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Comparative Study
. 2022 Sep 14;96(17):e0114022.
doi: 10.1128/jvi.01140-22. Epub 2022 Aug 24.

Characterization of Entry Pathways, Species-Specific Angiotensin-Converting Enzyme 2 Residues Determining Entry, and Antibody Neutralization Evasion of Omicron BA.1, BA.1.1, BA.2, and BA.3 Variants

Sabari Nath Neerukonda  1 , Richard Wang  1 , Russell Vassell  1 , Haseebullah Baha  1 , Sabrina Lusvarghi  1 , Shufeng Liu  1 , Tony Wang  1 , Carol D Weiss  1 , Wei Wang  1
Affiliations
Comparative Study

Characterization of Entry Pathways, Species-Specific Angiotensin-Converting Enzyme 2 Residues Determining Entry, and Antibody Neutralization Evasion of Omicron BA.1, BA.1.1, BA.2, and BA.3 Variants

Sabari Nath Neerukonda et al. J Virol. .

Abstract

The SARS-CoV-2 Omicron variants were first detected in November 2021, and several Omicron lineages (BA.1, BA.2, BA.3, BA.4, and BA.5) have since rapidly emerged. Studies characterizing the mechanisms of Omicron variant infection and sensitivity to neutralizing antibodies induced upon vaccination are ongoing by several groups. In the present study, we used pseudoviruses to show that the transmembrane serine protease 2 (TMPRSS2) enhances infection of BA.1, BA.1.1, BA.2, and BA.3 Omicron variants to a lesser extent than ancestral D614G. We further show that Omicron variants have higher sensitivity to inhibition by soluble angiotensin-converting enzyme 2 (ACE2) and the endosomal inhibitor chloroquine compared to D614G. The Omicron variants also more efficiently used ACE2 receptors from 9 out of 10 animal species tested, and unlike the D614G variant, used mouse ACE2 due to the Q493R and Q498R spike substitutions. Finally, neutralization of the Omicron variants by antibodies induced by three doses of Pfizer/BNT162b2 mRNA vaccine was 7- to 8-fold less potent than the D614G. These results provide insights into the transmissibility and immune evasion capacity of the emerging Omicron variants to curb their ongoing spread. IMPORTANCE The ongoing emergence of SARS-CoV-2 Omicron variants with an extensive number of spike mutations poses a significant public health and zoonotic concern due to enhanced transmission fitness and escape from neutralizing antibodies. We studied three Omicron lineage variants (BA.1, BA.2, and BA.3) and found that transmembrane serine protease 2 has less influence on Omicron entry into cells than on D614G, and Omicron exhibits greater sensitivity to endosomal entry inhibition compared to D614G. In addition, Omicron displays more efficient usage of diverse animal species ACE2 receptors than D614G. Furthermore, due to Q493R/Q498R substitutions in spike, Omicron, but not D614G, can use the mouse ACE2 receptor. Finally, three doses of Pfizer/BNT162b2 mRNA vaccination elicit high neutralization titers against Omicron variants, although the neutralization titers are still 7- to 8-fold lower those that against D614G. These results may give insights into the transmissibility and immune evasion capacity of the emerging Omicron variants to curb their ongoing spread.

