COVID-19 Mutations: Researchers Warn That New SARS-CoV-2 Mutation Strain N439K Could Be More Infectious And Antibody Resistant Than Wuhan Strain

COVID-19 Mutations: Researchers, virologists and genomic specialist from the Harbin Institute of Technology-China are warning that a new emerging mutated SARS-CoV-2 coronavirus strain N439K is more infectious and antibody resistant than the original Wuhan strains.


 
The SARS-CoV-2 virus causing the COVID-19 pandemic has been undergoing various mutations and in some cases becoming more infectious and even potent despite claims and so called manipulated studies by ‘garbage’ British researchers who have been basically been bought by the various vaccine manufacturers, and making claims that the virus has not mutated or that the mutations are harmless! It also ironical that despite their so called expertise, Britain itself is badly affected by the pandemic and many of its people are dying with more soon according to projections by certain epidemiology models.
 
The detailed analysis of the structural and energetic effects of mutations on protein-protein interactions between the receptor binding domain (RBD) of SARS-CoV-2 and angiotensin converting enzyme 2 (ACE2) or neutralizing monoclonal antibodies will be beneficial for epidemic surveillance, diagnosis, and optimization of neutralizing agents.
 
The study team warns that according to molecular dynamics simulation, a key mutation N439K in the SARS-CoV-2 RBD region created a new salt bridge which resulted in greater electrostatic complementarity.
 
Furthermore, the N439K-mutated RBD bound hACE2 with a higher affinity than wild-type, which leads to it being more infectious. In addition, the N439K-mutated RBD was markedly resistant to the SARS-CoV-2 neutralizing antibody REGN10987, which may lead to the failure of neutralization.
 
These study findings would offer guidance on the development of neutralizing antibodies and the prevention of COVID-19.
 
The study findings were published on a preprint server and are currently being peer reviewed. https://www.biorxiv.org/content/10.1101/2020.11.21.392407v1
 
The study team investigated the effect of different mutations of SARS-CoV-2 on its binding to the human angiotensin-converting enzyme 2 using molecular dynamic simulations. The team found the N439K mutant binds more strongly than the original Wuhan strain, which may have implications for therapeutics like monoclonal antibodies.
 
The receptor-binding domain of the spike proteins present on the virus envelope bind to the human angiotensin-converting enzyme 2 (ACE2), followed by fusion of the virus with the host cell membrane. The spike protein has two subunits: S1, which binds to host cells, and S2, which plays a role in membrane fusion.
 
It has been found that immunizing antibodies produced by the host immune system target the RBD and disrupt the virus's binding to ACE2. However, when there are mutations in the spike protein, it may affect the efficacy of the neutralizing antibodies. Currently, there are about 930 proper mutations reported worldwide. A mutation from ASP614 to GLY614 has made the virus more infectious, according to repor ts. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7366990/
 
At present most drugs, therapeutics, vaccines and antibody protocols being developed are based on the spike protein sequence of the Wuhan reference strain. Missense mutations in the previous infectious coronaviruses like MERS and SARS-CoV have been observed to become resistant to neutralizing antibodies for the original strain. Thus, mutations in SARS-CoV-2 may also lead to strains that are resistant to the antibody treatments being developed.
 
Importantly, it is necessary to monitor mutations in the circulating SARS-CoV-2 strains to develop better therapeutics.
 
The study team modeled the complexes formed between the RBD and the human ACE2 and monoclonal antibodies.


Structural and Energetic Details of Both Wild and Mutant RBD-mAbs Interactions (A) Crystal structures of RDB-CB6/REGN10987 complexes, the RBD is colored red, CB6 heavy and light chains are represented as marine and green respectively, REGN10987 heavy chain is colored yellow and light is blue, and the 439 residues are described as the sphere. (B) Characteristic dynamic fluctuations of both RBD-REGN10987 and RBD(N439K)-REGN10987 complexes. Mutant-type (100ns) and wild-type (100ns) are colored by orange and blue, respectively. (C) Dynamic conformations are projected on to the principal vectors (PC1 and PC2). Red and blue indicate mutant-type and wild-type 100ns MD trajectories respectively. (D) The RMSD of the receptor-binding motif in four complexes during the 100-ns MD simulations. (E) The binding free energies for both complexes of the mAb REGN10987 (including heavy and light chains), the color schemes are the same .The binding free energies of 200 configurations at an interval of 100ps from the last 20ns simulations. The t-test was conducted to check the statistical significance of the difference between two systems of binding free energies. A p-value of <0.05 indicates that the difference is statistically significant (95% confidence interval). 

