RBD-ACE2 interaction is a prerequisite for SARS-CoV-2 viral entry [
8,
9,
26]. Surprisingly, the binding of the Omicron RBD to the ACE2 receptor appears to be reduced in our settings compared to the currently dominant Delta variant, both in an ELISA assay as well as by affinity measurement using MST. Both techniques led to different absolute values caused by the different technique principles [
27] but resulted in the same trend. Still, the affinities measured by MST (40.7 nM for Wuhan RBD) were in the same range as the ACE2 affinities determined previously by surface plasmon resonance (44.2 nM) [
28]. Several Omicron RBD mutations are assumed to increase the binding to ACE2: G339D, S477N, T478K, Q493K, and N501Y; others are proposed to be neutral: S371L, S373P, G446S, E484A, Q493R, and Q498R, or are assumed to reduce the binding to ACE2: S375F, K417N, G496S, and Y505H according to yeast display experiments performed by Starr et al. [
26]. Hence, some bioinformatic models predicted an increase in the ACE2 binding affinity of Omicron RBD [
29] while other models rejected this scenario [
30,
31] and stated: “the Q493R/K mutations, in a combination with K417N and T478K, dramatically reduced the S1 RBD binding by over 100 folds” [
30]. However, the latter considerations are all based on in silico modeling. Thus, RBD-ACE2 interaction involving a heavily mutated RBD, such as the one of Omicron VOC, may deviate from predictions and requires empirical biochemical testing. To our knowledge, this is the first comprehensive empirical analysis of Omicron RBD binding efficacy to the ACE2 receptor. According to our data, the binding of Omicron RBD to human ACE2 was not increased, but rather decreased, especially when compared to Beta and Delta. Despite the observed reduced ACE2 Omicron RBD interaction, ACE2 remains necessary for cell entry shown by a recent study with Omicron pseudotyped viruses [
15]. An increased RBD ACE2 binding is leading to increased cell entry shown for Alpha, Beta, Gamma, and Delta [
32]. For Omicron, it was shown that the cell binding is reduced (weaker cell-cell fusion activity) when comparing Omicron to Delta [
33]. However, the decrease in RBD binding does not necessarily translate into reduced infectivity, as infectivity and replication are also defined by proteolytic spike processing, fusion efficacy, and RNA replication efficiency, just to name a few mechanisms [
34‐
36]. Furthermore, the severity of disease depends on several factors, e.g., Delta and Beta show the same affinity to ACE2, but Delta leads to a more severe disease compared to Beta [
37]. Nevertheless, our results argue that increased binding to the ACE2 receptors may be an unlikely cause of rapid Omicron spread. One has also to consider that we utilized in our work the originally available sequence with a Q493K mutation, whereas Q493R sequences have been published since. According to Starr et al. [
26], the K mutation has an even higher affinity as the R mutation in in vitro binding studies. The here measured reduced binding of Omicron RBD to ACE2 was later confirmed by other studies [
38,
39].
Importantly, RBD mutations may also lead to immune escape [
40]. The humoral immune answer is a key factor for the antiviral defense [
41] and the RBD is the main target of neutralizing antibodies [
10,
11,
42]. RBD binding and neutralization capacity do correlate [
43,
44]. The very low RBD binding in the Ad26.COV2.S compared to the COVID-19 patient group or the mRNA-vaccinated groups is in accordance with the former results [
45] but impaired definite conclusions on Omicron immune escape upon Ad26.COV2.S vaccination. The reduced binding of sera from COVID-19 patients and mRNA vaccinees to the Omicron RBD was in accordance with our and other recent results [
14‐
16,
46,
47] describing a highly reduced neutralization of the Omicron variant by human sera from vaccinated persons. However, a 2.6× reduction was observed in RBD binding while in this study the neutralization was under the detection limit for sera from 2× BNT162b2-vaccinated persons and other Omicron SARS-CoV-2 neutralization studies showing a reduction by a magnitude of 10× and more [
14‐
16,
46,
47]. Therefore, the Omicron mutations mainly reduce the SARS-CoV-2 neutralization but not the RBD binding in the same measure: This indicates an immune escape focused on serum neutralization evasion. Besides neutralization escape, the reduced affinity seems to be compensated by increased viral replication shown in ex vivo explant cultures of human bronchus [
48]. Sera from boost vaccine recipients showed a significant reduction in serum titers as well as 1–2 log fold reduced neutralization of the Omicron variant, whereas the titers and neutralization of the Delta variant did not significantly differ from the wild type. While we have measured a significant decrease of serum binding to Omicron RBD, the boost recipients still had a higher anti-Omicron RBD titer as well as at least a remaining neutralization activity compared to 2× vaccinated individuals. The efficacy of boost immunization in neutralization assays was also observed in other studies [
14‐
16]. The timepoint of sampling (duration from vaccination, respectively infection, to sampling) can influence the antibody response. In this study, we focused on a timepoint after vaccination/infection where high antibody titers are expected. Prospective studies with boosted individuals will be pursued in the near future when the availability of subjects with longer time spans after the third dose will be available.
The results of this Omicron RBD study are a snapshot of the current situation. According to the sequencing data deposited as GISAID (
https://www.gisaid.org/) and the analysis on
Outbreak.info [
49], the frequency of the 15 aa mutations in the RBD is very dynamic, e.g., K417N, described for the initial Omicron variant to occur in ~35% (status 2021-12-14, 2146 sequences) of all sequenced Omicron isolates, is now retrieved in above 55% of sequenced viruses (status 2022-02-07, 873.492 sequences). S477N, T478K, and E484A were initially at ~47% (status 2021-12-14, 2146 sequences), now instead above 88%, as N501Y (status 2022-02-07, 873.492 sequences). The K417N mutation is a key mutation also in the Beta variant, the T478K mutation instead in the Delta variant, and N501Y in the Alpha, Beta, and Gamma variants [
50]. All of these mutations may contribute to both ACE2 binding efficacy and immune escape. Therefore, Omicron variants with alternative mutations might evolve in the near future and alter the antibody recognition and/or the ACE2 binding efficacy. More comprehensive studies of various subvariants in the Omicron family may shed light on their biochemical and immunological properties and understand the potential for future SARS-CoV-2 evolution.