COVID-19 Mutations: International Study Warns Of More Strains Emerging That Can Evade Human Immune Response, New N439K Mutation Is A Concern.

COVID-19 Mutations: A new international study involving researchers from United Kingdom, United States, Switzerland, Australia and Italy from more than 23 institutions and entities are warning that more variants or strains that can evade human host immune response can emerge as a result of mutations in the spike proteins. One such spike protein mutation known as N439K is already becoming a major concern and if more strains emerge carrying that S protein mutation called N439K or if such strains become more prevalent in circulation, it could have a major impact on current vaccine developments and therapeutics and also implications for disease severity and reinfections.


 
The study team demonstrated that the immunodominant SARS-CoV-2 spike (S) receptor binding motif (RBM) is the most divergent region of S, and provide epidemiological, clinical, and molecular characterization of a prevalent RBM variant, N439K. The team demonstrated that N439K S protein has enhanced binding affinity to the hACE2 receptor, and that N439K virus has similar clinical outcomes and in vitro replication fitness as compared to wild- type. They observed that the N439K mutation resulted in immune escape from a panel of neutralizing monoclonal antibodies, including one in clinical trials, as well as from polyclonal sera from a sizeable fraction of persons recovered from infection. Immune evasion mutations that maintain virulence and fitness such as N439K can emerge within SARS-CoV-2 S, highlighting the need for ongoing molecular surveillance to guide development and usage of vaccines and therapeutics.
 
The research findings were published on a preprint server and are currently under peer review. https://www.biorxiv.org/content/10.1101/2020.11.04.355842v1
 
The study team showed that a variant in the viral spike protein called N439K enhanced binding affinity to the human host cell receptor angiotensin-converting enzyme 2 (ACE2) and resulted in immune escape from a panel of neutralizing monoclonal antibodies (mAbs) and from the polyclonal sera of recovered individuals.
 
Co-researcher Dr Gyorgy Snell from Vir Biotechnology in San Francisco, California told Thailand Medical News, “Immune evasion mutations that maintain virulence and fitness such as N439K can emerge within SARS-CoV-2 S [spike], highlighting the need for ongoing molecular surveillance to guide development and usage of vaccines and therapeutics.”
 
Ever since the first cases of COVID-19 were first identified in Wuhan, China, late last year (2019), more than 135,000 SARS-CoV-2 genomic sequences have been made publicly available via the GISAID Initiative.
 
These genomic data are key to monitoring the spread and evolution of the virus, particularly the evolution of the spike protein (or S-protein) - the protein the virus uses to interact with ACE2 and enable the delivery of the viral genome into host cells.
 
Currently the spike protein is the viral structure that is targeted by neutralizing antibodies following infection or vaccination and by the mAbs that are being tested in clinical trials.
 
The research team said that studies have shown that the spike variant D614G, which is now domin ant across most of the globe, may have greater infectivity, while maintaining efficient spread and virulence.
 
An important point however is that the amino acid 614 lies outside of the spike’s receptor-binding domain (RBD)  the region targeted by most of the neutralizing antibody activity that occurs in the serum of people who have survived infection.
 
The team added, “Initial studies suggest that D614G actually exhibits increased sensitivity to neutralizing antibodies, likely due to its effects on the molecular dynamics of the spike protein. Therefore, this dominant variant is unlikely to escape antibody-mediated immunity.”
 
However, as the number of SARS-CoV-2 infection cases continues to rise, the greater the likelihood that new variants will emerge that could impact on vaccine and therapeutic development, they warned.
 
It must be remembered that spike receptor binding motif (RBM) is main target of neutralizing antibodies.
 
By examining the SARS-CoV-2 receptor binding motif (RBM), the primary target of neutralizing antibodies within the spike RBD, the study team showed that the RBM is the most divergent region of spike and less conserved than the RBD or the entire protein.
 
In order to fully comprehend how this structural plasticity might influence immune evasion, the researchers investigated the clinical and epidemiological impact, molecular features, and immune response to an RBM variant called N439K.
 
Significantly, the N439K mutation, which is the second most commonly observed RBD variant globally, has arisen independently twice and, in each case, formed genomic lineages of more than 500 sequences.
 
The study team found that this variant had repeatedly emerged through convergent evolution, had spread to multiple countries and had significant representation in the SARS-CoV-2 sequence databases.
 
When compared with wildtype spike protein, the N439K variant increased binding affinity for ACE2, and was associated with slightly higher viral loads in vivo, similar clinical outcomes in vivo and similar replication fitness in cultured cells.
 
The mutated variant also resulted in immune escape from a panel of neutralizing mAbs and from polyclonal sera from a significant proportion of people who had recovered from infection.
 
Most worryingly, one of the mAbs the variant was resistant to is currently being tested in clinical trials of a two-mAb cocktail.
 
The study team warned, “If circulating viral strains already carry resistant mutations to one antibody in the cocktail, this could reduce the cocktail to a monotherapy.”
 
They also said that there is a high likelihood that immune-evading SARS-CoV-2 variants will continue to emerge.
 
The team stressed, “The fitness of N439K is consistent with our findings that the RBM is the most divergent region of S spike.”
 
The study team says this divergence suggests that SARS-CoV-2 is capable of accommodating mutations in the RBM, while retaining ACE2 binding affinity.
 
The team further warned, “The ability to accommodate mutations in the RBM indicates a high likelihood that immune-evading SARS-CoV-2 variants compatible with fitness will continue to emerge, with implications for reinfection, vaccines, and both monoclonal and polyclonal antibody therapeutics.”


