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. 2020 Oct 28:9:e61312.
doi: 10.7554/eLife.61312.

Escape from neutralizing antibodies by SARS-CoV-2 spike protein variants

Yiska Weisblum #  1 , Fabian Schmidt #  1 , Fengwen Zhang  1 , Justin DaSilva  1 , Daniel Poston  1 , Julio Cc Lorenzi  2 , Frauke Muecksch  1 , Magdalena Rutkowska  1 , Hans-Heinrich Hoffmann  3 , Eleftherios Michailidis  3 , Christian Gaebler  2 , Marianna Agudelo  2 , Alice Cho  2 , Zijun Wang  2 , Anna Gazumyan  2 , Melissa Cipolla  2 , Larry Luchsinger  4 , Christopher D Hillyer  4 , Marina Caskey  2 , Davide F Robbiani  2   5 , Charles M Rice  3 , Michel C Nussenzweig  2   6 , Theodora Hatziioannou  1 , Paul D Bieniasz  1   6
Affiliations

Escape from neutralizing antibodies by SARS-CoV-2 spike protein variants

Yiska Weisblum et al. Elife. .

Abstract

Neutralizing antibodies elicited by prior infection or vaccination are likely to be key for future protection of individuals and populations against SARS-CoV-2. Moreover, passively administered antibodies are among the most promising therapeutic and prophylactic anti-SARS-CoV-2 agents. However, the degree to which SARS-CoV-2 will adapt to evade neutralizing antibodies is unclear. Using a recombinant chimeric VSV/SARS-CoV-2 reporter virus, we show that functional SARS-CoV-2 S protein variants with mutations in the receptor-binding domain (RBD) and N-terminal domain that confer resistance to monoclonal antibodies or convalescent plasma can be readily selected. Notably, SARS-CoV-2 S variants that resist commonly elicited neutralizing antibodies are now present at low frequencies in circulating SARS-CoV-2 populations. Finally, the emergence of antibody-resistant SARS-CoV-2 variants that might limit the therapeutic usefulness of monoclonal antibodies can be mitigated by the use of antibody combinations that target distinct neutralizing epitopes.

Keywords: COVID19; SARS-CoV-2; VSV; antibody; immunology; infectious disease; inflammation; microbiology; virus.

Plain language summary

The new coronavirus, SARS-CoV-2, which causes the disease COVID-19, has had a serious worldwide impact on human health. The virus was virtually unknown at the beginning of 2020. Since then, intense research efforts have resulted in sequencing the coronavirus genome, identifying the structures of its proteins, and creating a wide range of tools to search for effective vaccines and therapies. Antibodies, which are immune molecules produced by the body that target specific segments of viral proteins can neutralize virus particles and trigger the immune system to kill cells infected with the virus. Several technologies are currently under development to exploit antibodies, including vaccines, blood plasma from patients who were previously infected, manufactured antibodies and more. The spike proteins on the surface of SARS-CoV-2 are considered to be prime antibody targets as they are accessible and have an essential role in allowing the virus to attach to and infect host cells. Antibodies bind to spike proteins and some can block the virus’ ability to infect new cells. But some viruses, such as HIV and influenza, are able to mutate their equivalent of the spike protein to evade antibodies. It is unknown whether SARS-CoV-2 is able to efficiently evolve to evade antibodies in the same way. Weisblum, Schmidt et al. addressed this question using an artificial system that mimics natural infection in human populations. Human cells grown in the laboratory were infected with a hybrid virus created by modifying an innocuous animal virus to contain the SARS-CoV-2 spike protein, and treated with either manufactured antibodies or antibodies present in the blood of recovered COVID-19 patients. In this situation, only viruses that had mutated in a way that allowed them to escape the antibodies were able to survive. Several of the virus mutants that emerged had evolved spike proteins in which the segments targeted by the antibodies had changed, allowing these mutant viruses to remain undetected. An analysis of more than 50,000 real-life SARS-CoV-2 genomes isolated from patient samples further showed that most of these virus mutations were already circulating, albeit at very low levels in the infected human populations. These results show that SARS-CoV-2 can mutate its spike proteins to evade antibodies, and that these mutations are already present in some virus mutants circulating in the human population. This suggests that any vaccines that are deployed on a large scale should be designed to activate the strongest possible immune response against more than one target region on the spike protein. Additionally, antibody-based therapies that use two antibodies in combination should prevent the rise of viruses that are resistant to the antibodies and maintain the long-term effectiveness of vaccines and therapies.

