1. Introduction
The appearance of the SARS-CoV-2 Omicron variant B.1.1.529 was reported in South Africa (SA) on 24 November 2021 by the World Health Organization (WHO) [
1]. Two days later, Omicron was designated as a new variant of concern (VOC) due to its potential to spread rapidly worldwide. The first known sample was collected in SA on 8 November 2021 and the first sample detected outside SA was found in a traveler arriving in Hong Kong from SA via Qatar on 11 November 2021. On 7 January 2022, Omicron was confirmed in 135 different countries worldwide [
2].
The WHO has estimated the danger from Omicron as potentially high due to the following reasons: the global risk of perpetuating the SARS-CoV-2 pandemic remains high; recent data have indicated that Omicron shows a significantly higher R-value than the Delta VOC with already enhanced transmission [
3,
4]. The R-value describes the average number of people that one person will pass on a virus. Increased numbers of cases have previously led to increased hospitalization and mortality, threatening to overwhelm healthcare systems. Preliminary evidence suggested an increased risk of reinfection with Omicron as compared to other VOCs [
2]. The concern was also growing because Omicron has spread amongst doubly vaccinated people [
5]. In turn, COVID-19 caused by Omicron appeared relatively mild and health care systems have remained functioning despite huge numbers of infected individuals, making it possible that this new variant may help end the pandemic by worldwide spreading and consequent broad immunization with significantly reduced disease severity and mortality.
Until recently, Delta (B.1.617.2) VOC was the predominant SARS-CoV-2 variant before the emergence of Omicron. Delta VOC was discovered in late 2020 in India and had spread to more than 163 nations by 24 August 2021. In June 2021, WHO stated, that the SARS-CoV-2 Delta strain would become the most prevalent strain in the world [
1]. Based on recent evidence, Delta strain is 40–60% more transmissible than the previous forms such as wild type (WT) Wuhan, or other recent VOCs such as Alpha (B.1.1.7). Delta has also been associated with an increased risk of hospitalization mostly for unvaccinated or partially vaccinated people [
6] which appears different for Omicron. For clarification, WHO has assigned the name Delta variant to lineage B.1.617.2 and it is one of the three Indian variants summarized under B.1.617. The Delta variant B.1.617.2 harbors seven mutations in the spike protein relative to the ancestor Wuhan SARS-CoV-2 strain; two of these mutations (L452R and T478K) are located in the receptor-binding domain (RBD) region [
7,
8]. Importantly, Delta VOC does not exhibit a mutation at position E484, which is usually associated with a strong reduction of recognition by antibodies [
9,
10,
11].
An unusually large number of thirty-seven mutations has been identified in Omicron in comparison to other VOCs. Most mutations are located in spike protein [
5]. Furthermore, fifteen mutations have been detected in RBD region which interacts with the main receptor angiotensin-converting enzyme 2 (ACE2) in comparison to only two mutations in Delta variant [
12]. The spike protein, particularly RBD, is the main target for COVID-19 vaccines [
13,
14]. Computational in silico modeling raised concerns about the possible ability of Omicron to dodge antibody-mediated immunity [
15]. Several reasons may account for the reduced ability of antibodies to neutralize emerging VOCs, including specificity alteration of antibody recognition (classical antibody specificity escape) as well as increased RBD-ACE2 affinity that partially outcompetes antibody binding (affinity escape) [
9]. So far it seems clear that alteration of antibody recognition of Omicron is related to its E484A mutation [
11,
16,
17,
18].
According to WHO, the global impact of Omicron will largely depend on four main aspects:
- (1)
the transmissibility of the variant,
- (2)
how well vaccinated and infected people are protected against Omicron,
- (3)
the virulence of Omicron, and finally
- (4)
the awareness of the population to take protective measures [
2]. In light of the importance of these key parameters, our study aimed at revealing the mechanisms underlying the overserved increased transmissibility and reduced protection by a vaccine- or infection-induced antibodies. Our data demonstrate an increased affinity of Omicron for its receptor ACE2 and reduced recognition by serum antibodies. These factors cause reduced viral neutralization. From a mechanistic point of view, our data demonstrate that Omicron avoids viral neutralization by both classical change of antibody specificity and affinity escape due to the high RBD-ACE2 affinity that outcompetes antibody binding.
2. Materials and Methods
2.1. Human Sera
Human sera were collected from COVID-19 convalescent patients (infected with wild type Wuhan in the first wave), vaccinated individuals who received two doses of mRNA vaccine (Moderna-mRNA-1273
® or Pfizer-BNT162b2
®), and vaccinated individuals who received a total of three doses of mRNA vaccine (Moderna-mRNA-1273
® or Pfizer-BNT162b2
®). Participants were recruited at the University Hospital of Bern, Bern, Switzerland and Qatar University, Doha, State of Qatar. Sera were collected within one-month post-vaccination [
16].
2.2. RBD-ACE2 Binding Kinetics
The binding kinetic was performed using Octet RED96E (Sartorius, Göttingen, Germany). SAX sensors were loaded with biotinylated ACE2 25 μg/mL and subsequently quenched with biocytin. RBD was serially diluted in kinetics buffer and dissociation was performed in 300 s. Kinetic buffer (KB) was used to dilute proteins. A loaded sensor run in KB served as a control. The resulting curves were aligned to the beginning of the association, and a 1:1 model was used for global fitting.
2.3. RBD Proteins
Recombinant SARS-CoV-2 spike S1 B.1.1.529-Omicron RBD was purchased from (antibodies-online GmbH, Aachen, Germany), recombinant SARS-CoV-2 Spike RBD, and recombinant SARS-CoV-2 Spike RBD B.1.617.2 L452R T478K were purchased from R&D Systems (Minneapolis, MN, USA). Proteins were reconstituted as per the manufacturer’s instructions.
