Vero E6-TMPRSS2-T2A-ACE2 cells (BEI NR-54970) were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C (Elsharkawy et al., 2024).

The SARS-CoV-2 Omicron subvariant hCoV-19/USA/MD-HP40900/2022 (XBB.1.5; NR-59104) was obtained from BEI Resources. Virus stocks were propagated in Vero E6-TMPRSS2-T2A-ACE2 cells as previously described (Elsharkawy et al., 2024). All animal experiments were conducted under biosafety level 3 (ABSL-3) containment at Georgia State University, following protocols approved by the Institutional Animal Care and Use Committee (IACUC Protocol Number: A24003). Wild-type C57BL/6 mice (JAX#0664) were purchased from The Jackson Laboratory. Six-week-old male and female mice were intranasally inoculated under anesthesia with 10⁵ plaque-forming units (PFU) of SARS-CoV-2 XBB.1.5 in phosphate-buffered saline (PBS), or with PBS alone for mock-infected controls. Mice were monitored daily for changes in body weight and clinical signs of disease. At designated time points post-infection, animals were euthanized, and respiratory tissues including nasal turbinates and lungs were collected for downstream virological, immunological, and histopathological analyses (Stone et al., 2025).

Frozen lung tissues were weighed and homogenized using a Fisherbrand™ Bead Mill 24 Homogenizer (Fisher scientific, catalog# 15-340-163). Virus titers in tissue homogenates were measured by plaque assay using Vero-hACE2-hTRMPSS2 cells as previously described (Rothan et al., 2022).

Nasal turbinates and lung tissues were lysed in RLT buffer. Total RNA was extracted using a Qiagen RNeasy Mini kit (Qiagen, Germantown, MD, USA). Viral RNA levels were measured using SARS-CoV-2 nucleocapsid gene primers (Integrated DNA Technologies, catalog#10006713) and SsoAdvanced universal probe supermix (BIO-RAD, catalog# 1725284). Viral genome copies were calculated using a standard curve and expressed viral genome copies per μg of total RNA (Jahantigh et al., 2025; Oh et al., 2024). Expression of host genes ( Table 1 ) were determined using a SsoAdvanced™ Universal SYBR^® Green Supermix (BIO-RAD, catalog# 1725271). Fold change for genes in virus-infected samples was calculated against mock-infected samples after normalization to the Gapdh gene (Auroni et al., 2023).

Lung tissue sections were stained with hematoxylin and eosin (H&E) for histopathological evaluation (Abcam, catalog# ab245880) (Kumari et al., 2021). For immunofluorescence staining, tissue sections were incubated with DsRNA (J2) mouse antibody (Absolute Antibody, catalog# Ab01299-2.0) overnight at 4°C, followed by Alexa Fluor 594 Goat anti-Mouse IgG (Life Technologies, catalog# A11005) antibodies for 30 min at room temperature. We acquired the images using a Zeiss LSM980 confocal microscope with ×10 (tiles) and ×20 objectives, then analyzed them using the ZEN 3.8 Blue software (Zhang et al., 2023). Lung tissue sections were also stained with CD45-Alexa Fluor^® 488 (Cell Signaling Technology, catalog# 59572) and Anti-Actin α-Smooth Muscle-Cy3™ antibodies (Sigma, catalog# C6198) overnight at 4°C (Stone et al., 2025). Additionally, lung tissue sections were stained with CD68-Alexa Fluor^® 488 (Cell Signaling Technology, catalog# 51644) and Anti-Actin α-Smooth Muscle-Cy3™ antibodies (Sigma, catalog# C6198) overnight at 4°C. We mounted the stained sections with ProlongTM Glass Antifade Mountant with NucBlue™ StainDAPI (Thermo Fisher Scientific, catalog# P36981). Images were acquired with the Invitrogen™- EVOS™ M5000 Cell Imaging System.

Spleen and lung single-cell suspensions were generated using the gentle MACS tissue dissociator (Miltenyi Biotec, catalog# 130-093-235). We incubated single-cell suspensions with Fc Block antibody (BD Pharmingen) in BD FACS™ Pre-Sort Buffer (BD Biosciences) for 10 min at room temperature. Cell suspensions from infected lung and spleen tissues were incubated with antibodies against the following markers: FITC Rat Anti-Mouse CD45 (BDB553080), APC Rat Anti-Mouse CD3 (BDB565643), PE Rat Anti-Mouse CD4 (BDB553730), PerCP-Cy5.5 Rat Anti-Mouse CD8β (BDB567597), and fixable Viability Stain 575V (BD Biosciences). Flow cytometry data was acquired on a BD LSRFortessa™ Cell Analyzer and was analyzed with the FlowJo software (Roe et al., 2012).

