYLTPGD246-253N mutation, we found that it increased after the emergence of the Lambda variant and closely associated with the increase in effective population size (Figure 1D). These results suggest that the acquisition of the RSYLTPGD246-253N mutation is associated with the outbreak of the Lambda variant in South America.

Enhanced infectivity and resistance to NAbs of the Lambda variant

To address the virological phenotype of the Lambda variant (hereafter, only genuine Lambda variant is referred to as “Lambda variant”), we prepared viruses pseudotyped with the S proteins of the Lambda variant as well as 4 VOCs, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1) and Delta (B.1.617.2) (Figure S1C). We also prepared pseudoviruses with the S protein of the Epsilon (B.1.427/429) variant (Figure S1C), which was used as a VOC/VOI and was reclassified on July 6, 2021 (WHO, 2021a). As the control of this experiment, the pseudoviruses with the S protein of the D614G-bearing isolate (B.1 lineage), which spread in the early pandemic (i.e., during early 2020 and before the emergence of VOCs/VOIs) (Ozono et al., 2021), were prepared, and this is referred to as “parental virus” in this study. As shown inFigure 2A, the infectivities of the Alpha and Beta variants were significantly lower than that of the parental virus (p = 0.0012, Alpha versus parental; p = 0.0012, Beta versus parental by Student’s t test), and the infectivities of the Gamma variant and the parental virus were comparable. Since the epidemic of SARS-CoV-2 in the early pandemic, which includes the B.1 lineage (parental virus in this study), was outcompeted by VOCs including the Alpha and Beta variants during late 2020 (WHO, 2021a), it was unexpected that the infectivities of Alpha and Beta variants were lower than that of the parental virus (Figure 2A). However, the lower infectivity of the Alpha variant compared to the D614G-bearing early pandemic virus, analyzed by pseudovirus assay (Figure 2A), is consistent with a previous report (Acevedo et al., 2021). In contrast, the infectivities of the Delta, Epsilon, and Lambda variants were significantly higher than that of the parental virus (Figure 2A: p = 0.017, Delta versus parental; p = 0.0031, Epsilon versus parental; p = 0.0004, Lambda versus parental by Student’s t test). This pattern was independent of the input dose of pseudoviruses used (Figure S2B).

To assess the effect of a characteristic mutation of the Lambda variant, the RSYLTPGD246-253N mutation (Figure 1), on viral infectivity, we prepared a pseudovirus with the Lambda S derivative with the recovery of this deletion (Lambda + N246-253RSYLTPGD) (Figure S2A). The infectivity of this mutant was comparable to that of the Lambda S (Figures 2A andS2B). To further verify the effect of the N246-253RSYLTPGD mutation on the infectivity of the Lambda variant, we prepared the fluorescent protein-expressing lentivirus vectors pseudotyped with Lambda S (expressing Crimson) and Lambda + N246-253RSYLTPGD S (expressing ZsGreen1). The competition assay using these fluorescent protein-expressing pseudoviruses showed that both viruses exhibit similar infectivities (Figure S2C), suggesting that the 7-amino acid deletion in the NTD does not affect viral infection.

Because NAb resistance is a notable phenotype of most VOCs (reviewed inHarvey et al., 2021), we next analyzed the sensitivity of Lambda S to NAbs induced by BNT162b2 vaccination (Table S4). As shown inFigure 2B, Lambda S was, on average, 1.5-fold (2.63-fold at a maximum) more resistant to BNT162b2-induced antisera than the parental S (p = 0.0077 by the Wilcoxon matched-pairs signed rank test). However, the neutralization level of Lambda + N246-253RSYLTPGD was similar to that of the parental S pseudovirus (P = 0.21 by the Wilcoxon matched-pairs signed rank test), and this recovered variant was 1.37-fold more sensitive to vaccine-induced neutralization than the Lambda variant (P = 0.016 by the Wilcoxon matched-pairs signed rank test) (Figures 2B andS2D). These results suggest that Lambda S is highly infectious and resistant to vaccine-induced humoral immunity, and the robust resistance of Lambda S to vaccine-induced neutralization is determined by a 7-amino acid deletion in the NTD.

Effect of consensus mutations in Lambda S on viral infectivity

We next examined 6 substitution mutations (G75V, T76I, L452Q, F490S, D614G, and T859N) and a deletion mutation (RSYLTPGD246-253N) of the Lambda variant in the structure of the SARS-CoV-2 S protein. Structural analysis showed that the 3 mutations, G75V, T76I, and RSYLTPGD246-253N, are in the NTD (Figure 2C), and the RSYLTPGD246-253N mutation is located in a loop structure, which was designated as loop 5 (residues 246–260) in a previous study (Chi et al., 2020) (Figure 2D). The G75V and T76I mutations are located at the flexible loop adjacent to loop 5 (Figure 2C). The L452Q and F490S mutations are situated in the receptor-binding motif in the RBD (Figures 2C and 2D), but neither residue is in direct contact with the angiotensin-converting enzyme 2 (ACE2) receptor (Figure S2E). The T859N mutation is in the heptad repeat 1 of the S2 subunit (Figure 2C).