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Conflict of interest statement

YW Rockefeller University has applied for a patent relating to the replication compentent VSV/SARS-CoV-2 chimeric virus on which YW is listed as an inventor (US patent 63/036,124), FS Rockefeller University has applied for a patent relating to the replication compentent VSV/SARS-CoV-2 chimeric virus on which FS is listed as an inventor (US patent 63/036,124), FZ, JD, DP, JL, FM, MR, HH, EM, CG, MA, AC, ZW, AG, MC, LL, CH, MC, CR No competing interests declared, DR Rockefeller University has applied for a patent relating to SARS-CoV-2 monoclonal antibodies on which DFR is listed as an inventor, MN Rockefeller University has applied for a patent relating to SARS-CoV-2 monoclonal antibodies on which MCN is listed as an inventor, TH Rockefeller University has applied for a patent relating to the replication compentent VSV/SARS-CoV-2 chimeric virus on which TH is listed as an inventor (US patent 63/036,124), PB Rockefeller University has applied for a patent relating to the replication compentent VSV/SARS-CoV-2 chimeric virus on which PDB is listed as an inventor (US patent 63/036,124)

Figures

Figure 1.
Figure 1.. Selection of SARS-CoV-2 S mutations that confer antibody resistance.
(A) Outline of serial passage experiments with replication-competent VSV derivatives encoding the SARS-CoV-2 S envelope glycoprotein and a GFP reporter (rVSV/SARS-CoV-2/GFP) in 293T/ACE2(B) cells in the presence of neutralizing antibodies or plasma. Each passage experiment was performed twice (once each with rVSV/SARS-CoV-2/GFP1D7 and rVSV/SARS-CoV-2/GFP2E1.) (B) Representative images of 293T/ACE2(B) cells infected with 1 × 106 PFU of rVSV/SARS-CoV-2/GFP in the presence or absence of 10 μg/ml of the monoclonal antibody C121. (C) Expanded view of the boxed areas showing individual plaques of putatively antibody-resistant viruses.
Figure 2.
Figure 2.. Analysis of S-encoding sequences following rVSV/SARS-CoV-2/GFP replication in the presence of neutralizing monoclonal antibodies.
(A–D) Graphs depict the S codon position (X-axis) and the frequency of non-synonymous substitutions (Y-axis) following the second passage (p2) of rVSV/SARS-CoV-2/GFP on 293T/ACE2(B) cells in the absence of antibody or plasma (A), or in the presence of 10 μg/ml C121 (B), C135 (C) or C144 (D). Results are shown for both rVSV/SARS-CoV-2/GFP variants (One replicate each for rVSV/SARS-CoV-2/GFP1D7 and rVSV/SARS-CoV-2/GFP2E1 - the frequency of 1D7 mutations is plotted as circles and 2E1 mutations as triangles).
Figure 2—figure supplement 1.
Figure 2—figure supplement 1.. Analysis of S-encoding sequences following rVSV/SARS-CoV-2/GFP replication in the presence of convalescent plasma COV-47 and COV-72.
(A–B) Graphs depict the S codon position (X-axis) and the frequency of non-synonymous substitutions (Y-axis) following the second, third or fourth passage (p2–p4) of rVSV/SARS-CoV-2/GFP on 293T/ACE2(B) cells in the presence of COV-47 plasma (A), or COV-72 plasma (B). Results are shown for both rVSV/SARS-CoV-2/GFP variants (the frequency of 1D7 mutations is plotted as circles and 2E1 mutations as triangles).
Figure 2—figure supplement 2.
Figure 2—figure supplement 2.. Analysis of S-encoding sequences following rVSV/SARS-CoV-2/GFP replication in the presence of convalescent plasma COV-107 and COV-NY.
(A–B) Graphs depict the S codon position (X-axis) and the frequency of non-synonymous substitutions (Y-axis) following the second, third or fourth passage (p2–p4) of rVSV/SARS-CoV-2/GFP on 293T/ACE2(B) cells in the presence of COV-107 plasma (A), or second passage in the presence of COV-NY plasma (B). Results are shown for both rVSV/SARS-CoV-2/GFP variants (the frequency of 1D7 mutations is plotted as circles and 2E1 mutations as triangles).
Figure 3.
Figure 3.. Characterization of mutant rVSV/SARS-CoV-2/GFP derivatives.
(A) Replication of plaque-purified rVSV/SARS-CoV-2/GFP bearing individual S amino-acid substitutions that arose during passage with the indicated antibody or plasma. 293T/ACE2cl.22 cells were inoculated with equivalent doses of parental or mutant rVSV/SARS-CoV-2/GFP isolates. Supernatant was collected at the indicated times after inoculation and number of infectious units present therein was determined on 293T/ACE2cl.22 cells. The mean of two independent experiments is plotted. One set of WT controls run concurrently with the mutants are replotted in the upper and lower left panels, A different set of WT controls run concurrently with the mutants is shown in the lower right panel (B) Infection 293T/ACE2cl.22 cells by rVSV/SARS-CoV-2/GFP encoding the indicated S protein mutations in the presence of increasing amounts of a chimeric ACE2-Fc molecule. Infection was quantified by FACS. Mean of two independent experiments is plotted. The WT controls are replotted in each panel.
Figure 3—figure supplement 1.
Figure 3—figure supplement 1.. Example of plaque purification of individual viral mutants from populations passaged in the presence of antibodies.
The upper panels show sequence traces from amplicons obtained from viral populations following replication in the presence of monoclonal antibodies, the bottom panels show sequence traces of amplicons obtained from mutants isolated by limiting dilution of the viral populations.
Figure 4.