To deplete CD4⁺ or CD8⁺ T cell populations, mice were administered intraperitoneal injections of monoclonal antibodies (mAbs) targeting either CD4 or CD8β. Specifically, 100 μL of anti-mouse CD4 mAb (clone GK1.5, IgG2b; BioXcell) or anti-mouse CD8β mAb (clone 53–5.8, IgG1; BioXcell) was administered at a dose of 200 μg/mouse. To confirm depletion of targeted cells, splenic cell suspensions were incubated with antibodies against the following markers: FITC Rat Anti-Mouse CD45 (BDB553080), PE-Cy™7 Anti-Mouse CD3 (BDB552774), PE Rat Anti-Mouse CD4 (BDB553730), PerCP-Cy5.5 Rat Anti-Mouse CD8β (BDB567597) following the protocol described above. Treatments were performed on days -3 and -1 prior to XBB.1.5 infection and on days 1, 3, 6, and 12 post-infection. Control animals received 100 μL of PBS via intraperitoneal injection following the same schedule. The CD8β-specific antibody (clone 53–5.8) has been validated to achieve complete depletion of CD8⁺ T cells, while the CD4-specific antibody (clone GK1.5) effectively binds to and depletes CD4⁺ T cells by competitive binding to the CD4 molecule. Successful depletion using these antibodies has been confirmed in multiple prior studies (Ferrari de Andrade et al., 2020; Hollern et al., 2019; Shi et al., 2024).

We performed the statistical analyses using the GraphPad Prism software, version 10 and considered results statistically significant at P-values of p < 0.05. Statistical tests are indicated in figure legends.

To assess the susceptibility of C57BL/6 wild-type mice to the SARS-CoV-2 Omicron subvariant XBB.1.5, six-week-old mice were intranasally inoculated with 10⁵ PFU of XBB.1.5. Following infection, mice were monitored daily for clinical signs of disease and body weight changes for up to 12 days post-inoculation (dpi). Throughout the observation period, XBB.1.5-infected mice exhibited no significant weight loss and did not develop severe clinical symptoms ( Figure 1A ), suggesting that infection with this variant results in a mild disease phenotype in this mouse model.

In independent experiments, mice were intranasally inoculated with 10⁵ PFU of the XBB.1.5 variant and euthanized at 3 and 6 dpi for analysis. Viral RNA levels were quantified by RT-qPCR. High viral RNA loads were detected in the nasal turbinates at 3 dpi (mean = 7.2 × 10³ copies), followed by a significant reduction at 6 dpi (mean = 3.2 × 10² copies) ( Figure 1B ). Similarly, viral RNA levels in the lungs were elevated at 3 dpi (mean = 4.5 × 10⁴ copies) but declined markedly by 6 dpi (mean = 8.2 × 10¹ copies) ( Figure 1C ). In addition, plaque assay analysis confirmed the presence of infectious virus in lung homogenates at 3 dpi (mean = 1.5 × 10⁵ PFU). However, no infectious virus was detected in the lungs by 6 dpi ( Figure 1D ).

During viral infection, the accumulation of double-stranded RNA (dsRNA) serves as a hallmark of active viral replication (Li et al., 2021). To assess viral replication within lung tissues, we performed immunofluorescence staining to detect dsRNA. Consistent with the viral loads quantified by RT-qPCR and plaque assay, lungs from XBB.1.5-infected mice exhibited a markedly increased dsRNA signal compared to mock-infected controls at 3 dpi that waned by 6 dpi ( Figure 1E ). These findings further corroborate active viral replication in the pulmonary tissues of C57BL/6 wild-type mice following XBB.1.5 infection.

To characterize the host immune response to XBB.1.5 infection, we quantified the expression of key inflammatory genes in lung tissue using RT-qPCR. Irf7, a central regulator of the type I interferon response (Qing and Liu, 2023), was upregulated 8.9-fold at 3 dpi and 2.4-fold at 6 dpi compared to mock-infected controls. It was reported that Zbp1 expression was upregulated in the lungs following infection with major SARS-CoV-2 variants and contributed to lung inflammation and immune cell infiltration (Elsharkawy et al., 2025). Similarly, Zbp1 showed an 8.3-fold increase at 3 dpi and decreased significantly to 2.0-fold by 6 dpi in XBB.1.5-infected lungs. Ccl2, previously reported to be elevated in SARS-CoV-2-infected lungs (Ranjbar et al., 2022), showed a 19-fold increase at 3 dpi and remained elevated (mean = 7.9-fold) at 6 dpi. Expression of Cxcl10, a biomarker associated with severe COVID-19 outcomes (Gudowska-Sawczuk and Mroczko, 2022), was also significantly induced, with 20.4-fold and 6.2-fold increases at 3 and 6 dpi, respectively ( Figure 2A ).