To investigate the effects of these seven consensus mutations in Lambda S on viral infectivity and NAb sensitivity, we prepared viruses pseudotyped with parental S-based derivatives that have mutations of the Lambda variant.Figure 2E shows that the G75V mutation significantly reduces viral infectivity (p = 0.0004, by Student’s t test), while the T76I and GT75-76VI mutations significantly increase viral infectivity (p = 0.0011, T75I versus parental; p = 0.012, GT75-76TI versus parental by Student’s t test). In addition, 91.5% (1,746/1,908) of the Lambda variant sequences had these 2 mutations, and the phylogenetic tree of the Lambda variant indicated that the variant harboring either G75V or T76I sporadically emerged during the epidemic of the Lambda variant (Figure S1A). However, there were no viral sequences harboring the G75V mutation without the T76I mutation (Figure S1A). These findings suggest that T76I is a compensatory mutation to recover the decreased infectivity by the G75V mutation.

Similar to the results from the experiment using pseudoviruses with Lambda S and Lambda + N246-253RSYLTPGD (Figure 2A), the insertion of the RSYLTPGD246-253N mutation did not affect viral infectivity (Figure 2E). The infectivity of the T859N mutation was also similar to that of the parental pseudovirus (Figure 2E). When we focused on the effect of the mutations in the RBD, the L452Q and L452Q/F490S mutations significantly increased viral infectivity (p = 0.0011, L452Q versus parental; p = 0.0003, L452Q/F490S versus parental by Student’s t test), while the F490S mutation did not (Figure 2E). In addition, the binding affinity of the SARS-CoV-2 RBD to soluble ACE2 was significantly increased by the insertion of the L452Q mutation, but not the F490S mutation (Figure 2F: p = 0.0004, L452Q versus parental; p = 0.92, F490S versus parental; p = 0.0002, L452Q/F490S versus parental by Student’s t test). These results suggest that the T76I and L452Q mutations are responsible for the higher infectivity of Lambda S (Figure 2E). The effect of each mutation was similar with different amounts of pseudoviruses used and in the target cells without TMPRSS2 expression (Figure S2F).

Evasion of vaccine-induced cellular immunity induced by the L452Q mutation

Previous studies showed that the 9-mer peptide spanning the 448–456 residues of the SARS-CoV-2 S protein (designated NF9) is an immunodominant epitope presented by human leukocyte antigen (HLA)-A24 in convalescent individuals with SARS-CoV-2 infection (Gao et al., 2021;Hu et al., 2021;Kared et al., 2021). Interestingly, we recently demonstrated that the L452R mutation, a hallmark mutation in the Delta and Epsilon variants, could induce evasion of the antiviral effects triggered by HLA-A24-restricted cytotoxic T lymphocytes (CTLs) (Motozono et al., 2021). To determine whether the L452Q mutation in the Lambda variant can contribute to evasion of HLA-A24-restricted anti-SARS-CoV-2 cellular immunity, as in the case of the L452R mutation (Motozono et al., 2021), we obtained peripheral blood mononuclear cells (PBMCs) from HLA-A^∗24:02⁺ individuals vaccinated with BNT162b2 and stimulated these cells with the NF9 peptide (Table S5). As shown inFigure S2G, the NF9 peptide activated the PBMCs from HLA-A^∗24:02⁺ vaccinated individuals, but not those from HLA-A^∗24:02⁻ vaccinated individuals or HLA-A^∗24:02⁺ unvaccinated individuals, suggesting that the NF9 peptide is an immunodominant epitope for PBMCs from HLA-A^∗24:02⁺ BNT162b2-vaccinated individuals. We then assessed whether the L452Q mutation evades the HLA-A24-restricted cellular immunity induced by BNT162b2 vaccination. Consistent with our previous study (Motozono et al., 2021), the NF9 peptide significantly induced interferon-γ (IFN-γ) production (Figures 2G andS2H: p = 0.031 by the Wilcoxon matched-pairs signed rank test). In sharp contrast, 2 substitutions of the NF9 peptide, NF9-L452R and NF9-L452Q, faintly activated the NF9-specific CTLs (Figures 2G andS2H). These results suggest that the NF9 peptide is an immunodominant epitope in HLA-A^∗24:02⁺ vaccinated individuals and that the L452Q mutation potentially results in evasion of HLA-A24-restricted cellular immunity.

Evasion of vaccine-induced humoral immunity

We next assessed the sensitivity of these pseudoviruses with the mutated S proteins to BNT162b2-induced antisera. As shown inFigure 2H, the G75V, T76I, GT75-76VI, and T859N mutations did not affect vaccine-induced neutralization. In contrast, the RSYLTPGD246-253N mutant exhibited significant resistance to the vaccine-induced neutralization (Figure 2H: p = 0.027 by the Wilcoxon matched-pairs signed rank test), which is relevant to the experiment with the S proteins of the Lambda variant and the Lambda + N246-253RSYLTPGD derivative (Figure 2B). In addition, we found that the L452Q and F490S mutations confer resistance to vaccine-induced antisera (Figure 2H). The finding that the F490S mutation does not affect viral infectivity (Figure 2E) but confers the resistance to the vaccine-induced antisera (Figure 2H) suggests that this mutant has acquired resistance to antiviral humoral immunity. In contrast, the L452Q mutation not only increases viral infectivity (Figure 2E) and affinity to ACE2 (Figure 2F) but also augments resistance to the vaccine-induced antiviral cellular (Figure 2G) and humoral immunities (Figure 2H), suggesting that this mutation is critical for the viral dissemination in the human population.