2.4. Anti-RBD Titers
ELISA assays were performed as follows; 96 half-well plates were coated overnight with 1 μg/mL of RBD proteins. Plates were blocked for 2 h with 0.15% casein in PBS and bound for 1 h with 1:20 sera diluted in 1:3 steps. Bound IgG antibodies were detected using goat anti-human IgG-POX antibody (Nordic MUbio, Susteren, The Netherlands). ELISA plates were developed using tetramethylbenzidine (TMB) and stopped with 1 M H2SO4. Absorbance was read at 450 nm and curves were generated using OD450 and OD50. OD50 refers to the measure of the half-max response and OD450 refers to an optical density at 450 nm wavelength.
2.5. BLI-Based Competitive Assay
The ability of the human sera to compete with ACE2 for binding to RBD
WT, RBD
Delta, and RBD
Omicron was tested as previously described by Vogel et al. [
11].
2.6. Neutralization Assay (Cytopathic Effect-Based Neutralization Assay)
Sera samples were heat-inactivated for 30 min at 56 °C and diluted from 1:20 to 1:320. 100 TCID50 of WT (SARS-CoV-2/ABS/NL20), Delta (SARS-CoV-2/ABSD/NL21), and Omicron (SARS-CoV-2/hCoV-19/NH-RIVM-72291/2021) was added to each well and incubated for 1 h at 37 °C. Following incubation, the mixture was added to a monolayer of Vero cells and incubated for an additional four days at 37 °C. Afterward, wells were inspected for the presence of cytopathic effect (CPE) and titers were expressed as the highest serum dilution which fully inhibits CPE formation (100% inhibition). Sera samples were analyzed as described before [
17,
19].
2.7. Statistics
GraphPad Prism 9.0 (GraphPad Software, Inc, San Diego, CA, USA) was used to perform statistics. Paired or Unpaired Student’s t-test was performed to test the statistical significance between the two groups (as indicated in the figure’s legend). Statistical significance is displayed as p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), p ≤ 0.0001 (****).
4. Discussion
In this report, we have immunologically and mechanistically assessed the difference between Delta VOC (B.1.617.2) and Omicron (B.1.1.529) VOC using three different groups of sera. Omicron harbors fifteen mutations in RBD region besides other mutations in its spike protein. In contrast, Delta has only two critical mutations, L452R and T478K. The latter mutation has also been detected in Omicron. Both L452R and T478K mutations are located in RBD, the essential region for viral binding to ACE2 and entry into receptor cells. The exact function of the remaining mutations in Omicron has yet to be revealed; nevertheless, the unusually large number of mutations in Omicron raised serious concerns about the reduced efficacy of the licensed COVID-19 vaccines to induce Omicron-neutralizing antibodies.
Recent studies have assessed the binding affinity of RBD
Omicron to human ACE2 receptors in comparison to the ancestral Wuhan strain, via atomistic molecular dynamics simulation [
23] or surface plasmon resonance [
5]. The results revealed stronger binding to human ACE2 receptor, indicating that Omicron infects cells by a similar mechanism to the WT (via ACE2) and suggesting that the clinically observed increased infectivity might be due to stronger binding interaction with ACE2. By performing Biolayer Interferometry (BLI) we demonstrate here definitively at the biophysical level that both RBD
Omicron, as well as RBD
Delta variant, have a well-defined two-fold increased affinity to ACE2 compared to RBD
wt.
Our data show that Omicron has developed two mechanisms that account for the escape from neutralizing antibodies: First, RBD mutations result in a partial mismatch with the initial antibody response, such that antibodies generated by RBDs from previous infection or vaccination are less well-blocking due to their specificity for previous RBD versions. Particularly, E484A mutation accounts for this type of specificity-escape, as it prominently changes an important epitope for neutralizing antibodies. Second, Omicron’s increased RBD-ACE2 affinity represents a challenge for neutralizing antibodies, as the virus’ RBD binds so strongly to ACE2 that antibodies become unable to compete and thus cannot block the virus. We call this phenomenon ‘affinity escape’ as this type of immune escape is clearly different from the more classical antibody specificity escape.
Our findings are consistent with the fact that Coronaviruses do not form serotypes, in contrast -for example- to Polio and Dengue viruses [
24]. Coronaviruses are large viruses with about three times more RNA nucleotides than most other RNA viruses. Despite the many small mutations that accumulate over time, Coronaviruses depend on a relatively high degree of genetic and structural stability which is assured by the RNA proofreading system of Coronaviruses [
25]. For enhancing transmission, an apparently better strategy than novel serotypes is to increase receptor affinity, a phenomenon that is now well documented for SARS-CoV-2 [
9,
11,
26,
27]. The new phenomenon of ‘affinity escape’ from neutralizing antibodies allows for a better understanding of the interactions of the virus with the immune system. We suggest that vaccines may be rendered more efficient by optimization for inducing large numbers of neutralizing antibodies with high affinity. It is well known that booster vaccinations significantly contribute to this aim, as continued affinity maturation in germinal centers of secondary lymphoid organs selectively generates and promotes high-affinity antibodies [
28]. Indeed, individuals who have received three rather than only two RNA vaccinations reach relatively high protection from Omicron [
29,
30,
31], although these vaccinations are still based on RBD
WT (Wuhan), supporting the notion that antibody specificity may not be the major reason for a breakthrough in infections. Overall, the net effect of the antibody response is a combination of specificity, activity, and titer. Mass action effect (i.e., a high magnitude response) can compensate for some shortcomings in affinity.
In summary, both Delta and Omicron VOCs show enhanced receptor affinity translated into reduced neutralization (affinity escape). For Omicron, reduction in a neutralization is, however, much more pronounced as its affinity escape is combined with specificity escape.