SARS-CoV-2 N escape mutations do not affect the rapid antigen test


The ongoing Coronavirus 2019 (COVID-19) pandemic has killed more than 6.4 million people, with new cases continuing to emerge. With widespread vaccination campaigns, the spread of the virus was expected to decrease. The occurrence of solitary infection and re-infection proved this hope wrong.

The study: Deep Mutagenesis Scan identifies escape mutations from SARS-CoV-2 Nucleocapsid for currently available rapid antigen tests.  Image Credit: eakasarn / Shutterstock
Stady: Deep Mutagenesis Scan identifies escape mutants of SARS-CoV-2 Nucleocapsid for currently available rapid antigen tests. Image Credit: eakasarn / Shutterstock

A new study identifies escape mutations found in different strains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that can cause them to escape neutralization by antibodies or detection by rapid antigen binding-dependent tests. to get a positive result.

an introduction

Diagnostic tests for COVID-19 were launched as early as April 2020, using in the laboratory Reporting using viral sequencing of a virus ancestral variant, the Wuhan variant. With the emergence of new variants, questions have arisen about the performance of these tests, as they exploit the ability of antibodies to recognize and bind to the virus. Antigens. This recognition is subverted in the presence of escape mutants.

The current study is published in the journal cella new method for assessing the effect of such N-target mutations on antigen recognition by diagnostic antibodies used in rapid antigen tests.

Rapid antigen tests help detect the presence of SARS-CoV-2 quickly and easily. In most cases, they use the viral nucleocapsid antigen (N), which is abundantly present in viral particles as well as in infected individuals. Protein N is key to the viral life cycle, including viral replication and packaging. It contains an RNA-binding domain (N-RBD) and a dimerization domain (N-DD), around which there are three disordered regions.

Antibodies bind to epitopes, which are antigen-specific regions that have a structure complementary to the antibody-binding domain. Epitope mapping is a field that uses multiple techniques, such as structure determination, mass spectrometry, or site-directed mutagenesis, to help identify the locations of antigen-escape mutations. However, none of the currently used methods can directly measure the effect of a particular mutation on antibody binding.

Many researchers are turning to deep mutational scanning (DMS), a method that screens most or all of the mutations in a protein via a library of unique mutations or sequences. These sequences can be checked simultaneously using in the laboratory Selection techniques to enrich for related mutations.

When used in conjunction with cell surface views, DMS has aided in the study of SARS-CoV-2 Spike Protein Interactions with the angiotensin-converting enzyme 2 (ACE2) receptor and key mutations that disrupt antibody binding when RBD is elevated.

In the current study, mammalian cells are used for N-protein surface presentation to achieve a direct quantitative antibody binding assay. By combining this with DMS using a library of all possible amino acid substitutions along the full length of the protein N, the researchers were able to fully test the effects of all potential mutations on antigen binding with the 17 diagnostic antibodies currently used to test them. This virus.

The result is a complete picture of the possible escape mutations for each antibody, scored for the escape probability in each region. The scores show the abundance of the particular mutation in the population of cells expressing escape mutations. Thus, it shows how each mutation affects antibody binding.

The scores thus help identify both the epitope and the diagnostic antibody’s susceptibility to mutations in or near the epitope. The data helps understand how each mutation at these sites affects antibody recognition. In this way, the binding strength of the antibody over the entire N mutational sequence library was measured, taking into account each potential point mutation.

What did the study show?

As expected, most mutations do not reduce antibody binding, and this effect is limited to a small group of mutations at well-defined sites. This includes epitopes bound to antibodies such as R040, C706 and 3C3. In the first case, a mutation at three different loci results in a reduction in binding, but only changes in charged or polar amino acids at another locus produce this effect.

The latter two are examples of antibodies bound to 3D epitopes where only a few mutations are closely related to significantly reduced antibody binding. With 3C3, substitution of E323 always has a significant effect, but not in V324, where mutations only in charged or aromatic amino acids result in reduced binding.

In general, this shows that a particular epitope is sensitive to some substitutions but not others and highlights the detailed information available with that platform.

Any mutation at three sites in the R040 epitope, and four sites in the C706 epitope, abolished the association, but outside of these epitopes, the mutations were insignificant. Interestingly, with 3C3, two mutations abolished the association and one reduced the association affinity by two orders of magnitude. The fourth had only a mild effect, but the other antibodies picked up this mutation at normal binding levels.

This indicates that the partial misfolding of the protein caused by the last mutation was not sufficient to reduce the binding affinity of these antibodies. Long-term effects on association were also seen with some N-RBD mutants, indicating that the association might decline without a noticeable change in affinity.

The study yielded a wealth of information about where antibodies bind to the N protein. It shows how different antibodies evade through their distinct combinations of escape mutants, helping to reveal the underlying mechanism of escape and allowing antibodies to be distinguished even when they bind to intervening loops.

Rapid antigen tests work on the basis of two antibodies, one in a solid and one in a mobile phase, both of which are required to bind to the antigen to generate the signal. Interestingly, the data from this study indicate that these diagnostic groups of antibodies bind to different epitopes, so it is unlikely that they both have the same high degree of escape in a specific region of the N-protein. This means that all antibodies used in these tests can To be connected simultaneously, verify the reliability of these tests.

Together, these data demonstrate that DMS can be used to guide the selection of appropriate antibody pairs in the design of new antigen assays. “

repercussions

The results demonstrate the value of rapid antigen tests in capturing mutations in both current and older variants of SARS-CoV-2. It is unlikely that the currently known N-protein mutations would cause the test to fail.

Second, by providing association test results for all potential point mutations, including those that could emerge in the future, they are important to further trace the evolution of this virus and the evolution of the epidemic. This has tremendous value for clinical and public health management.

Again, this method that combines DMS and mammalian cell surface viewing is superior to the current gold standard, structural epitope mapping, as it directly assesses antibody recognition of a full-length antigen while using a mutagenesis library to measure association with any potential mutation.

Although it does not directly identify the epitope, it provides the fingerprint of an antibody-specific escape mutation. This can be used more broadly to study how antigenic mutations affect antibody binding. It can be adapted to study protein-protein interactions in general, provided that a system for expressing proteins on both sides can be designed.

Moreover, it can help understand other processes such as affinity maturation in the germinal center of B cells, including increased antibody affinity with increased specificity and resistance to antigen mutations.



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