Figure 4.. Neutralization of rVSV/SARS-CoV-2/GFP RBD mutants by monoclonal antibodies.
(A) Examples of neutralization resistance of rVSV/SARS-CoV-2/GFP mutants that were isolated following passage in the presence of antibodies. 293T/ACE2cl.22 cells were inoculated with WT or mutant rVSV/SARS-CoV-2/GFP in the presence of increasing amount of each monoclonal antibody, and infection quantified by FACS 16 hr later. Mean and SD from two technical replicates, representative of two independent experiments. (B) Neutralization sensitivity/resistance of rVSV/SARS-CoV-2/GFP mutants isolated following replication in the presence of antibodies. Mean IC50 values were calculated for each virus-monoclonal antibody combination in two independent experiments. (C) Position of neutralization resistance-conferring substitutions. Structure of the RBD (from PDB 6M17 Yan et al., 2020) with positions that are occupied by amino acids where mutations were acquired during replication in the presence of each monoclonal antibody or COV-NY plasma indicated.
Figure 5.
Figure 5.. Loss of binding to monoclonal antibodies by neutralization escape mutants.
(A) Schematic representation of the binding assay in which NanoLuc luciferase is appended to the C-termini of a conformationally stabilized S-trimer. The fusion protein is incubated with antibodies and complexes captured using protein G magnetic beads (B) Bound Nanoluc luciferase quantified following incubation of the indicated WT or mutant Nanoluc-S fusion proteins with the indicated antibodies and Protein G magnetic beads. Mean of three technical replicates at each S-Nanoluc concentration.
Figure 6.
Figure 6.. Neutralization of rVSV/SARS-CoV-2/GFP RBD mutants by convalescent plasma.
(A, B) Neutralization of rVSV/SARS-CoV-2/GFP mutants isolated following replication in the presence COV-47 plasma (A) or COV-NY plasma (B). 293T/ACE2cl.22 cells were inoculated with WT or mutant rVSV/SARS-CoV-2/GFP in the presence of increasing amounts of the indicated plasma, and infection quantified by flow cytometry, 16 hr later. Mean of two technical replicates, representative of two independent experiments (C) Plasma neutralization sensitivity/resistance of rVSV/SARS-CoV-2/GFP mutants isolated following replication in the presence of monoclonal antibodies or convalescent plasma. Mean NT50 values were calculated for each virus-plasma combination from two independent experiments.
Figure 7.
Figure 7.. Effects of naturally occurring RBD amino-acid substitutions on S sensitivity to neutralizing monoclonal antibodies.
(A–C) Neutralization of HIV-based reporter viruses pseudotyped with SARS-CoV-2 S proteins harboring the indicated naturally occurring substitutions. 293T/ACE2cl.22 cells were inoculated with equivalent doses of each pseudotyped virus in the presence of increasing amount of C121 (A) C135 (B) or C144 (C). Mean IC50 values were calculated for each virus-antibody combination from two independent experiments. (D) Position of substitutions conferring neutralization resistance relative to the amino acids close to the ACE2 binding site whose identity varies in global SARS-CoV-2 sequences. The RBD structure (from PDB 6M17 Yan et al., 2020) is depicted with naturally varying amino acids close to the ACE2 binding site colored in yellow. Amino acids whose substitution confers partial or complete (IC50 > 10 μg/ml) resistance to each monoclonal antibody in the HIV-pseudotype assays are indicated for C121 (red) C135 (green) and C144 (purple). (E) Binding of S-NanoLuc fusion protein in relative light units (RLU) to 293T or 293T/ACE2cl.22 cells after preincubation in the absence or presence of C121, C135, and C144 monoclonal antibodies. Each symbol represents a technical replicate.
Figure 8.
Figure 8.. Position and frequency of S amino-acid substitutions in SARS-CoV-2 S.
Global variant frequency reported by CoV-Glue in the SARS-CoV-2 S protein. Each individual variant is indicated by a symbol whose position in the S sequence is indicated on the X-axis and frequency reported by COV-Glue is indicated on the Y-axis. Individual substitutions at positions where mutations conferring resistance to neutralizing antibodies or plasma were found herein are indicated by enlarged and colored symbols: red for C121 and C144, green for C135, purple for COV-47 plasma and orange for COV-NY plasma. The common D/G614 variant is indicated.
Figure 9.
Figure 9.. Suppression of antibody resistance through the use of antibody combinations.
(A) Representative images of 293T/ACE2 (B) cells infected with the equivalent doses of rVSV/SARS-CoV-2/GFP in the absence or presence of 10 μg/ml of one (C144) or 5 μg/ml of each of two (C144 +C135) neutralizing monoclonal antibodies. (B) Expanded view of the boxed areas containing individual plaques from the culture infected in the presence of 10 μg/ml C144. (C) Expanded view of the boxed areas in A containing infected cells from the culture infected in the presence of 5 μg/ml each of (C144 and C135). (D) Infectious virus yield following two passages of rVSV/SARS-CoV-2/GFP in the absence or presence of individual neutralizing antibodies or combinations of two antibodies. Titers were determined on 293T/ACE2cl.22 cells. Each symbol represents a technical replicate and results from two independent experiments using rVSV/SARS-CoV-2/GFP1D7 and rVSV/SARS-CoV-2/GFP2E1 are shown.
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