The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to the coronavirus disease 2019 (COVID-19) pandemic. Despite the rapid rollout of vaccines, the emergence of newer variants of the virus, some with higher transmissibility and others with immune escape capabilities, led to the continuation of the viral spread.
A new paper in the journal Cell Reports discusses the role of mutations in the receptor-binding domain (RBD) of the spike protein in the generation of antibody resistance and loss of vaccine efficacy.
Introduction
Given the many variants that have been identified so far, the need to study the antigenic landscape in relation to the tropism of the virus, and other biological effects, is obvious. The current study focused on the numerous RBD variants deposited in the public SARS-CoV-2 genome database, 129 in all, along with 24 double-mutant RBDs, and 11 variants of the virus, either variants of concern or variants of interest (VOC/VOI), respectively.
These were expressed in lentiviral-pseudotyped particles (LVpp), and their infectivity potential measured against cells expressing heterologous angiotensin converting enzyme 2 (ACE2) receptors from 18 animal species. The ability to neutralize these various particles was measured, using human convalescent plasma samples (HCP), and polyclonal antibodies from vaccine or vaccine-candidate-derived sera, and monoclonal antibodies (mAbs).
The scientists also tracked the correlation between the mutations in the circulating RBD variants and the potential for cross-species infection and antibody escape.
What did the study show?
The results showed that of the 129 RBD mutants, 17 showed a reduction in spike expression by over 50%, compared to the wildtype D614G strain, while ~5% had significantly reduced spike expression. This translated to >5-fold loss of infectivity to the wildtype variant, while others showed a slight increase.
The SARS-CoV-2 spike bound efficiently to 13 of 18 ACE2 orthologs, with further analysis showing potent ACE2-spike binding. The pseudovirus infection assays confirmed that the wild-type virus could easily infect most cell lines except for the 5 with poor ACE2-spike binding. Thus, ACE2-spike binding correlates well with infectivity in ACE2-expressing cells.
Most RBD mutants also showed comparable binding to ACE2 in cells that supported spike binding by the wild-type virus, excluding the 17 with reduced spike expression. Certain mutations conferred a significant improvement or loss of infectivity. For instance, in cells expressing mouse ACE2, the presence of K417N, E484K, and N501Y led to a steeply increased infectivity.
With the horseshoe bat ACE2, E484K was the major influencer, among other mutations like V382L, N440K, G476S, P521R, and A522S, with >3-fold increase in infectivity. Other mutations conferred improved infection rates with whale ACE2 expression.
All variants with the N501Y mutant, especially the Alpha, Beta, Gamma, and Omicron variants, showed 100-fold increased infectivity with mouse ACE2-expressing cells. Other variants with E484K but not N501Y had 10-30-times higher infectivity in these cells. Certain mutations like L452R appeared to produce a synergistic effect since they increased infectivity when present with other mutations.
The Omicron showed increased infectivity, probably due to the N440K mutation, in ferretACE2 expressing cells.
Checking for neutralizing activity, they used 9 mAbs against 48 mutants with receptor binding motif (RBM) mutations. Only one of these mAbs, the 4A8, binds the N-terminal domain (NTD) of the spike, the others interacting with the RBD.
The antibodies included human, humanized mice, and mouse mAbs, belonging to Classes 1 through 4 according to their mode of recognition of the antigen. Immune escape was considered to have occurred once the concentration required to inhibit the entry of 50% of particles (IC50) dropped by four times or more, compared to that against the wild-type spike.
The findings showed that the relative neutralizing potency was reduced up to a hundred times by the E484K mutation, including that of the Regeneron mAb REGEN10933, and the human antibody COVA2-15. Similar effects were seen with the E484A mutation, except with the latter antibody.
The N501Y mutations reduced the relative neutralizing potency with COVA2-15 by 14 times. As for K417N, it reduced the neutralizing potency of REGEN10933 by over 40 times. The mutations in aa 444-456 and aa 484-494 regions also reduced the neutralizing potency enough to facilitate immune escape from neutralizing antibodies targeting the RBD.
Neutralization by HCP
HCPs from the first wave of COVID-19 were found to have varying cross-reactivity against the variants of the virus. Mutations surrounding aa439-448 and aa484 seem to markedly impact neutralization by HCP antibodies, showing them to be two essential antigenic regions.
All three HCP antibodies completely lost neutralizing potency against Omicron, while the Beta, Gamma, and other variants containing E484K became resistant to P020.
According to the authors,
The Omicron showed the most striking resistance among all variants.”
Neutralization by vaccine sera
Vaccine-elicited antisera in mice and monkeys appeared to have similar cross-neutralizing profiles to human HCP antibody P020. The greatest escape occurred, again, with Omicron, with E484K-bearing mutants showing neutralization resistance.
The third booster shot explains why vaccine antisera seem to have higher neutralizing titers for VOC/VOI than HCP, since it increased the titer of nAbs against most variants except, notably, the Delta variant.
These data appeared to be consistent with the findings that the 3-dose mRNA vaccinations help to elicit cross-variants neutralization antibodies in humans.”
Nonetheless, nAbs against the variants with a distinct antigen profile remained several-fold lower than against the wildtype.
Prevalence associated with cross-species infectivity
Interestingly, the changes in infectivity with each single-mutation RBD variant for other animal species, relative to the wildtype, were associated with the cumulative prevalence of the variant.
The cross-species-infectivity (CSI) score, derived from relative infectivity in ferret and mouse ACE-bearing cells, correlated with the cumulative prevalences in human-derived viral sequences. Further analysis showed that the most frequent mutations in human-derived viral RBD sequences were the T478K (cumulative prevalence=59%), L452R (59%), N501Y (22%), E484K and S477N (<5% each).
The greater the infection potential across species, the higher the cumulative frequencies of the RBD mutations, showing that spillover across species may be important in maintaining the transmission of the virus in humans and animals.
Implications
In this fascinating study, the researchers showed that the circulating variants of SARS-CoV-2 can infect cells bearing various forms of the ACE2 receptor in other species, making the virus tropic for other mammals, including aquatic.
The least readily infected were ACE2 from the ferret, horseshoe bat, mouse, tupala and brown trout, but this can be overcome, especially for the first three species and orders, by RBD mutations such as K417N, E484K, and N501Y. These are found in several variants.
Both mouse and ferret, especially, are abundant and inhabit settings in close proximity to humans, making spillover to these species a matter of high epidemiological concern. These mutations have increased mouse susceptibility to the Alpha, Beta and Gamma variants, besides Omicron, compared to the early resistance to the ancestral variant.
Mink susceptibility was demonstrated earlier, with back-and-forth infections between farmed mink and farmworkers. The Y453F mutation in mink-derived SARS-CoV-2 indicated adaptation by these animals to the virus, enhancing infectivity. The VOCs/VOIs of today are also more infective in ferrets, and perhaps also in minks, this study suggests.
The increased infection potentials of RBD mutants in animals may expand the host range of SARS-CoV-2 and possibly cause cross-species spillover.”
These mutations also appear to facilitate immune evasion in many cases, as shown by the multi-fold increase in neutralization resistance with the Omicron VOC. Even with three doses of vaccine, serum samples continued to show much lower neutralizing antibody titers against Omicron compared to other variants. Newer vaccines should take advantage of current knowledge of the antigenic profile of these new variants, using mutation patches to elicit broadly neutralizing antibodies capable of neutralizing multiple variants.
These findings highlight the antigenic drift and the possible cross-species spillover driven by viral genetic changes and will guide the prediction and surveillance of SARS-CoV-2 spike mutations.”
