Identification of antibody-resistant SARS-CoV-2 spike protein mutations

Researchers have used different sets of monoclonal antibodies to identify different mutations in the SARS-CoV-2 spike protein that lead to resistance. Understanding these resistance mutations is important in developing effective therapeutic strategies.

Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), the cause of the COVID-19 pandemic, infects host cells via spike proteins on the surface of the virus. The N-terminal subunit (S1) plays a role in receptor binding, and the C-terminal subunit (S2) aids in membrane fusion between the virus and the host cell.

The receptor binding domain (RBD) of S1 binds to human angiotensin converting enzyme 2 (ACE2). SARS-CoV-2 antibody protects against infection by targeting RBD.

RNA viruses such as SARS-CoV-2 exist as a group of genomic sequences around the core sequence. Mutants may escape from this herd and become resistant in the presence of antibodies or drugs.

Currently, more than 2,700 mutations have been identified in the SARS-CoV-2 virus spike protein. There may be several mechanisms for these mutations, including host adaptation and immune selection during natural infection.Additional variants include vaccines, therapeutic antibodies, and Convalescent plasma Treatments that may reduce the effectiveness of these treatments.

Mutations resistant to antibodies

In the preprint paper published in bioRxiv* Researchers at Washington University in St. Louis, Harvard, and May York Clinic report on the status of resistance mutations in SARS-CoV-2RBD using a variety of monoclonal antibodies (mAbs).

They used a chimeric infectious vesicular stomatitis virus (VSV), a SARS-CoV-2 mimic, to replace glycoproteins with SARS-CoV-2 spike proteins. The authors used mAb 2B04 to confirm that the unneutralized SARS-CoV-2 mutant had a mutation in the RBD associated with residues involved in ACE2 binding.

Further testing with the other nine mAbs revealed similar results. For mAb 2H04, resistance mutations were present outside the ACE2 binding site, on the sides and base of the RBD. This suggests that viral neutralization can be caused by alternative adherent factors.

Some resistance mutations are common among the various mAbs tested, suggesting that they represent important antigenic sites on RBD.

Resistance mutations originating from different mAbs resulted in resistance to other mAbs in the selected mAb array. Substitutions with S477 and E484 resulted in broad tolerance, and substitutions at several other sites resulted in resistance to multiple mAbs.

Soluble human ACE2 receptors, which do not attach to cells and can compete with receptors on host cells to bind to the virus, are another strategy being investigated to combat the virus. The authors tested the resistance of VeroE6 cells to ACE2, which is soluble in humans and mice. Human soluble ACE2 neutralized all escaped mutants, but some mutations required higher ACE2 concentrations to neutralize.

The authors also used the sera of four convalescent COVID-19 patients to test whether serum antibodies neutralized the escape mutant virus. They found many mutations that were resistant to serum neutralization. In particular, the mutation at residue E484 is resistant to all four sera, suggesting that this is the major neutralizing epitope. However, substitutions at this position were very rare and were found only in about 0.05% of the sequenced strains.

VSV-SARS-CoV-2 avoids mutant isolation. (A) Outline of escape mutant selection experiment. 2B04 and control anti-influenza mAbs were tested for neutralizing activity against VSV-SARS-CoV-2. The concentration of 2B04 added to the overlay completely suppressed the virus infection (center panel). The data are representative of two independent experiments. A plaque assay was performed to isolate VSV-SARS-CoV-2 escape mutants on Vero E6 TMPRSS2 cells (shown by red arrows). Plaque assay using 2B04 for overlay (bottom plaque on right panel). Plaque assay without Ab in overlay (upper plaque on right panel). The data are representative images of three independent experiments. (B) Schematic diagram of the S gene that has undergone sanger sequencing to identify mutations (left panel). For validation, each VSV SARS-CoV-2 mutant was tested in a plaque assay with and without 2BO4 on Vero cell overlays (right panel). Representative images of two independent experiments are shown.

Resistance mutations found in virus isolates from humans

The authors also tested additional mutations by adding up to 48 different escape mutants. The team also edited the available genomic sequences of the virus and compared them to the genomic sequences of the mutants to see if any of these mutants were present in human isolates of SARS-CoV-2. Was investigated. They found that 27 of the 48 mutations circulate in humans, the most common mutation being D614G, which was observed in 86% of isolates.

Substitution with S477N, which conferred some resistance to all mAbs, was the second most abundant mutant in human isolates.

The author also notes some limitations of the study. VSV is an effective mimic of the SARS-CoV-2 virus, but 27 escape variants were found only in human isolates of the virus. They are also looking at some human sera tested. More human serum samples may help determine the degree of neutralization and escape variants present.

If such changes occur in the virus after vaccination, they may be restricted Effectiveness of processing. “Relatively low genetic barriers to resistance combined with knowledge of the presence of relevant substitutions in clinical isolates suggest that effective mAb therapy is likely to require at least two combinations. Neutralizing antibodyDetermining residues that are resistant to a particular antibody may help select combinations based on non-overlapping resistance mutations.

*Important Notices

bioRxiv Publish preliminary scientific reports that should not be considered definitive as they are not peer-reviewed, guide clinical / health-related behaviors, and should not be treated as established information.