SARS-CoV-2 escape mutations identified for monoclonal antibodies and polyclonal immune sera

The 2019 coronavirus disease (COVID-19) pandemic shows no signs of ending soon, and the definitive solution is to achieve herd immunity through vaccination or spontaneous infection.Development of effective vaccines that can be induced by unacceptably high mortality associated with the latter pathway Neutralizing antibody Countermeasures against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the disease, are an urgent issue.

Therapeutic antibodies and antibodies induced by current vaccines are effective in neutralizing viral antigens belonging to. Spike protein Of SARS-CoV-2. However, as new mutants emerge, understanding the epitopes to which these protective antibodies bind is an important area of ​​research. Due to these fluctuations, it is also necessary to investigate the development of natural tolerance to neutralization.

New papers published in the journal Cell hosts and microorganisms It describes the selection of SARS-CoV-2 mutants related to resistance to antibodies and has important implications for vaccine development and the use of therapeutic antibodies.

Mutation of SARS-CoV-2

Every RNA virus has multiple variant genome sequences that form a herd or quasi-species around the core consensus genome. When a neutralizing antibody or drug acts on a virus, several mutants are selected for resistance to neutralization. If they have a high fitness, this leads to the emergence of resistant variants.

The· Coronavirus The mutation rate is relatively low among RNA viruses. This is due to the presence of calibrators to correct random changes in the RNA genome introduced by viral RNA-dependent RNA polymerase (RdRp).

However, the SARS-CoV-2 spike gene still shows over 4,000 mutations, which causes over 1,200 amino acid substitutions. Of these, nearly 190 are in the receptor binding domain (RBD). The presence of a large number of circulating viral variants indicates retention of virus fitness.

Such non-synonymous mutations persist due to host adaptation, selection of escape mutations by neutralizing antibodies formed during spontaneous infection, and possibly reinfection of immunocompromised individuals. There is a possibility.

Selection of escape mutants

Current research uses a panel of 19 antibodies to identify escape mutations. The researchers used the vesicular stomatitis virus (VSV), which expresses the SARS-CoV-2 spikes indicated by VSV-SARS-CoV-2. This replication virus may form high titers while binding to the human angiotensin converting enzyme 2 (ACE2) receptor. It is also neutralized with an anti-spike monoclonal antibody.

After treatment with neutralizing antibodies, the researchers sequenced the plaque-forming mutant. The cells were then infected with these isolates in the presence and absence of the antibody to confirm that they were resistant.

Researchers have been able to isolate 50 escaped variants with multiple substitutions in RBD, often within areas of direct contact with ACE2. This suggests that for many neutralizing antibodies that bind to the aspects of RBD, the mechanism of action is due to allosteric interference of spike-ACE2 binding. Another possible mechanism is by blocking the interaction of the virus with other attachment factors.

For mutations at the RBD base, allosteric inhibition of the “up” conformation required for binding to the ACE2 receptor may contribute to resistance.

Escape from multiple monoclonal antibodies

They also found that some mutations conferred resistance to multiple antibodies. Each of these changes exhibited a unique pattern of resistance when multiple substitutions were possible in different side chains for a given residue. S477N and S477G conferred resistance on all antibodies used.

Researchers recommend further study of the S477N mutation, a fairly common spike variant, due to its broad resistance to the panel of monoclonal antibodies studied here. However, this mutation is still susceptible to neutralization by human immune serum, indicating that this epitope is not immune-dominant in humans.

Escape from inhibition by polyclonal human serum

E484 substitution with any of the four possible residues was associated with escape from neutralization with four polyclonal human sera. One of the four sera could only escape with a change in this position. Substitution with E484 increased infectivity even in the presence of multiple sera.

Several other substitutions allowed escape from inhibition by two or three sera.

Escape from soluble ACE2 decoy inhibition

On the other hand, the F486S mutation was associated with escape from inhibition by multiple antibodies and inhibition by soluble recombinant human ACE2, which is expected to act as a decoy for the virus and prevent host cells from binding to the ACE2 receptor. ..

When they compared 50 resistant mutants to clinical isolates, they found that some of the mutants already in circulation were resistant to both monoclonal and polyclonal antibodies. “Neutralizing mAbs against RBD allows you to choose mutations or changes at locations that already exist in the population.”

Selection of escape mutants

Researchers have also been able to demonstrate that the use of monoclonal antibody cocktails can delay the emergence of such escaped variants, even if they are not completely blocked. Such combinations bind to different epitopes of peplomer. However, if the circulating strain already contains an escape mutation for one of the antibodies in the cocktail, you can choose an additional escape mutation.

What is the impact?

This study shows that some escape mutants, such as S477N, are resistant to neutralization by multiple monoclonal antibodies. Others, such as the E484K, show an escape from neutralization by human immune serum. The latter finding indicates that the polyclonal neutralization response in at least some convalescent patients is due to antibodies that bind only a few epitopes.

A significant number of escape variants identified here, such as E484, indicate substitutions at residues where the circulating isolate is already mutated. This means that escape variants can appear in the presence of such a limited polyclonal response. This indicates that the usefulness of vaccines that induce these neutralizing antibodies should be limited and the spectrum of neutralizing antibody responses should be increased.

Second, escape mutations are very easily selected and are already frequently present in clinical isolates, so effective therapeutic antibodies will probably be needed in the form of cocktails. Accurate knowledge of the association between different residues and resistance to each monoclonal antibody helps in choosing the correct combination and avoids duplicate escape mutations.

However, resistance to such cocktails can manifest through sequential escaping if a mutant that evades neutralization by one monoclonal antibody subsequently acquires an escape mutation in another monoclonal antibody. This process has been demonstrated in current studies and will help identify such mutants.