Effects of NTD-targeting monoclonal antibodies on Lambda variant infection

To further assess the effect of the mutations in Lambda S, particularly those in the NTD, we used 2 monoclonal antibodies that target the NTD: an NTD-targeting NAb, clone 4A8 (Chi et al., 2020), and an EAb, clone COV2-2490, that recognizes the NTD and enhances viral infectivity (Liu et al., 2021c). As shown inFigures 2I and 2J, the 4A8 antibody detected the cell surface expression of S proteins of the parental virus, Lambda + N246-253RSYLTPGD derivative, G75V, T76I, and GT75-76VI and inhibited the infections of these pseudoviruses in a dose-dependent manner. Intriguingly, the 4A8 antibody failed to detect the surface expression of the S proteins of the Lambda and the RSYLTPGD246-253N mutant (Figure 2I), and the pseudoviruses with the S proteins of the Lambda and the RSYLTPGD246-253N mutant were resistant to the antiviral effect mediated by the 4A8 antibody (Figure 2J). Because a polyclonal antibody targeting the SARS-CoV-2 S protein detected these mutants, similar to the parental S (Figure S2I), our results suggest that the RSYLTPGD246-253N mutation critically affects the sensitivity to certain NAbs targeting the NTD, such as the 4A8 antibody.

For the COV2-2490 antibody, flow cytometry analysis showed that this antibody more efficiently detects the Lambda S than the parental S (Figure 2K). Although this antibody enhanced the infectivities of all of the pseudoviruses tested (Figure 2L), it is important to note that the infectivity of the Lambda S was more significantly enhanced than that of the parental S (Figure 2L: p = 0.032 at 0.010 μg/mL; p = 0.013 at 0.1 μg/mL; p = 0.024 at 1.0 μg/mL by Student’s t test). These data suggest that Lambda S is more susceptible to EAb-mediated enhancement of viral infection. Because an apparent enhancement of viral infectivity compared to the parental S pseudovirus was not observed in the substituted mutants (G75V, T76I, GT75-76VI, and RSYLTPGD246-253N) (Figure 2L), our data suggest that multiple mutations in the NTD of Lambda S are crucial for the augmented enhancement of viral infectivity. Although the surface detection level of the Lambda variant was comparable to those of the Lambda + N246-253RSYLTPGD derivative and other NTD mutants tested (T76I, GT75-76VI, and RSYLTPGD246-253N) (Figure 2K), the augmented enhancement of viral infectivity was specific for the Lambda S (Figure 2L). These observations suggest that the detection level of the S protein on the cell surface by flow cytometry does not necessarily reflect the augmentation level of viral infection by the COV2-2490 antibody, and further actions of the S protein following the binding of the COV2-2490 antibody, such as the Lambda S-specific conformational change, may be needed to lead to the augmented enhancement of viral infectivity.

Association of a nascent N-linked glycosylation site (NLGS) by the RSYLTPGD246-253N mutation with sensitivity to NTD-targeting antibodies

We revealed that Lambda S is more resistant to vaccine-induced antiviral humoral immunity (Figures 2B and 2H) as well as an NTD-targeting NAb (Figure 2J) and that the RSYLTPGD246-253N mutation is responsible for NAb resistance. In addition, the sensitivity of Lambda S to EAb was higher than that of the parental S and was also attributed to the multiple mutations in the NTD, including the RSYLTPGD246-253N mutation (Figure 2L). These data suggest the importance of the RSYLTPGD246-253N mutation in determining the virological phenotype of Lambda S; however, it remains unclear how this large mutation affects sensitivity to neutralizing/enhancing antibodies. When we further assessed the amino acid sequences of the S proteins of the parental and Lambda variant, we found that the RSYLTPGD246-253N mutation generates an NLGS (N-X-S/T consensus sequence)de novo (Figure 3A). Because emergent N-linked glycans in viral spike proteins, such as HIV-1 envelope protein (Li et al., 2008;Quinones-Kochs et al., 2002;Reitter et al., 1998;Wagh et al., 2020), and influenza A virus hemagglutinin protein (Abe et al., 2004;Kobayashi and Suzuki, 2012;Skehel et al., 1984;Wang et al., 2009), can shield the viral particles from NAbs, we hypothesized that this property of Lambda S is attributed to the nascent N-glycan conferred by the RSYLTPGD246-253N mutation. To address this possibility, we prepared two Lambda S derivatives, N246A and N246Q, which abolished the NLGS (Figure 3A). We also prepared two parental S RSYLTPGD246-253N mutant-based derivatives, RSYLTPGD246-253A and RSYLTPGD246-253Q (Figure 3A). As shown inFigure 3B, the infectivities of the parental S RSYLTPGD246-253N mutant-based pseudoviruses (p < 0.0001, RSYLTPGD246-253A versus parental; p < 0.0001, RSYLTPGD246-253Q versus parental by Student’s t test) but not that of the Lambda S-based pseudoviruses (p = 0.081, RSYLTPGD246-253A versus Lambda; p = 0.26, RSYLTPGD246-253Q versus Lambda by Student's t test) were significantly increased by the NLGS disruption. However, NLGS disruption did not affect the sensitivity to vaccine-induced NAbs (Figure 3C). As in the cases of the Lambda S and the parental S RSYLTPGD246-253N mutant (Figure 2I), flow cytometry analysis showed that the 4A8 antibody faintly detected the NLGS-disrupted mutants on the cell surface (Figure 3D). In addition, a neutralization assay revealed that the NLGS disruption did not affect the sensitivity to the 4A8 antibody (Figure 3E). For an NTD-targeting EAb, the COV2-2490 antibody, the detection on the cell surface by flow cytometry (Figure 3F), and the enhancement of the pseudovirus infectivity (Figure 3G) were comparable between the NLGS-disrupted mutants and the Lambda S and the parental S RSYLTPGD246-253N mutants. These results suggest that the addition of an NLGS does not affect the sensitivity of Lambda S to the antibodies targeting the NTD.

