Omicron subvariant BA.5 efficiently infects lung cells

The Omicron variant (B.1.1.529) emerged in late 2021 with extensive spike mutations enabling marked escape from neutralizing antibodies and rapid global spread in populations with preexisting immunity. Despite its transmissibility, Omicron (particularly BA.1 and BA.2) generally caused less severe disease than prior variants and showed attenuated infection and cell–cell fusion in lung-derived cell lines, associated with decreased reliance on TMPRSS2 and increased dependence on endosomal activation pathways. BA.4 and BA.5 subsequently displaced BA.1/BA.2 in many regions. Whether these later Omicron subvariants retained the attenuated lung cell tropism of BA.1/BA.2 was unclear. The study aims to determine if BA.5 has increased capacity for lung cell entry and replication relative to BA.1/BA.2 and to elucidate spike determinants and entry pathway usage underpinning any change in tropism.

Prior work demonstrated that Omicron spike mutations confer broad neutralization escape and reduced fusogenicity compared to earlier VOCs (Alpha–Delta). BA.1/BA.2 spike-mediated entry is diminished in lung-derived cells (Calu-3) and less TMPRSS2-dependent, aligning with lower pathogenicity. The S1/S2 furin cleavage site and protease usage (TMPRSS2 vs endosomal cathepsins) modulate SARS-CoV-2 entry route, fusogenicity, and pathogenesis. Recurrent spike deletions (e.g., H69/V70) have been linked to increased infectivity (e.g., Alpha). Reports on BA.5 suggested evolving tropism and immune evasion, but the mechanistic basis for any regained lung tropism versus BA.1/BA.2 remained debated, with some studies indicating increased TMPRSS2 usage and others not, likely reflecting cell-type and expression context differences.

- Cell lines: 293T, Vero, A549-ACE2, Caco-2, Calu-3 maintained under standard conditions; A549-ACE2 stably expressing ACE2. - Pseudovirus system: VSV-based particles bearing SARS-CoV-2 S proteins (B.1, BA.1, BA.2, BA.2.12.1, BA.4/BA.5) used to assess entry. Spike expression plasmids generated via Gibson assembly; point mutations introduced by overlap-extension PCR. - Spike cleavage and incorporation: Immunoblot of concentrated pseudoparticles probed with anti-S2 and anti–VSV-M to quantify total S and S1/S2 processing. - Cell–cell fusion: Quantitative beta-galactosidase complementation assay using S-expressing 293T effector cells and ACE2-expressing 293T or A549-ACE2 targets; luminescence readout. - ACE2 binding and blockade: Flow cytometry of S-expressing 293T cells incubated with soluble ACE2-Fc; anti-ACE2 neutralizing antibody used to block pseudovirus entry (dose-response) and compute AUC. - Protease usage: Target cells pretreated with cathepsin L inhibitor MDL28170 and TMPRSS2 inhibitor camostat to assess impact on S-mediated entry, including concentration-response in Vero cells; two-way ANOVA for significance. - Mutational analysis: BA.2 and BA.4/BA.5 spike mutants focusing on BA.4/BA.5-specific changes (including H69/V70 region, L452R, F486V, R493Q) tested for effects on cell–cell fusion and entry into Vero and Calu-3 cells. - Authentic virus replication: Vero and Calu-3 cells infected with B.1, BA.1, BA.2, BA.4, BA.5 (MOI 0.01); supernatant titers quantified by plaque assay over time; AUC computed. - Mouse infections: Female BALB/c mice intranasally infected with BA.1, BA.4, BA.5; monitored for weight and clinical signs; lung viral titers by plaque assay; viral RNA and cytokine transcripts (e.g., IL-6, TNF-α) quantified by qPCR at defined days post infection. - Ferret infections: Female ferrets intranasally inoculated with BA.5; serial nasal swabs/washes collected; viral RNA quantified by qRT-PCR; serology by RBD-ELISA at 21 dpi; clinical monitoring including body weight and lethargy. - Statistics: Student’s t-tests with Welch correction, two-way ANOVA with Dunnett’s post-hoc test; p ≤ 0.05 considered significant.