They compared the complexes formed by the wild-type virus and the mutated virus and performed molecular dynamics simulations along with molecular mechanics/Poisson-Boltzmann surface area scheme. The mutations in SARS-CoV-2 were seen mainly in the open reading frame (ORF) regions, which encode for non-structural proteins, nucleocapsid protein, and spike protein.

It was found that the ORF3a protein, which can change the environment inside the infected cell and make holes on the host cell membranes, has a higher mutation rate in North America and Oceania, and may allow the virus to spread better.
 
However in the case of the N439K strains, they are found to be circulating at present in North America, parts of Europe including UK, Spain, Italy and also in Indonesia, Malaysia and Philippines.
 
The study team found that mutations were not on single sites. On the spike protein, the D614G variant was the majority mutation, followed by D936Y. The N439K variant, where asparagine at the 439th site is replaced by lysine, is the most dominant in the spike protein RBD.
 
Importantly the molecular dynamics simulations showed more flexibility changes in the N439K variant, which could result in structural rearrangements in the SARS-CoV-2 RBD-ACE2 complex that lead to a stronger binding. Furthermore, the complex with the mutated virus forms more hydrogen bonds than the wild-type complex.
 
Interestingly the binding energy of the N439K complex was also higher than that of the wild-type complex. This suggests that the mutant virus has a stronger association with human ACE2. The stronger binding could be because the replacement of asparagine with lysine forms a new salt bridge in the complex with human ACE2, which could increase electrostatic interactions. Apart from this interaction, the complexes are also bound by van der Waals interactions and polar solvation free energy.
 
Despite the fact that certain substandard studies by unqualified British researchers have suggested mutant versions of the virus may be less infectious, the stronger binding of human ACE2 with the N439K mutant suggests this mutant strain may be more infectious. The N439K mutation is completely included in the D614G samples, which have been observed to be more infectious than the original strain.
 
The study team also performed simulations of the N439K mutant of human ACE2 complexes with two neutralizing monoclonal antibodies, REGN10987 and CB6. REGN10987 binds to CR2 and CR3 regions of the RBD where N439K is located, while CB6 binds to CR1 and CR2. The analysis indicated that the N439K mutation decreased sensitivity to CB6 antibodies.   
   
Significantly, although CB6 could neutralize the N439K mutant, the strain was quite resistant to REGN10987 antibodies.
 
Hence, as new antiviral strategies are being developed based on the Wuhan strain, given the possible mutations in SARS-CoV-2 that could become resistant to antibodies developed for this strain, "it is necessary to consider the impact of different mutations on the effectiveness of neutralizing antibodies," the study team warns.
 
The team concluded, “The SARS-CoV-2 virus is expected to continue evolving, some mutations only appear in a certain period and other mutations may appear in an unpredictable way. It is necessary to continuously analyze the SARS-CoV-2 virus according to the mutation frequency and time pattern. Meanwhile, COVID-19 antibodies are developed based on the Wuhan reference genome, it is necessary to consider the impact of different mutations on the effectiveness of neutralizing antibodies. In summary, the team have counted the amino acid changes in a variety of mutation strains and laid special stress on analyzing the infectivity of the variant N439K in the SARS-CoV-2 virus and the effectiveness of well-characterized neutralizing mAbs using MD simulations. The variant N439K enhanced the infectivity of the virus with hACE2 and altered antigenicity to some neutralizing mAbs. Taken together, the study findings shed light on the influence of N439K Variant on SARS-CoV-2 infection efficiency and antigenicity.”
 
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