D614G Spike Mutation Increases SARS CoV-2 Susceptibility to Neutralization

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein acquired a D614G mutation early in the pandemic that confers greater infectivity and is now the globally dominant form. To determine whether D614G might also mediate neutralization escape that could compromise vaccine efficacy, sera from spike-immunized mice, nonhuman primates, and humans were evaluated for neutralization of pseudoviruses bearing either D614 or G614 spike. In all cases, the G614 pseudovirus was moderately more susceptible to neutralization. The G614 pseudovirus also was more susceptible to neutralization by receptor-binding domain (RBD) monoclonal antibodies and convalescent sera from people infected with either form of the virus. Negative stain electron microscopy revealed a higher percentage of the 1-RBD "up" conformation in the G614 spike, suggesting increased epitope exposure as a mechanism of enhanced vulnerability to neutralization. Based on these findings, the D614G mutation is not expected to be an obstacle for current vaccine development.

Keywords: COVID-19; D614G; LNP; SARS-CoV-2; Spike; electron micrograph; mRNA; neutralization; nucleoside-modified; vaccine.

Graphical abstract

Figure 1

The G614 Spike Is Neutralized More Potently than the D614 Spike by Mouse Sera The G614 spike is neutralized more potently than the D614 spike by sera from mice immunized twice at a four-week interval with nucleoside-modified mRNA-LNPs encoding the Wuhan sequence of spike (D614) with diProline stabilization mutations. Sera (10 animals/group) obtained four weeks after the second immunization were tested for neutralization against pseudoviruses with the D614 and G614 variants of spike. Log₁₀ of the inhibitory dose to reduce infection by 50% (ID50) and by 80% (ID80) are shown; higher values indicate increased neutralization activity. Each pair of bars represents serum from one animal; for each serum, the blue bar shows the neutralization titer against the G614 form, and the orange bar is the titer against the original D614 form. Top panels are ID50; bottom panels are ID80. Summary statistics for each group are shown at the bottom. The geometric means for the ratio of G614:D614 neutralizing antibodies are shown. Log₁₀ of values of the ID50 and ID80 titers were used in a paired t test to calculate the p value and the 95% CI of geometric mean for the ratio of G614:D614.

Figure 2

The G614 Spike Is Neutralized More Potently than the D614 Spike by NHP Sera The G614 spike is neutralized more potently than the D614 spike by sera from NHPs (rhesus macaques) immunized with nucleoside-modified mRNA-LNPs encoding RBD and full-length spike immunogens and humans immunized with RBD trimers. Sera from macaques immunized twice at a four-week interval with the Wuhan sequence of spike (D614) with a mutated furin cleavage site (n = 5) or secreted RBD monomers (n = 6) obtained four weeks after the second immunization were tested for neutralization against pseudoviruses with the D614 and G614 variants of spike (A). Also shown are sera from five humans immunized twice at a three-week interval with nucleoside-modified mRNA-LNPs encoding a secreted RBD trimer (B). Each pair of bars represents one macaque or human. Top panels are ID50; bottom panels are ID80. For each serum, the blue bar shows the neutralization sensitivity of the G614 form, and the orange bar shows the original D614 form. The geometric means for the ratio of G614:D614 neutralizing antibody titers measured in sera are provided in the summary at the bottom. Log₁₀ of values of the ID50 and ID80 titers were used in a paired t test to calculate the p value and the 95% CI of geometric mean for the ratio of G614:D614. Overall response levels were comparable between the two different immunogens in the NHP and between NHPs and humans.

Figure 3

G614 Spike-Pseudotyped Virus Is Neutralized More Potently than D614 Spike-Pseudotyped Virus by Human Sera and mAbs G614 spike-pseudotyped virus is neutralized more potently than D614 spike-pseudotyped virus by sera from people infected with either the D614 or G614 variant of spike or RBD-specific mAbs. (A) Each blue/orange pair of bars represents convalescent serum sampled from one person. Left, people infected with D614. Right, people infected with G614. (B) MAbs were assayed at 3-fold dilutions starting at 50 μg/mL for a total of eight dilutions. IC50 and IC80 values are in μg/mL, where a lower bar height corresponds to greater neutralization potency. MPI was calculated as the percentage of neutralization at the highest mAb concentration tested.

Figure 4

Negative Stain Electron Microscopy Reconstructions and 3D Classification of Expressed Spike Constructs Negative stain electron microscopy reconstructions of expressed spike constructs after 3D classification. View is looking down the 3-fold trimer axis onto the S1 domain. (A) D614 variant showing the 3-RBD-down structure on the left with individual RBDs labeled (R), and the 1-RBD-up structure on the right with the up RBD labeled (asterisk). The fraction of particle images that sorted into each class is indicated below, expressed as average ± standard deviation, n = 3 each. (B) G614 variant also showing 3-down and 1-up structures.