- BA.4/BA.5 spike proteins display increased S1/S2 cleavage relative to earlier Omicron subvariants, consistent with enhanced activation potential. - Cell–cell fusion: BA.4/BA.5 S drives substantially greater syncytia formation than BA.1/BA.2 and approaches B.1 levels; BA.2.12.1 is intermediate in some target cell contexts. - ACE2 engagement: All Omicron S variants (BA.1, BA.2, BA.2.12.1, BA.4/BA.5) bind ACE2 with comparable efficiency. Anti-ACE2 antibody blocks entry across variants, with slightly reduced blockade efficacy for BA.4/BA.5. - Pseudovirus entry: BA.4/BA.5 shows significantly increased entry into Calu-3 lung cells versus BA.1/BA.2 (approximately 1.7-fold on average), while entry into 293T, Vero, and A549-ACE2 is similar to or slightly higher than BA.1/BA.2. - Protease preference: Sensitivity patterns to MDL28170 (cathepsin L inhibitor) and camostat (TMPRSS2 inhibitor) indicate BA.4/BA.5 retains the Omicron-like preference for cathepsin L–mediated activation; no evidence for a major shift toward TMPRSS2 usage relative to BA.1/BA.2. - Determinants of lung cell entry: The H69/V70 region is required for robust Calu-3 entry mediated by BA.4/BA.5 spike; reversion abrogates the enhanced lung cell entry phenotype. Other substitutions (L452R, F486V, R493Q) did not alone account for increased Calu-3 entry. - Authentic virus replication in vitro: BA.5 replicates efficiently in Calu-3 lung cells, in contrast to BA.1/BA.2, indicating regained lung cell tropism. - Mouse model: BA.5 replicates in lungs ~100-fold more efficiently than BA.1; BA.4 replicates robustly but roughly 10-fold less than BA.5. BA.5 induces higher expression of inflammatory cytokines (e.g., IL-6) than BA.1. - Ferret model: BA.5 achieves robust replication in the nasal cavity; one animal exhibited severe lethargy, indicating increased upper respiratory tract fitness versus BA.1. - Overall: BA.5 has acquired efficient lung cell infectivity and replication in vivo, suggesting partial loss of attenuation seen with earlier Omicron subvariants.

The study addresses whether BA.5 maintains the attenuated lung tropism characteristic of BA.1/BA.2. Functional assays show that BA.5 spike is more effectively cleaved and more fusogenic, translating into significantly enhanced entry into polarized lung cells (Calu-3) without a shift toward TMPRSS2 dependence. Mutation analyses implicate the H69/V70 region in mediating the improved lung cell entry. Authentic virus data corroborate pseudovirus results: BA.5 replicates efficiently in lung-derived cells and in vivo in mouse lungs and ferret nasal epithelium, markedly exceeding BA.1. These findings indicate that BA.5’s evolution restored properties conducive to lower airway infection, potentially increasing pathogenic capacity relative to BA.1/BA.2. Nevertheless, clinical data remain mixed: populations with high preexisting immunity (e.g., South Africa) did not observe increased severity, whereas some studies in Denmark and Canada detected higher hospitalization risk with BA.5 compared to BA.1/BA.2. Thus, while BA.5 exhibits biological features compatible with greater virulence, the realized disease severity in humans likely depends on host immunity and demographics.

This work demonstrates that Omicron BA.5 has regained efficient lung cell entry and replication capacities relative to BA.1/BA.2, driven by enhanced spike cleavage, increased fusogenicity, robust ACE2 engagement, and determinants including the H69/V70 region. In vivo, BA.5 shows substantially higher replication in mouse lungs and ferret nasal epithelium, consistent with partial loss of attenuation. These insights underscore that ongoing Omicron evolution can modulate tropism and potentially pathogenicity. Future research should: (1) define the full set of genetic determinants (in spike and non-spike genes) governing BA.5 lung tropism; (2) clarify entry pathway usage across diverse primary respiratory cell models; (3) assess the impact of innate restriction factors and endosomal barriers; and (4) integrate virological findings with clinical outcomes across populations with varying immunity to refine risk assessments for emerging subvariants.

- Some results rely on pseudotyped VSV particles, which model entry but not full replication cycles. - Cell line models (Calu-3, Caco-2, 293T, Vero) differ in receptor and protease expression; protease dependence may vary in primary human airway tissues. - The mutational analysis, while implicating the H69/V70 region, may not capture epistatic effects among multiple spike changes. - Animal studies used small cohorts; species differences limit direct extrapolation to human disease severity. - Discrepancies between in vitro human lung cell replication and robust mouse lung replication suggest additional viral genetic factors outside spike may contribute; these were not dissected here. - Comparisons to other reports of TMPRSS2 usage indicate context dependence; definitive conclusions on protease preference in vivo require primary tissue studies.