Limitations of the study

The experiments using pseudoviruses showed that the Lambda variant is more infectious than the parental virus (Figure 2A). However, it remains unaddressed whether the live SARS-CoV-2 Lambda variant is more replicative than the live parental SARS-CoV-2 (B.1 lineage). Although our data showed that the Alpha pseudovirus is less infectious than the parental (B.1 lineage) pseudovirus, which is consistent with a previous study (Acevedo et al., 2021),Touret et al. (2021) showed that the growth capacity of the live Alpha variant is comparable to that of the B.1 lineage virus. Because the cells used for the infection experiments are different, the discrepancy between the pseudovirus assay and the experiment using live virus may be due to the differences in the experimental setup. Further investigation will be needed to evaluate the replication capacity of SARS-CoV-2 variants using live viruses and multiple cell types. However, the use of live SARS-CoV-2 variants for in-depth experiments is still limited by experimental, processing, and safety limitations.

Consistent with a previous report (Liu et al., 2021c), we showed that an EAb, COV2-2490, enhanced viral infectivity inin vitro cell culture experiments (Figures 2L and3G). In addition, COV2-2490 confers greater benefit to the Lambda pseudovirus (Figures 2L and3G). However,Li et al. (2021) reported that the EAbs do not necessarily promote viral expansion in anin vivo animal model. Because the effect of EAbs on viral replicationin vitro andin vivo may be inconsistent, the possibility of the contribution of EAbs for the expansion of Lambda variant as well as other VOCs/VOCsin vivo should be addressed in the future.

Key resources table

Resource availability

Experimental model and subject details

Method details

Quantification and statistical analysis

Data analyses were performed using Prism 9 (GraphPad Software). Data are presented as average with SD.

Author contributions

I.K., Y. Kosugi, J.Z., D.Y., E.P.B., Y.L.T., K.U., N.M., C.M., A.S., and K. Sato performed the experiments. K. Shirakawa, Y. Kazuma, R.N., Y.H., and A.T.-K. collected the clinical samples. Y.L. and H.A. prepared the reagents. I.K., Y. Kosugi, J.Z., D.Y., E.P.B., Y.L.T., K.U., K.T., T.U., G.S., C.M., A.S., and K. Sato designed the experiments and interpreted the results. J.W. and S.N. performed the molecular phylogenetic analysis. K. Sato wrote the original manuscript. All of the authors reviewed and proofread the manuscript. The Genotype to Phenotype Japan (G2P-Japan) Consortium contributed to the project administration.

Declaration of interests

The authors declare no competing interests.