The RBM exhibits significant natural diversity in circulating SARS-CoV-2 isolates (GISAID, Oct 7, 2020, n≈130,000). (A) RBD variants with a minimum of 10 observed isolates in the RBM (blue) and outside the RBM (gray) mapped onto an X-ray structure of the SARS-CoV-2 RBD (PDB ID: 6M0J). (B) Number of observed variants in three non-overlapping S regions (RBM, non-RBM RBD, non-RBD S) normalized by the total number of residues in each region, where the number of observed isolates required to define a variant is varied. Inset: barplot representation where at least 10 isolates are observed per variant. (C) Heatmap of Deep Mutational Scanning (DMS) hACE2 binding and expression data for RBM residues (Starr et al., 2020). DMS score is the binding or expression fold change over WT on a Log10 scale. Aggregated DMS data is shown for each residue by taking the minimum (most disruptive variant) or the average score across all possible variants of a residue, except for the reference residue and the stop codon (‘mutagenesis’ columns). Alternatively, minimum and average scores are computed only across variants that have naturally occurred (‘observed variants’ columns). When no natural variants have been observed, cells are grey. The heatmap is annotated with frequency of non-reference amino acids in deposited sequences (at least 4 sequences were required to call a variant), in Log10 scale; number of countries in which a variant was observed; and percentage of total binding energy between RBD and hACE2 computed from an X-ray crystal structure. Data were sorted on the leftmost DMS column.

The team said that the evolution of the SARS-CoV-2 RBM, a critical epitope for vaccine response and therapeutic mAbs, will depend on the fitness of RBM variants. The study findings herein describe an example of a naturally-occurring RBM variant which can evade antibody-mediated immunity while maintaining fitness.
 
Fitness of this variant, N439K, was demonstrated by repeated emergence by convergent evolution, spread to multiple countries and significant representation in the SARS-CoV-2 sequence databases, the fact that the N439K RBD retains a high affinity interaction with the hACE2 receptor, efficient viral replication in cultured cells, and no disease attenuation in a large cohort of infected individuals. The fitness of N439K is consistent with the study findings that the RBM is the most divergent region of S. This divergence indicates an ability of SARS-CoV-2 to accommodate mutations at the RBM while retaining the functional requirement of hACE2 binding, and is likely to be linked to immune pressure from neutralizing Ab responses.
 
There is precedent for the most immunogenic region of a viral surface protein to be the fastest mutating despite harboring the receptor binding site; for example, the immunogenic globular head domain of the influenza virus hemagglutinin surface protein, which contains the sialic acid receptor binding site, evolves faster than the stalk region. https://www.nature.com/articles/s41467-018-03665-3
 
https://pubmed.ncbi.nlm.nih.gov/29992986/
 
The ability to accommodate mutations in the RBM indicates a high likelihood that immune-evading SARS-CoV-2 variants compatible with fitness will continue to emerge, with implications for reinfection, vaccines, and both monoclonal and polyclonal antibody therapeutics. In our profile of immune escape from the N439K variant, the team observed resistance to a mAb currently being evaluated in clinical trials as part of a two-mAb cocktail.
 
The promise of using cocktails of mAbs is that they should significantly lower the likelihood of drug-induced selection of resistant viruses. https://science.sciencemag.org/content/369/6506/1014
 
However, if circulating viral strains already carry resistant mutations to one antibody in the cocktail, this could reduce the cocktail to a monotherapy.
 
Additionally, considering the high level of plasticity of the RBM demonstrated in the present study, there could be many combinations of RBM mutations compatible with viral fitness while leading to immune escape. This is supported by our result that N439K can compensate for a mutation (K417V) that otherwise decreases receptor binding affinity. This particular combination of mutations is plausibly compatible with fitness as it parallels SARS-CoV RBM:hACE2 interactions (salt bridge at SARS-CoV RBD position R426 and no salt bridge at V404, ). Notably, several mAbs which were not sensitive to these mutations individually were sensitive to them in combination, including the two-mAb cocktail.
 
The team propose two approaches that will be critical for minimizing the impact of mAb escape mutations. One is to develop mAbs with epitopes that are highly resistant to viral escape. This may include epitopes outside of the RBM and/or epitopes that are crossreactive across SARS-CoV and SARS-CoV-2, indicating conserved epitopes with a low tolerance for mutatio. A comparison of epitopes of RBM-targeting mAbs with the most conserved regions of the RBM may also identify RBM mAbs with a higher barrier to escape. The second approach is to screen patients, likely at the population level, for the presence of potential resistance variants prior to drug administration. The availability of multiple different mAb therapeutics in the clinic could provide the opportunity to tailor the choice of therapeutic to local circulating variants.
 
In general, given that access to therapeutic monoclonal antibodies via clinical trials and emergency use authorization is expanding, and as more people develop immune responses to the wildtype virus, monitoring the evolution of SARS-CoV-2 will be increasingly critical. Although SARS-CoV-2 is evolving slowly and at present should be controllable by a single vaccine, variation accumulating in the RBM could put this at risk, especially for individuals with a moderate Ab response to vaccination or infection. While we only report on evasion of antibody-mediated immunity here, it would be surprising to us if similar changes are not observed to evade T cell immunity and innate immunity.
 
The study team cautioned that RBM variants must be evaluated when considering vaccines and the therapeutic or prophylactic use of mAbs.
 
They said, “Long term control of the pandemic will require systematic monitoring of immune escape variants and selection of strategies that address the variants circulating in targeted populations.”
 
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