As vaccines roll out against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the agent behind the devastating coronavirus disease 2019 (COVID-19) pandemic, new variants have emerged with increased infectivity and, in some cases, virulence. More importantly, they display partial or complete resistance to the antibodies elicited by natural infection with older strains, or by vaccines, as well as therapeutic monoclonal antibodies such as those in the Regeneron cocktail.
A new study, published in Cell Reports, suggests that SARS-CoV-2 may have acquired such immune evasion mutations during its transmission in farmed mink.
The Dutch and Danish outbreaks among farmed mink and farmworkers last year showed that not only was the virus amplified in mink, but these mutant strains were then transmitted back into humans.
When human-to-human spread of this variant was observed, it stirred up fears that mink could be a potential reservoir of infection, which led to the culling of 17 million mink.
Spike mutations in mink
The viral spike protein is present on the viral envelope and mediates viral entry into the host cell via the angiotensin-converting enzyme 2 (ACE2) receptor. This interaction involves the receptor-binding domain (RBD) and requires that the spike protein be primed first via the serine protease TMPRSS2 on the host cell.
The spike protein in mink-derived variants showed a distinct cluster of mutations than those seen in human-derived SARS-CoV-2, namely, deletion of H69/70, Y453F substitution in the RBD, I692V, S1147L in the S2 spike subunit, and M1229I in the spike transmembrane domain. All five mutations are present in cluster 5 variants.
The current study examined the role of Y453F in viral biology, including spike-mediated entry and inhibition of neutralization. The use of pseudoviruses expressing the SARS-CoV-2 spike protein enabled the researchers to avoid using the much more dangerous SARS-CoV-2 for their experiments.
Two sets of controls were used, one the wildtype spike and the other the D614G spike. The mink-associated mutant spikes were also studied.
These analyses showed that none of these mutations interfered with the expression of the spike protein or with cleavage at the furin cleavage site at the S1/S2 interface. ACE2 interactions were also efficiently carried out, with viral entry into the cells in culture.
Robust viral entry
The D614G spike showed increased capability for spike-mediated cell entry. This mutation is now globally dominant among human SARS-CoV-2 as well as among mink variants. When combined with the Y453F mutation, either alone or with the deletion H69/70, the efficiency of entry did not increase further.
When cluster 5 variants with the D614G mutation were observed, cell entry was reduced, but the virus continued to enter human intestinal and lung cell lines efficiently.
Preserved inhibition of entry by ACE2 inhibition
The study also shows that the presence of soluble ACE2 blocks the entry of spike-bearing pseudoviruses irrespective of these mutations. These alterations do not block the inhibitory activity of the TMPRSS2 inhibitor camostat. The most marked inhibition was with the cluster of five mutations along with D614G.
The scientists examined the efficacy of the Y453F mutation on the ability of the virus to evade neutralizing antibodies generated by natural infection, by vaccination or those available in commercial monoclonal antibody cocktails. With this mutation being in the RBD, it is quite likely to alter the antibody-binding epitope.
The mutation inhibited neutralization by most convalescent plasma or serum samples, increasing the 50% inhibitory concentration (IC50) by a median of 1.62, though the increase varied between samples.
It also prevented neutralization by casirivimab/REGN10933, one of two Regeneron antibodies in the approved combination. The anti-neutralization effect appears to be because the position 453 is on the binding surface between the antibody and the spike protein.
The other antibody in this cocktail, imdevimab/REGN10987, bound to a different spike region and preserved its efficiency against spike-mediated viral entry. This cocktail also blocked viral entry into cells in culture even with this mutation.
“Mutation Y453F observed in mink may be an adaptation to efficient use of mink ACE2 for entry, since amino acid 453 is known to make direct contact with human ACE2.” This mutation also arose in ferrets during the course of infection with this virus, in the laboratory, indicating an adaptation to the ferret ACE2.
The other explanation is that it might be a mechanism of immune evasion, supported by the recent finding of its emergence in a patient with prolonged COVID-19.
What are the implications?
The Y453F mutation affects viral entry into a few cell lines, when present along with the other cluster 5 mutations, which may be why this cluster became extinct soon after it emerged. Cluster 5 spike protein is also more easily inhibited by soluble ACE2, which may indicate that the binding affinity is altered in the presence of all five mutations.
The study also suggests that the mutation reduces the efficacy of convalescent plasma against the virus after its amplification in mink in a fraction of cases. “This means that people who were infected with SARS-CoV-2 may have reduced protection against mink variants of the virus.”
However, since most of the samples completely block entry even at the lowest titers, high antibody titers are protective even against mink-derived virus.
Our results suggest that the introduction of SARS-CoV-2 into mink allows the virus to acquire mutations that may compromise viral control by the humoral immune response in humans. “
The same mutation has also been identified in the virus during human infections, though not as the result of exposure to the mink variant. Instead, it has been observed to arise in prolonged infection.
The identification of SARS-CoV-2 in wild mink is also a threat to human health. It may indicate the potential for establishing a permanent natural reservoir for such viruses, both wildtype and new variants, that may repeatedly spill over into humans and other animals. The occurrence of such amplification must be detected by continuous monitoring, as well as the changes this brings about in viral biology.