Abstract
Background
The COVID-19 pandemic is caused by the betacoronavirus SARS-CoV-2. In November 2021, the Omicron variant was discovered and immediately classified as a variant of concern (VOC), since it shows substantially more mutations in the spike protein than any previous variant, especially in the receptor-binding domain (RBD). We analyzed the binding of the Omicron RBD to the human angiotensin-converting enzyme-2 receptor (ACE2) and the ability of human sera from COVID-19 patients or vaccinees in comparison to Wuhan, Beta, or Delta RBD variants.
Methods
All RBDs were produced in insect cells. RBD binding to ACE2 was analyzed by ELISA and microscale thermophoresis (MST). Similarly, sera from 27 COVID-19 patients, 81 vaccinated individuals, and 34 booster recipients were titrated by ELISA on RBDs from the original Wuhan strain, Beta, Delta, and Omicron VOCs. In addition, the neutralization efficacy of authentic SARS-CoV-2 wild type (D614G), Delta, and Omicron by sera from 2× or 3× BNT162b2-vaccinated persons was analyzed.
Results
Surprisingly, the Omicron RBD showed a somewhat weaker binding to ACE2 compared to Beta and Delta, arguing that improved ACE2 binding is not a likely driver of Omicron evolution. Serum antibody titers were significantly lower against Omicron RBD compared to the original Wuhan strain. A 2.6× reduction in Omicron RBD binding was observed for serum of 2× BNT162b2-vaccinated persons. Neutralization of Omicron SARS-CoV-2 was completely diminished in our setup.
Conclusion
These results indicate an immune escape focused on neutralizing antibodies. Nevertheless, a boost vaccination increased the level of anti-RBD antibodies against Omicron, and neutralization of authentic Omicron SARS-CoV-2 was at least partially restored. This study adds evidence that current vaccination protocols may be less efficient against the Omicron variant.
Background
SARS-CoV-2 is the etiological agent of the severe pneumonia COVID-19 (coronavirus disease 2019) [1, 2]. A new variant B.1.1.529 of the betacoranavirus SARS-CoV-2 was identified in late November 2021 and has rapidly been classified as a variant of concern (VOC) by the WHO and named Omicron [3]. The Omicron variant shows a high number of mutations in the SARS-CoV-2 spike protein in comparison to the previously described VOCs Alpha [4], Beta (B.1.351) [5], Gamma (P.1) [6], and the currently dominating Delta variant (B.1.617.2) [7]. The first sequenced Omicron variant (GISAID accession ID EPI_ISL_6913995, collection date 2021-11-08, South Africa) contains a total of 36 mutations compared to the original Wuhan strain and includes 29 amino acid (aa) changes, six aa deletions, and one aa insertion. Fifteen of these mutations are concentrated in the N-terminal receptor-binding domain (RBD) of the spike protein which binds to the human zinc peptidase angiotensin-converting enzyme 2 (ACE2) for cell entry [8, 9].
Importantly, the RBD is targeted by more than 90% of the neutralizing serum antibodies, making it the most relevant target for SARS-CoV-2 neutralization [10, 11]. Consequently, the majority of therapeutic antibodies for the treatment of COVID-19 are designed to interact with this part of the SARS-CoV-2 spike protein [12, 13]. The abundant mutations in spike might indicate that the Omicron variant may bind with a different affinity to the ACE2 receptor, therefore may be altering its cell entry characteristics. Simultaneously, the mutations may help the virus to escape the immune recognition by antibodies, facilitating viral spread in a seropositive population.
While initial studies have shown a severe reduction in serum neutralizing capacity of vaccinated and convalescent patients against the Omicron variant [14,15,16], it is unclear to which extent the RBD domain mutations contribute to this loss in neutralization activity. Additionally, while several mutations present in Omicron are computationally predicted to increase ACE2 binding affinity, others are predicted to reduce its affinity [17].
The aim of this study is the analysis of the new Omicron RBD and unravel why the Omicron variant is displacing other variants. Therefore, the binding of ACE2 to the new Omicron RBD was determined in comparison to the original Wuhan strain and the Beta and Delta variants by two different techniques. Moreover, we tested the binding of human sera from COVID-19 hospitalized patients or vaccinated persons with 2× BNT162b2, 1× Ad26.COV2.S, or 2× mRNA1273 vaccines, as well as boost vaccinated persons, to the RBD of the original Wuhan strain, the Beta, the Delta, and the Omicron VOC. Furthermore, the neutralization efficacy of sera of 2× BNT161b2 and boost vaccinees was analyzed using the authentic SARS-CoV-2 virus.
Methods
Serum samples
Blood samples were obtained from non-vaccinated, intensive care unit (ICU) patients with severe symptoms from the second (pre-Alpha) and third (Alpha variant) pandemic wave in Croatia (Rijeka, sampling December 2020 to April 2021) and Italy (Pavia, sampling March 2020 to February 2021) or from vaccinated people in Germany (Braunschweig, sampling June 2021 to December 2021), Sweden (Stockholm, May 2021 to November 2021), and Italy (Pavia, February 2021 to January 2022) as indicated. While all voluntary donors were informed about the project and gave their consent for the study, consent requirement was waived by the ethical committee in Rijeka for patients in intensive care where sampling was a part of routine diagnostics. Ten out of the 17 patients of the Croatian cohort died by COVID-19 infection. The sampling was performed in accordance with the Declaration of Helsinki. The donors included adults of both sexes. The first WHO International Standard for anti-SARS-CoV-2 immunoglobulin (NIBSC code: 20/136) was used as positive control serum, and pre-pandemic negative control sera were provided by the LADR Braunschweig and did not bind to any RBD variant (data not shown). Approval was given from the ethical committee of the Technische Universität Braunschweig (Ethik-Kommission der Fakultät 2 der TU Braunschweig, approval number FV-2020-02). The study in Croatia was approved by the Ethics committee of the Rijeka Clinical Hospital Center (2170-29-02/1-20-2). The study in Italy was performed under the approval of the Institutional Review Board of Policlinico San Matteo (protocol number P_20200029440). The study in Sweden was approved by the ethics committee in Stockholm (Dnr 2020-02646).