Keywords: SARS-CoV-2 Omicron variants; animal ACE2 receptors; breakthrough infections; neutralization resistance; transmembrane serine protease 2; vaccine booster; virus entry pathways.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
Omicron lineage substitutions in spike. Substitutions in Omicron spikes are shown in a primary structure of the SARS-CoV-2 spike protein, with various domains and cleavage sites indicated. SP, signal peptide; NTD, N-terminal domain; RBD, receptor binding domain; RBM, receptor binding motif; C, domain C; D, domain D; S1/S2, furin cleavage junction of S1/S2 subunits; UH, upstream helix; FP, fusion peptide; HR1/2, heptad repeat 1/2; CH, central helix; BH, beta hairpin; CD, connector domain; SH, stem helix; TM, transmembrane domain. Substitutions common to Omicron (BA) are shown in black. Substitutions unique to BA.1 are shown in red. R346K substitution additionally present in BA.1.1 is shown in light blue. Substitutions unique to BA.2 are shown in green. Substitutions in BA.1 and BA.2 shared by BA.3 are shown as residues marked with an asterisk.
FIG 2
FIG 2
Infection and cell entry mechanisms of Omicron lineage pseudoviruses. Each cell type was infected with D614G and Omicron lineage pseudoviruses. (A) Infectivity of D614G and Omicron (BA) lineage pseudovirus stocks on 293T-ACE2 and 293T-ACE2-TMPRSS2 cells. (B) Relative infectivity of D614G and Omicron lineage pseudoviruses in stable 293T-ACE2 cells compared to stable 293T-ACE2-TMPRSS2 cells. (C and D) Chloroquine sensitivity of D614G and Omicron lineage pseudoviruses in stable 293T-ACE2 (C) and 293T-ACE2-TMPRSS2 cells (D). Cells were pretreated with the indicted concentration of chloroquine for 2 h prior to infections with pseudoviruses in medium containing the inhibitor. (E and F) Camostat sensitivity of D614G and Omicron lineage pseudoviruses in stable 293T-ACE2-TMPRSS2 (E) and 293T-ACE2 cells (F). Cells were pretreated with the indicted concentration of camostat mesylate for 2 h prior to infection with pseudoviruses in medium containing the inhibitor. The x axis indicates the concentration of inhibitor. The y axis indicates the percentage of inhibition compared to pseudovirus infection without inhibitor treatment. Results shown are the average of three independent experiments. Asterisks (*) denote significance: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.
FIG 3
FIG 3
The neutralization of soluble ACE2 to Omicron lineage pseudoviruses. The sensitivity of D614G and Omicron lineage pseudoviruses to soluble human ACE2 was evaluated on stable 293T-ACE2-TMPRSS2 cells. Results shown are representative of three independent experiments.
FIG 4
FIG 4
Thermal stability of Omicron pseudoviruses. The temperature stability of D614G and Omicron lineage pseudoviruses on 293T-ACE2-TMPRSS2 and 293T-ACE2 cells was evaluated. (A and C) Pseudoviruses were untreated or subjected to various temperatures for an hour prior to infectivity on 293T-ACE2-TMPRSS2 (A) and 293T-ACE2 (C) cells. (B and D) Pseudoviruses were untreated or subjected to 50°C for different durations prior to infectivity on 293T-ACE2-TMPRSS2 (B) and 293T-ACE2 (D) cells. Results shown are the average of three independent experiments.
FIG 5
FIG 5
pH stability of Omicron pseudoviruses. The pH stability of D614G and Omicron lineage pseudoviruses on 293T-ACE2-TMPRSS2 and 293T-ACE2 cells was evaluated. (A and B) Pseudoviruses were untreated or subjected to various pHs for an hour prior to adjusting the pH to 7.0 and infectivity on 293T-ACE2-TMPRSS2 (A) and 293T-ACE2 (B) cells. Results shown are the average of three independent experiments.
FIG 6
FIG 6
Omicron lineage pseudovirus entry into cells expressing ACE2 from different species. (A to J) Infectivity of D614G (A), BA.1 (B) BA.2 (C) BA.3 (D) BA.1.1 (E), Q493R (F), Q498R (G), BA.2_R493Q (H), S373P (I), and Y453F (J) pseudoviruses on 293T cells transiently transfected with ACE2 orthologs of the indicated species. The ACE2 of African green monkey is denoted as AGM. 293T cells expressing ACE2 of the indicated species, as well as control 293T-ACE2 cells stably expressing human ACE2, were simultaneously infected by the indicated pseudoviruses with titers of ~106 RLU/mL on 293T-ACE2 cells. Luciferase activities were determined 48 h postinfection. ns, not significant. Significant differences in infectivity between each species’ ACE2 compared to the pcDNA3.1 control for pseudoviruses are denoted by asterisks: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****,: P ≤ 0.0001. The dashed line indicates maximum background level infection on 293T cells. Results shown are the average of three independent experiments with eight intra-assay replicates. (K to N) The SARS-CoV-2 Omicron RBD-ACE2 interface (PDB: 7WBP) is shown with contacting residues as sticks at the RBD-ACE2 interface. The SARS-CoV-2 Omicron RBD and ACE2 are colored in gray and cyan, respectively. Positions in RBD (blue) that contact ACE2 (red) residues are highlighted. Residue positions are indicated by arrows. The SARS-CoV-2 RBD/ACE2 interactions between 493R/E35 (K), R493/K35 (L), Q493/K35 (M), and R498/D38-H353 (N) are shown.
FIG 7
FIG 7
The sensitivity of Omicron lineage pseudoviruses to post-vaccine booster and vaccine breakthrough infection sera. (A) Neutralization of D614G and Omicron lineage pseudoviruses to vaccine booster-elicited sera obtained from individuals that received primary two-dose series and a third booster dose of Pfizer/BNT162b2 mRNA vaccine. (B) Sera neutralization titers pre- and post-booster receipt in individuals. (C) Titers of five vaccine breakthrough cases that experienced BA.1 or BA.1.1 infection. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. NS, not significant. Results shown are average two independent experiments.
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