In vitro data suggest that Indian delta variant B.1.617 of SARS-CoV-2 escapes neutralization by both receptor affinity and immune evasion

Abstract

Background: Emerged mutations can be attributed to increased transmissibility of the B.1.617 and B.1.36 Indian delta variants of SARS-CoV-2, most notably substitutions L452R/E484Q and N440K, respectively, which occur in the receptor-binding domain (RBD) of the Spike (S) fusion glycoprotein.

Objective: We aimed to assess the effects of mutations L452R/E484Q and N440K (as well as the previously studied mutation E484K present in variants B.1.351 and P.1) on the affinity of RBD for ACE2, SARS-CoV-2 main receptor. We also aimed to assess the ability of antibodies induced by natural infection or by immunization with BNT162b2 mRNA vaccine to recognize the mutated versions of the RBD, as well as blocking the interaction RBD-ACE2, an important surrogate readout for virus neutralization.

Methods: To this end, we produced recombinant wild-type RBD, as well as RBD containing each of the mutations L452R/E484Q, N440K, or E484K (the latest present in variants of concern B.1.351 and P.1), as well as the ectodomain of ACE2. Using Biolayer Interferometry (BLI), we measured the binding affinity of RBD for ACE2 and the ability of sera from COVID-19 convalescent donors or subjects immunized with BNT162b2 mRNA vaccine to block this interaction. Finally, we correlated these results with total anti-RBD IgG titers measured from the same sera by direct ELISA.

Results: The binding assays showed L452R/E484Q double-mutant RBD to interact with ACE2 with higher affinity (K_D = 4.6 nM) than wild-type (K_D = 21.3 nM) or single mutants N440K (K_D = 9.9 nM) and E484K (K_D = 19.7 nM) RBDs. Meanwhile, the anti-RBD IgG titration resulted in lower recognition of mutants E484K and L452R/E484Q by infection-induced antibodies, whereas only mutant E484K was recognized less by antibodies induced by vaccination. More interestingly, sera from convalescent as well as immunized subjects showed reduced ability to block the interaction between ACE2 and RBD mutants E484K and L452R/E484Q, as shown by the inhibition assays.

Conclusion: Our data suggest that the newly emerged SARS-CoV-2 variant B.1.617, as well as the better-studied variants B.1.351 and P.1 (all containing a mutation at position E484) display increased transmissibility both due to their higher affinity for the cell receptor ACE2 and their ability to partially bypass immunity generated against the wild-type virus. For variant B.1.36 (with a point mutation at position N440), only increased affinity seems to play a role.

Keywords: RBD; SARS-CoV-2; affinity; neutralization; vaccine.

FIGURE 1

Positions of mutations N440K, L452R, and E484Q. (A) S monomer (blue ribbon and surface) bound to ACE2 ectodomain (yellow surface). (B) This interaction is shown in detail, highlighting residues N440, L452, and E484 (green sticks). Interestingly, both residues L452 and E484 are found in close contact with ACE (ie, at the RBM), whereas residue N440 is relatively distant from the interaction surface (ie, out of the RBM). (C) Distance from the nearest residue for N440, L452, and E484 (residues from the RBD shown in green and from ACE2 in orange). From PDB files 6ACG and 2AJF

FIGURE 2

BLI sensorgrams illustrating the interaction between ACE2 and (A) wild‐type RBD (RBD_WT); (B) E484K single‐mutant RBD (RBD_E484K); (C) L452R/E484Q double‐mutant RBD (RBD_L452R/E484Q; and (D)) N440 single‐mutant RBD (RBD_N440K)

FIGURE 3

Comparison of total anti‐RBD IgG and RBD‐ACE2‐blocking antibodies between wild‐type RBD and mutants E484K, L452R/E484Q, and N440K. (A) Total anti‐RBD IgG titers of sera obtained from COVID‐19‐convalescent donors. (B) Inhibition of RBD‐ACE2 binding by 1:20 dilution of the same samples; (C) Total anti‐RBD IgG titers of for sera obtained from donors immunized with two doses of BNT162b2 mRNA COVID‐19 vaccine (Pfizer); (D) Inhibition of RBD‐ACE2 binding by 1:20 dilution of the same samples