Details about study participants are shown in Table 1.
Construction of the expression vectors
All sequences of the RBD variants (319-541 aa of GenBank: MN908947) were inserted in a NcoI/NotI compatible variant of the OpiE2 expression vector [18] containing an N-terminal signal peptide of the mouse Ig heavy chain and a C-terminal 6xHis-tag. Single-point mutations to generate the Beta and Delta variants of RBD were inserted into the original Wuhan strain through site-directed mutagenesis using overlapping primers according to Zheng et al. [19] with slight modifications: S7 fusion polymerase (Mobidiag) with the provided GC buffer and 3% dimethyl sulfoxide was used for the amplification reaction. The RBD Omicron variant was ordered as GeneString from GeneArt (Thermo Fisher) according to EPI_ISL_6590608 (partial RBD Sanger sequencing from Hong Kong), EPI_ISL_6640916, EPI_ISL_6640919, and EPI_ISL_6640917 including Q493K which was corrected later to Q493R. Table 2 gives an overview about the used variants.
Expression and purification of the RBD variants
The different RBD variants were produced in the baculovirus-free High Five cell system [20] and purified as described before [21]. Briefly, High Five cells (Thermo Fisher Scientific) were cultivated at 27°C, 110–115 rpm in EX-CELL 405 media (Sigma Aldrich) at a cell density between 0.3 and 5.5 × 106 cells/mL. On the day of transfection, cells were centrifuged and resuspended in fresh media to a density of 4 × 106 cells/mL before 4 μg expression plasmid/mL and 16 μg/mL of linear PEI 40 kDa (Polysciences) was pipetted directly into the cell suspension. After 4–24 h, cells were supplemented with fresh media to dilute the cells ~1 × 106 cells/mL, and 48 h after transfection, culture volume was doubled. Cell supernatant was harvested 4 to 5 days after transfection by a two-step centrifugation (4 min at 180 ×g and 20 min at >3500 ×g) and then 0.2 μm filtered for purification. Immobilized metal ion affinity chromatography (IMAC) His tag purification of insect cell supernatant was performed with a HisTrap excel column (Cytiva) on Äkta system (Cytiva) according to the manufacturer’s manual. In a second step, the RBD domains were further purified by size exclusion chromatography (SEC) by 16/600 Superdex 200 kDa pg column (Cytiva).
Expression and purification of ACE2-hFc
The extracellular domain of ACE2 receptor (GenBank NM_021804.3) was produced in pCSE2.6-hFc expression vector in Expi293F cells (Thermo Fisher Scientific) as described before [22]. In brief, Expi293F cells were cultivated at 37°C, 110 rpm, and 5% CO2 in Gibco FreeStyle F17 expression media (Thermo Fisher Scientific) supplemented with 8 mM Glutamine and 0.1% Pluronic F68 (PAN Biotech). For transfection, 1 μg DNA and 5 μg of 40 kDa PEI (Polysciences) per mL transfection volume were diluted separately in 5 transfection volumes and then mixed for the formation of complexes (20–30 min). Afterwards, PEI:DNA complexes were added to 1.5–2 × 106 cells/mL. Forty-eight hours later, the culture volume was doubled by feeding HyClone SFM4Transfx-293 media (GE Healthcare) supplemented with 8 mM Glutamine and HyClone Boost 6 supplement (GE Healthcare) with 10% of the end volume. One week after transfection, the supernatant was harvested by 15 min centrifugation at 1500 ×g. Purification was performed on a 1-mL HiTrap Fibro PrismA (Cytiva) column on Äkta go (Cytiva) according to the manufacturer’s manual.
ACE2 binding to RBD analyzed by titration ELISA
ACE2 binding to the produced RBD variant antigens was analyzed in ELISA in triplicates where 300 ng RBD per well was immobilized on a Costar High binding 96-well plate (Corning, Costar) at RT for 1 h. Next, the wells were blocked by 330 μL 2% MPBST (2% (w/v) milk powder in PBS; 0.05% Tween20) for 1 h at RT and then washed 3 times with H2O and 0.05% Tween20 (BioTek Instruments, EL405). ACE2-hFc was titrated from 0.01 mg/mL down to 1 ng/mL and incubated 1 h at RT prior to another 3× times washing step. Detection was performed by goat-anti-hIgG(Fc) conjugated with HRP (1:70,000, A0170, Sigma) and visualized with tetramethylbenzidine (TMB) substrate (20 parts TMB solution A (30 mM potassium citrate; 1% (w/v) citric acid (pH 4.1)) and 1 part TMB solution B (10 mM TMB; 10% (v/v) acetone; 90% (v/v) ethanol; 80 mM H2O2 (30%)) were mixed). After addition of 1 N H2SO4 to stop the reaction, absorbance at 450 nm with a
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