1. Abe Y., Takashita E., Sugawara K., Matsuzaki Y., Muraki Y., Hongo S. Effect of the addition of oligosaccharides on the biological activities and antigenicity of influenza A/H3N2 virus hemagglutinin. J. Virol. 2004;78:9605–9611. doi: 10.1128/JVI.78.18.9605-9611.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
2. Acevedo M.L., Alonso-Palomares L., Bustamante A., Gaggero A., Paredes F., Cortés C.P., Valiente-Echeverría F., Soto-Rifo R. Infectivity and immune escape of the new SARS-CoV-2 variant of interest Lambda. MedRxiv. 2021:21259673. [Google Scholar]
3. Chen R.E., Zhang X., Case J.B., Winkler E.S., Liu Y., VanBlargan L.A., Liu J., Errico J.M., Xie X., Suryadevara N., et al. Resistance of SARS-CoV-2 variants to neutralization by monoclonal and serum-derived polyclonal antibodies. Nat. Med. 2021;27:717–726. doi: 10.1038/s41591-021-01294-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
4. Chi X., Yan R., Zhang J., Zhang G., Zhang Y., Hao M., Zhang Z., Fan P., Dong Y., Yang Y., et al. A neutralizing human antibody binds to the N-terminal domain of the Spike protein of SARS-CoV-2. Science. 2020;369:650–655. doi: 10.1126/science.abc6952. [DOI] [PMC free article] [PubMed] [Google Scholar]
5. Collier D.A., De Marco A., Ferreira I., Meng B., Datir R., Walls A.C., Kemp S.S., Bassi J., Pinto D., Fregni C.S., et al. Sensitivity of SARS-CoV-2 B.1.1.7 to mRNA vaccine-elicited antibodies. Nature. 2021;593:136–141. doi: 10.1038/s41586-021-03412-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
6. Ferreira I., Kemp S.A., Datir R., Saito A., Meng B., Rakshit P., Takaori-Kondo A., Kosugi Y., Uriu K., Kimura I., et al. SARS-CoV-2 B.1.617 mutations L452R and E484Q are not synergistic for antibody evasion. J. Infect. Dis. 2021;224:989–994. doi: 10.1093/infdis/jiab368. [DOI] [PMC free article] [PubMed] [Google Scholar]
7. Fiser A., Do R.K., Sali A. Modeling of loops in protein structures. Protein Sci. 2000;9:1753–1773. doi: 10.1110/ps.9.9.1753. [DOI] [PMC free article] [PubMed] [Google Scholar]
8. Gao A., Chen Z., Amitai A., Doelger J., Mallajosyula V., Sundquist E., Segal F.P., Carrington M., Davis M.M., Streeck H., et al. Learning from HIV-1 to predict the immunogenicity of T cell epitopes in SARS-COV-2. iScience. 2021:102311. doi: 10.1016/j.isci.2021.102311. [DOI] [PMC free article] [PubMed] [Google Scholar]
9. Garcia-Beltran W.F., Lam E.C., St Denis K., Nitido A.D., Garcia Z.H., Hauser B.M., Feldman J., Pavlovic M.N., Gregory D.J., Poznansky M.C., et al. Multiple SARS-CoV-2 variants escape neutralization by vaccine-induced humoral immunity. Cell. 2021;184:2372–2383.e9. doi: 10.1016/j.cell.2021.03.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
10. Gobeil S.M., Janowska K., McDowell S., Mansouri K., Parks R., Stalls V., Kopp M.F., Manne K., Li D., Wiehe K., et al. Effect of natural mutations of SARS-CoV-2 on spike structure, conformation, and antigenicity. Science. 2021;373:1353–1360. doi: 10.1126/science.abi6226. [DOI] [PMC free article] [PubMed] [Google Scholar]
11. Gonzalez-Galarza F.F., McCabe A., Santos E., Jones J., Takeshita L., Ortega-Rivera N.D., Cid-Pavon G.M.D., Ramsbottom K., Ghattaoraya G., Alfirevic A., et al. Allele frequency net database (AFND) 2020 update: gold-standard data classification, open access genotype data and new query tools. Nucleic Acids Res. 2020;48:D783–D788. doi: 10.1093/nar/gkz1029. [DOI] [PMC free article] [PubMed] [Google Scholar]
12. Hacisuleyman E., Hale C., Saito Y., Blachere N.E., Bergh M., Conlon E.G., Schaefer-Babajew D.J., DaSilva J., Muecksch F., Gaebler C., et al. Vaccine breakthrough infections with SARS-CoV-2 variants. N. Engl. J. Med. 2021;384:2212–2218. doi: 10.1056/NEJMoa2105000. [DOI] [PMC free article] [PubMed] [Google Scholar]
13. Harvey W.T., Carabelli A.M., Jackson B., Gupta R.K., Thomson E.C., Harrison E.M., Ludden C., Reeve R., Rambaut A., Consortium C.-G.U., et al. SARS-CoV-2 variants, spike mutations and immune escape. Nat. Rev. Microbiol. 2021;19:409–424. doi: 10.1038/s41579-021-00573-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
14. Hoffmann M., Arora P., Gross R., Seidel A., Hornich B.F., Hahn A.S., Kruger N., Graichen L., Hofmann-Winkler H., Kempf A., et al. SARS-CoV-2 variants B.1.351 and P.1 escape from neutralizing antibodies. Cell. 2021;184:2384–2393.e12. doi: 10.1016/j.cell.2021.03.036. [DOI] [PMC free article] [PubMed] [Google Scholar]
15. Hu C., Shen M., Han X., Chen Q., Li L., Chen S., Zhang J., Gao F., Wang W., Wang Y., et al. Identification of cross-reactive CD8+ T cell receptors with high functional avidity to a SARS-CoV-2 immunodominant epitope and its natural mutant variants. Genes Dis. 2021;9:216–229. doi: 10.1016/j.gendis.2021.05.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
