As the COVID-19 pandemic spread, scientists accelerated their efforts to find a vaccine that could put an end to lockdowns and social distancing for good. However, the emergence of the new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutation D614G has raised many questions, including whether it will cause the virus to become resistant to the vaccines produced against earlier versions of the virus. A new study published in the journal npj Vaccines in October 2020 shows that this may be an unfounded fear.
This transmission electron microscope image shows SARS-CoV-2—also known as 2019-nCoV, the virus that causes COVID-19. isolated from a patient in the U.S., emerging from the surface of cells cultured in the lab.Image captured and colorized at NIAID's Rocky Mountain Laboratories (RML) in Hamilton, Montana. Credit: NIAID
Why is the D614G Mutation Important?
Most candidate vaccines target the spike (S) protein, which exists as a homotrimer on the surface of the virus envelope. The spike glycoprotein is the factor that enables the virus to engage with the angiotensin-converting enzyme 2 (ACE2) receptor on the host cell to enter and infect the cell.
Soon after the pandemic began to spread, a single A-to-G nucleotide change was noticed at position 23,403 in the Wuhan-Hu-1 reference genome, resulting in an aspartate residue being replaced by glycine at position 614 of the spike protein.
Since the emergence of this mutation, the DG614 variant has become dominant over earlier variants, being observed in over 85% of uploaded sequences from all over the world, counting up to July 1, 2020. This has led to the hypothesis that viral strains with this mutation have a structural advantage.
Possibly, say some scientists, such isolates bind better to the furin cleavage site on the S1 subunit. This allows the DG614 variant to be more transmissible, to replicate more efficiently, and to produce higher viral loads. Also, DG614 are better adapted to enter the human host cell, are more pathogenic, and may be associated with higher mortality.
Modeling Vaccine Efficacy in D614G Strains
The current study led by Australia's national science agency, the Commonwealth Scientific and Industrial Research Organisation (CSIRO), explored the possible impact of this mutation on vaccine efficacy.
The researchers used serum from ferrets to which the COVID-19 vaccine candidate was designed to counter the DG614 variant of the S protein. These sera were tested for neutralization capacity against SARS-CoV-2 isolates with and without the D614G mutation, and otherwise comparable in respect of cell binding and entry.
The researchers used the Australian isolates' VIC01' and 'SA01' (which are D614) and 'VIC31' (which is DG614) in the neutralization assays. They found that the ferrets had a median neutralizing antibody titer of 6.3 log against all three isolates. Overall, the mean titer of neutralizing antibodies against the G614 variant resembled that of the D614 variants.
They then modeled the spike protein at the molecular level to uncover the structural dynamics of the mutation, to study the possible effect of the mutation on the vaccine efficacy, and whether the mutation conferred the advantages proposed by other researchers.
The mutation site is upstream of the furin cleavage site at the S1/S2 interface and is buried below the surface and shielded by a sugar molecule at the N616 position. Thus, it is probably not a neutralizing epitope component, and unlikely to affect the neutralizing efficiency of the antibodies generated by the D614-based vaccines. Though there are two neutralizing epitopes in this location, the S614 position is distinct from them.
Modeling Rules out Cleavage Site Advantage with D614G
Earlier studies show that the D614G mutation produces an elastase cleavage site. Still, the molecular dynamics simulations in the current study show that this does not produce elastase-mediated S1/S2 cleavage, and thus does not affect replication efficiency. Even though furin can gain access to the cleavage site at the interface, elastase cannot probe the newly introduced site due to its buried position and shielding, as described above, without the separation of the S1 trimer cap and the S2.
Again, the D614 site on S1 interacts with S2 through hydrogen bond interactions and a salt bridge. The G614 mutation abolishes the salt-bridge, and thus, rather than improving the interaction between S1 and S2, it may reduce the stability.
The researchers simulated these interactions in the 'up' orientation of the receptor-binding domain (RBD), but also found that in a short simulation, the ACE2 tilted to come into contact with the RBD on the adjacent monomer, through contacts between three residues.
This indicates the possibility that the adjacent RBD in the 'down' conformation plays a role in ACE2 binding and specificity. In fact, this agrees with earlier studies showing the flexible nature of spike protein interactions, where the spike protein was able to form a complex with ACE2 in three different conformations.
RdRp Site Mutation Increases Infectivity
Another relevant finding is that the D614G mutation is also associated with a C-to-U mutation at position 14,408, causing a Pro-Leu 314 mutation in orf1b that encodes RNA-dependent polymerase (RdRp/nsp12). This may be a reason for the increased infectivity of the new variant.
This mutation is in a hydrophobic cleft near the enzyme's active site. This cleft is, according to the model. lined with arginine residues and these may promote nucleotide binding. This may stabilize the conformation locally, by its effects on the backbone constraints. Its impact on virulence is unknown at present, indicating the need for more work.
Future studies must include antibody protection assays to understand the effects of these mutations in living organisms.
Says senior author Professor Seshadri Vasan, "Despite this D614G mutation to the spike protein, we confirmed through experiments and modeling that vaccine candidates are still effective." This provides the intensive vaccine development efforts a clear rationale to proceed to completion.
Secondly, he says, "We've also found the G-strain is unlikely to require frequent 'vaccine matching' where new vaccines need to be developed seasonally to combat the virus strains in circulation."
Moreover, the researchers say, given the wide availability of modeling approaches, and to ensure public confidence in the vaccines once available, "it would be desirable to analyze the impact of identified mutations, in collaboration with such organizations, before speculating on potential adverse effects on vaccines."