To assess virus-induced lung pathology following XBB.1.5 infection, histological examination of lung tissues was performed. Hematoxylin and eosin (H&E) staining revealed significant pathological alterations in infected lungs as early as 3 dpi. Compared to mock-infected controls, lungs from infected mice exhibited marked immune cell infiltration into perivascular and alveolar regions, consolidation of alveolar spaces, and evidence of vascular congestion ( Figure 2B ). These histopathological changes are indicative of acute lung injury and reflect robust inflammatory responses induced by XBB.1.5 infection.

Previous studies have reported elevated levels of CD45⁺ immune cells in the lungs of COVID-19 patients (Won et al., 2022). To assess immune cell infiltration in response to XBB.1.5 infection, we performed immunofluorescence staining for CD45. Compared to mock-infected controls, XBB.1.5-infected mice exhibited a marked increase in CD45⁺ cells in the lungs at 3 dpi ( Figure 3A ). Similarly, postmortem analyses of COVID-19 patients have revealed increased numbers of CD68⁺ macrophages in the lungs (Barbosa et al., 2022; Cao et al., 2022), and their activation has been implicated in pulmonary tissue damage (Ziablitsev et al., 2023). To investigate macrophage infiltration, we performed immunofluorescence staining for CD68. Our data showed a substantial accumulation of CD68⁺ macrophages in the lungs of XBB.1.5-infected mice compared to controls ( Figure 3B ). These cells were primarily located in the alveolar spaces, with some clusters also observed around blood vessels. Collectively, these findings indicate that XBB.1.5 infection induces robust immune cell infiltration in the lungs of C57BL/6 wild-type mice.

Early and robust T cell responses have been associated with effective viral control in mild cases of COVID-19, whereas impaired T cell responses are often observed in severe cases (Le Bert et al., 2020; Tan et al., 2021). To characterize the cellular immune response in XBB.1.5-infected mice, we performed flow cytometric analysis on spleen and lung homogenates at 6 dpi. The gating strategy is presented in Figure 4A . No significant differences were detected in the frequencies of splenic CD4⁺ and CD8⁺ T cells between mock- and XBB.1.5-infected mice ( Figures 4B, C ). In contrast, a significant increase in lymphoid cell populations was observed in the lungs of XBB.1.5-infected mice. Specifically, the mean number of CD4⁺ T cells was 5,859 in infected lungs compared to 748 in mock-infected controls, and the mean number of CD8⁺ T cells was 1,874 compared to 474 in mock-infected lungs ( Figures 4D, E ).

To assess the role of T cells in viral clearance during SARS-CoV-2 infection, we employed antibody-mediated depletion of CD4⁺ and CD8⁺ T cell populations. To confirm the depletion of targeted cells, spleens were collected a day following PBS, Anti-CD4, and Anti-CD8 intraperitoneal treatment ( Figure 5A ). Flow cytometric analysis confirmed successful depletion of CD4⁺ and CD8⁺ T cells following Anti-CD4 and Anti-CD8 treatment, respectively. XBB.1.5-infected mice were intraperitoneally injected with depleting antibodies (anti-CD4 or anti-CD8; 200 µg/mouse) on days -3 and -1 prior to infection, and subsequently on days 1, 3, 6, and 12 post-infection. As a control, a group of XBB.1.5-infected mice were injected intraperitoneally with 100 μL of PBS following the same schedule ( Figure 5B ). Animals were sacrificed at 3-, 6-, and 16 dpi, and nasal turbinates and lung tissues were collected for viral load quantification.

CD4⁺ T cell depletion resulted in a significant increase in viral load in the nasal turbinates at both 3 and 6 dpi compared to PBS-treated controls ( Figures 5C, D ). In contrast, CD8⁺ T cell depletion caused a modest, but not statistically significant, elevation in viral loads at the same time points ( Figures 5C, D ). Importantly, viral RNA persisted in the nasal turbinates of CD4⁺ T cell-depleted mice at 16 dpi ( Figure 5E ), whereas only minimal viral RNA was detectable in CD8⁺ T cell-depleted and PBS-treated mice. In the lungs, CD4⁺ T cell-depleted mice similarly exhibited significantly higher viral loads at 3 and 6 dpi compared to PBS controls ( Figures 5F, G ). CD8⁺ T cell depletion led to a slight, non-significant increase in lung viral loads at 6 dpi ( Figure 5G ). By 16 dpi, viral RNA levels were negligible across all groups in lung tissues ( Figure 5H ). These findings indicate that CD4⁺ T cells play a critical role in the clearance of SARS-CoV-2 from the upper respiratory tract.