16. Jackson B., Boni M.F., Bull M.J., Colleran A., Colquhoun R.M., Darby A., Haldenby S., Hill V., Lucaci A., McCrone J.T., et al. Generation and transmission of interlineage recombinants in the SARS-CoV-2 pandemic. Cell. 2021;184:5179–5188.e8. doi: 10.1016/j.cell.2021.08.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
17. Jacobson K.B., Pinsky B.A., Montez Rath M.E., Wang H., Miller J.A., Skhiri M., Shepard J., Mathew R., Lee G., Bohman B., et al. Post-vaccination SARS-CoV-2 infections and incidence of presumptive B.1.427/B.1.429 variant among healthcare personnel at a northern California academic medical center. Clin. Infect. Dis. 2021 doi: 10.1093/cid/ciab554. [DOI] [PMC free article] [PubMed] [Google Scholar]
18. Jara A., Undurraga E.A., Gonzalez C., Paredes F., Fontecilla T., Jara G., Pizarro A., Acevedo J., Leo K., Leon F., et al. Effectiveness of an inactivated SARS-CoV-2 vaccine in Chile. N. Engl. J. Med. 2021;385:946–948. doi: 10.1056/NEJMoa2107715. [DOI] [PMC free article] [PubMed] [Google Scholar]
19. Karaki S., Kariyone A., Kato N., Kano K., Iwakura Y., Takiguchi M. HLA-B51 transgenic mice as recipients for production of polymorphic HLA-A, B-specific antibodies. Immunogenetics. 1993;37:139–142. doi: 10.1007/BF00216838. [DOI] [PubMed] [Google Scholar]
20. Kared H., Redd A.D., Bloch E.M., Bonny T.S., Sumatoh H., Kairi F., Carbajo D., Abel B., Newell E.W., Bettinotti M.P., et al. SARS-CoV-2-specific CD8+ T cell responses in convalescent COVID-19 individuals. J. Clin. Invest. 2021;131 doi: 10.1172/JCI145476. [DOI] [PMC free article] [PubMed] [Google Scholar]
21. Katoh K., Standley D.M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 2013;30:772–780. doi: 10.1093/molbev/mst010. [DOI] [PMC free article] [PubMed] [Google Scholar]
22. Kobayashi Y., Suzuki Y. Evidence for N-glycan shielding of antigenic sites during evolution of human influenza A virus hemagglutinin. J. Virol. 2012;86:3446–3451. doi: 10.1128/JVI.06147-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
23. Li D., Edwards R.J., Manne K., Martinez D.R., Schafer A., Alam S.M., Wiehe K., Lu X., Parks R., Sutherland L.L., et al. In vitro and in vivo functions of SARS-CoV-2 infection-enhancing and neutralizing antibodies. Cell. 2021;184:4203–4219.e32. doi: 10.1016/j.cell.2021.06.021. [DOI] [PMC free article] [PubMed] [Google Scholar]
24. Li Q., Wu J., Nie J., Zhang L., Hao H., Liu S., Zhao C., Zhang Q., Liu H., Nie L., et al. The impact of mutations in SARS-CoV-2 spike on viral infectivity and antigenicity. Cell. 2020;182:1284–1294 e9. doi: 10.1016/j.cell.2020.07.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
25. Li Y., Cleveland B., Klots I., Travis B., Richardson B.A., Anderson D., Montefiori D., Polacino P., Hu S.L. Removal of a single N-linked glycan in human immunodeficiency virus type 1 gp120 results in an enhanced ability to induce neutralizing antibody responses. J. Virol. 2008;82:638–651. doi: 10.1128/JVI.01691-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
26. Liu J., Liu Y., Xia H., Zou J., Weaver S.C., Swanson K.A., Cai H., Cutler M., Cooper D., Muik A., et al. BNT162b2-elicited neutralization of B.1.617 and other SARS-CoV-2 variants. Nature. 2021;596:273–275. doi: 10.1038/s41586-021-03693-y. [DOI] [PubMed] [Google Scholar]
27. Liu Y., Liu J., Xia H., Zhang X., Fontes-Garfias C.R., Swanson K.A., Cai H., Sarkar R., Chen W., Cutler M., et al. Neutralizing activity of BNT162b2-elicited serum. N. Engl. J. Med. 2021;384:1466–1468. doi: 10.1056/NEJMc2102017. [DOI] [PMC free article] [PubMed] [Google Scholar]
28. Liu Y., Soh W.T., Kishikawa J.I., Hirose M., Nakayama E.E., Li S., Sasai M., Suzuki T., Tada A., Arakawa A., et al. An infectivity-enhancing site on the SARS-CoV-2 spike protein targeted by antibodies. Cell. 2021;184:3452–3466 e3418. doi: 10.1016/j.cell.2021.05.032. [DOI] [PMC free article] [PubMed] [Google Scholar]
29. Lok S.M. An NTD supersite of attack. Cell Host Microbe. 2021;29:744–746. doi: 10.1016/j.chom.2021.04.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
30. McCallum M., De Marco A., Lempp F.A., Tortorici M.A., Pinto D., Walls A.C., Beltramello M., Chen A., Liu Z., Zatta F., et al. N-terminal domain antigenic mapping reveals a site of vulnerability for SARS-CoV-2. Cell. 2021;184:2332–2347.e6. doi: 10.1016/j.cell.2021.03.028. [DOI] [PMC free article] [PubMed] [Google Scholar]
31. Meng B., Kemp S.A., Papa G., Datir R., Ferreira I., Marelli S., Harvey W.T., Lytras S., Mohamed A., Gallo G., et al. Recurrent emergence of SARS-CoV-2 spike deletion H69/V70 and its role in the Alpha variant B.1.1.7. Cell Rep. 2021;35:109292. doi: 10.1016/j.celrep.2021.109292. [DOI] [PMC free article] [PubMed] [Google Scholar]
32. Minh B.Q., Schmidt H.A., Chernomor O., Schrempf D., Woodhams M.D., von Haeseler A., Lanfear R. IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era. Mol. Biol. Evol. 2020;37:1530–1534. doi: 10.1093/molbev/msaa015. [DOI] [PMC free article] [PubMed] [Google Scholar]
33. Mlcochova P., Kemp S.A., Dhar M.S., Papa G., Meng B., Ferreira I., Datir R., Collier D.A., Albecka A., Singh S., et al. SARS-CoV-2 B.1.617.2 Delta variant replication and immune evasion. Nature. 2021;599:114–119. doi: 10.1038/s41586-021-03944-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
34. Motozono C., Toyoda M., Zahradnik J., Saito A., Nasser H., Tan T.S., Ngare I., Kimura I., Uriu K., Kosugi Y., et al. SARS-CoV-2 spike L452R variant evades cellular immunity and increases infectivity. Cell Host Microbe. 2021;29:1124–1136. doi: 10.1016/j.chom.2021.06.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
35. Niwa H., Yamamura K., Miyazaki J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene. 1991;108:193–199. doi: 10.1016/0378-1119(91)90434-d. [DOI] [PubMed] [Google Scholar]
36. Nixon D.F., Ndhlovu L.C. Vaccine breakthrough infections with SARS-CoV-2 variants. N. Engl. J. Med. 2021;385:e7. doi: 10.1056/NEJMc2107808. [DOI] [PubMed] [Google Scholar]
37. Ozono S., Zhang Y., Ode H., Sano K., Tan T.S., Imai K., Miyoshi K., Kishigami S., Ueno T., Iwatani Y., et al. SARS-CoV-2 D614G spike mutation increases entry efficiency with enhanced ACE2-binding affinity. Nat. Commun. 2021;12:848. doi: 10.1038/s41467-021-21118-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
38. Ozono S., Zhang Y., Tobiume M., Kishigami S., Tokunaga K. Super-rapid quantitation of the production of HIV-1 harboring a luminescent peptide tag. J. Biol. Chem. 2020;295:13023–13030. doi: 10.1074/jbc.RA120.013887. [DOI] [PMC free article] [PubMed] [Google Scholar]
39. Peleg Y., Unger T. Application of the restriction-free (RF) cloning for multicomponents assembly. Methods Mol. Biol. 2014;1116:73–87. doi: 10.1007/978-1-62703-764-8_6. [DOI] [PubMed] [Google Scholar]
40. Piccoli L., Park Y.J., Tortorici M.A., Czudnochowski N., Walls A.C., Beltramello M., Silacci-Fregni C., Pinto D., Rosen L.E., Bowen J.E., et al. Mapping neutralizing and immunodominant sites on the SARS-CoV-2 spike receptor-binding domain by structure-guided high-resolution serology. Cell. 2020;183:1024–1042 e1. doi: 10.1016/j.cell.2020.09.037. [DOI] [PMC free article] [PubMed] [Google Scholar]
41. Planas D., Bruel T., Grzelak L., Guivel-Benhassine F., Staropoli I., Porrot F., Planchais C., Buchrieser J., Rajah M.M., Bishop E., et al. Sensitivity of infectious SARS-CoV-2 B.1.1.7 and B.1.351 variants to neutralizing antibodies. Nat. Med. 2021;27:917–924. doi: 10.1038/s41591-021-01318-5. [DOI] [PubMed] [Google Scholar]
42. Plante J.A., Liu Y., Liu J., Xia H., Johnson B.A., Lokugamage K.G., Zhang X., Muruato A.E., Zou J., Fontes-Garfias C.R., et al. Spike mutation D614G alters SARS-CoV-2 fitness. Nature. 2021;592:116–121. doi: 10.1038/s41586-020-2895-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
43. Quinones-Kochs M.I., Buonocore L., Rose J.K. Role of N-linked glycans in a human immunodeficiency virus envelope glycoprotein: effects on protein function and the neutralizing antibody response. J. Virol. 2002;76:4199–4211. doi: 10.1128/JVI.76.9.4199-4211.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
44. Rambaut A., Holmes E.C., O'Toole A., Hill V., McCrone J.T., Ruis C., du Plessis L., Pybus O.G. A dynamic nomenclature proposal for SARS-CoV-2 lineages to assist genomic epidemiology. Nat. Microbiol. 2020;5:1403–1407. doi: 10.1038/s41564-020-0770-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
45. Rana K., Mohindra R., Pinnaka L. Vaccine breakthrough infections with SARS-CoV-2 variants. N. Engl. J. Med. 2021;385:e7. doi: 10.1056/NEJMc2107808. [DOI] [PubMed] [Google Scholar]
46. Reitter J.N., Means R.E., Desrosiers R.C. A role for carbohydrates in immune evasion in AIDS. Nat. Med. 1998;4:679–684. doi: 10.1038/nm0698-679. [DOI] [PubMed] [Google Scholar]
47. Saito A., Irie T., Suzuki R., Maemura T., Nasser H., Uriu K., Kosugi Y., Shirakawa K., Sadamasu K., Kimura I., et al. Enhanced fusogenicity and pathogenicity of SARS-CoV-2 Delta P681R mutation. Nature. 2021 doi: 10.1038/s41586-021-04266-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
48. Sato K., Misawa N., Fukuhara M., Iwami S., An D.S., Ito M., Koyanagi Y. Vpu augments the initial burst phase of HIV-1 propagation and downregulates BST2 and CD4 in humanized mice. J. Virol. 2012;86:5000–5013. doi: 10.1128/JVI.07062-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
49. Sato K., Misawa N., Iwami S., Satou Y., Matsuoka M., Ishizaka Y., Ito M., Aihara K., An D.S., Koyanagi Y. HIV-1 Vpr accelerates viral replication during acute infection by exploitation of proliferating CD4+ T cells in vivo. PLOS Pathog. 2013;9 doi: 10.1371/journal.ppat.1003812. [DOI] [PMC free article] [PubMed] [Google Scholar]
50. Sato K., Misawa N., Nie C., Satou Y., Iwakiri D., Matsuoka M., Takahashi R., Kuzushima K., Ito M., Takada K., et al. A novel animal model of Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis in humanized mice. Blood. 2011;117:5663–5673. doi: 10.1182/blood-2010-09-305979. [DOI] [PubMed] [Google Scholar]
51. Skehel J.J., Stevens D.J., Daniels R.S., Douglas A.R., Knossow M., Wilson I.A., Wiley D.C. A carbohydrate side chain on hemagglutinins of Hong Kong influenza viruses inhibits recognition by a monoclonal antibody. Proc. Natl. Acad. Sci. U S A. 1984;81:1779–1783. doi: 10.1073/pnas.81.6.1779. [DOI] [PMC free article] [PubMed] [Google Scholar]
52. Suchard M.A., Lemey P., Baele G., Ayres D.L., Drummond A.J., Rambaut A. Bayesian phylogenetic and phylodynamic data integration using BEAST 1.10. Virus Evol. 2018;4:vey016. doi: 10.1093/ve/vey016. [DOI] [PMC free article] [PubMed] [Google Scholar]
53. Touret F., Luciani L., Baronti C., Cochin M., Driouich J.S., Gilles M., Thirion L., Nougairede A., de Lamballerie X. Replicative fitness of a SARS-CoV-2 20I/501Y.V1 variant from lineage B.1.1.7 in human reconstituted bronchial epithelium. mBio. 2021;12 doi: 10.1128/mBio.00850-21. [DOI] [PMC free article] [PubMed] [Google Scholar]
54. Wagh K., Hahn B.H., Korber B. Hitting the sweet spot: exploiting HIV-1 glycan shield for induction of broadly neutralizing antibodies. Curr. Opin. HIV AIDS. 2020;15:267–274. doi: 10.1097/COH.0000000000000639. [DOI] [PMC free article] [PubMed] [Google Scholar]
55. Wall E.C., Wu M., Harvey R., Kelly G., Warchal S., Sawyer C., Daniels R., Adams L., Hobson P., Hatipoglu E., et al. AZD1222-induced neutralising antibody activity against SARS-CoV-2 Delta VOC. Lancet. 2021;398:207–209. doi: 10.1016/S0140-6736(21)01462-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
56. Wang C.C., Chen J.R., Tseng Y.C., Hsu C.H., Hung Y.F., Chen S.W., Chen C.M., Khoo K.H., Cheng T.J., Cheng Y.S., et al. Glycans on influenza hemagglutinin affect receptor binding and immune response. Proc. Natl. Acad. Sci. U S A. 2009;106:18137–18142. doi: 10.1073/pnas.0909696106. [DOI] [PMC free article] [PubMed] [Google Scholar]
57. Wang P., Casner R.G., Nair M.S., Wang M., Yu J., Cerutti G., Liu L., Kwong P.D., Huang Y., Shapiro L., et al. Increased resistance of SARS-CoV-2 variant P.1 to antibody neutralization. Cell Host Microbe. 2021;29:747–751 e744. doi: 10.1016/j.chom.2021.04.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
58. Wang P., Nair M.S., Liu L., Iketani S., Luo Y., Guo Y., Wang M., Yu J., Zhang B., Kwong P.D., et al. Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7. Nature. 2021;593:130–135. doi: 10.1038/s41586-021-03398-2. [DOI] [PubMed] [Google Scholar]
59. WHO Tracking SARS-CoV-2 variants. 2021.https://www.who.int/en/activities/tracking-SARS-CoV-2-variants/
60. WHO WHO coronavirus (COVID-19) dashboard in Chile. 2021.https://covid19.who.int/region/amro/country/cl
61. Wolfl M., Kuball J., Ho W.Y., Nguyen H., Manley T.J., Bleakley M., Greenberg P.D. Activation-induced expression of CD137 permits detection, isolation, and expansion of the full repertoire of CD8+ T cells responding to antigen without requiring knowledge of epitope specificities. Blood. 2007;110:201–210. doi: 10.1182/blood-2006-11-056168. [DOI] [PMC free article] [PubMed] [Google Scholar]
62. Wrobel A.G., Benton D.J., Xu P., Roustan C., Martin S.R., Rosenthal P.B., Skehel J.J., Gamblin S.J. SARS-CoV-2 and bat RaTG13 spike glycoprotein structures inform on virus evolution and furin-cleavage effects. Nat. Struct. Mol. Biol. 2020;27:763–767. doi: 10.1038/s41594-020-0468-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
63. Yan R., Zhang Y., Li Y., Xia L., Guo Y., Zhou Q. Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2. Science. 2020;367:1444–1448. doi: 10.1126/science.abb2762. [DOI] [PMC free article] [PubMed] [Google Scholar]
64. Yurkovetskiy L., Wang X., Pascal K.E., Tomkins-Tinch C., Nyalile T.P., Wang Y., Baum A., Diehl W.E., Dauphin A., Carbone C., et al. Structural and functional analysis of the D614G SARS-CoV-2 spike protein variant. Cell. 2020;183:739–751 e8. doi: 10.1016/j.cell.2020.09.032. [DOI] [PMC free article] [PubMed] [Google Scholar]
65. Zahradník J., Dey D., Marciano S., Kolářová L., Charendoff C.I., Subtil A., Schreiber G. A protein-engineered, enhanced yeast display platform for rapid evolution of challenging targets. Acs. Synth. Biol. 2021;10:3445–3460. doi: 10.1021/acssynbio.1c00395. [DOI] [PMC free article] [PubMed] [Google Scholar]
66. Zahradnik J., Marciano S., Shemesh M., Zoler E., Harari D., Chiaravalli J., Meyer B., Rudich Y., Li C., Marton I., et al. SARS-CoV-2 variant prediction and antiviral drug design are enabled by RBD in vitro evolution. Nat. Microbiol. 2021;6:1188–1198. doi: 10.1038/s41564-021-00954-4. [DOI] [PubMed] [Google Scholar]

Supplementary Materials

Data Availability Statement

• •

  The data reported in this paper will be shared by the lead contact upon request.

• •

  This paper does not report original code.

• •

  Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.