HEp-2 cell lines were gifted by Enmei Liu’s group from Children’s Hospital of Chongqing Medical University (Chongqing, China), and were maintained in DMEM containing 10% heat-inactivated FBS. THP-1 cell lines were purchased from Procell Life Science & Technology Co.,Ltd (Wuhan, China), and were maintained in RPMI 1640 medium containing 10% FBS.

RSV A2 was gifted by Enmei Liu’s group and recombinant red fluorescent protein (RFP)-expressing RSV A2 (rrRSV) was provided by Yuqing Wang’s group from Children’s Hospital of Suzhou Medical University (Suzhou, China). Both RSV A2 and rrRSV were grown on HEp-2 cells, purified via sucrose gradient ultracentrifugation, and stored at -80°C until use. Virus titers were determined by plaque assays.

Nirsevimab, nirsevimab Fab, and palivizumab, provided by TRINOMAB BIOTECH CO., LTD. (Zhuhai, China), were serially diluted (10-fold gradient) from original concentrations (1 µg/µL). RSV A2 was incubated with these antibody dilutions at 37°C for 1 h. Subsequently, HEp-2 cells (MOI=1) and THP-1 cells (MOI=10) were infected and incubated at 37°C for 1 h. Afterward, the medium was replaced, and the cells continued to incubate at 5% CO2, 37°C for 24 h. These cells were then used for flow cytometric analysis.

To prevent non-specific binding of antibodies to Fc receptors, cells were incubated with Fc block reagent (Biolegend, 422301) at 4°C for 15 min. Cells were then stained with FITC-conjugated goat polyclonal antibody to RSV (Invitrogen, PA1-73017) at 4°C for 30 min. Flow cytometry was performed on the BD FACSCanto (BD Bioscience), with data collected from 20,000 cells per sample. Data were analyzed using FlowJo V10 software (FLOWJO, LLC, OR). Cells were considered RSV-positive if they exhibited fluorescence more intense than that of 99.5% of the mock-infected cells.

RSV A2 was incubated with antibodies at 37°C for 1 h, followed by addition of cells on ice for 1 h. Then, cells were collected, washed twice with PBS, fixed with 4% formaldehyde, blocked with Fc receptor block reagent, stained, and finally detected by flow cytometry.

RSV A2 was incubated with antibodies at 37°C for 1 h, followed by the addition of cells and continued incubation at 37°C for 1 h. Cells were washed twice with PBS and fresh medium was added to the incubator for continued incubation for 1 h. Cells were then collected for the following assays.

Flow Cytometry: Cells were washed twice with PBS and incubated with 0.5% trypsin on ice for 10 min to remove surface-attached viruses (25). Then, Fc receptors were blocked, and intracellular staining was performed after using Fixating/Permeabilization Kit (BD Bioscience, 554714). Finally, cells were detected by flow cytometry.

TEM (Transmission Electron Microscopy): Cells were collected and centrifuged in 1.5 ml EP tubes at 1000 rpm for 5 min. The supernatant was discarded, and a fixed solution diluted 1:5 (3% glutaraldehyde: 0.1 mol/L PBS buffer) was added slowly along the wall of the tube. The cells were then resuspended and allowed to stand for 5 min at 4°C. Afterward, cells were centrifuged at 12,000 rpm for 10 min, supernatant was gently discarded, and 3% glutaraldehyde fixative was slowly added along the inner wall to avoid dispersing cells. Finally, the samples were sent to Chengdu Lilai Biotechnology Co., Ltd (Chengdu, China) for testing.

RSV A2 or rrRSV was incubated with the antibodies at 37°C for 1 h, followed by the addition of cells and continued incubation at 37°C for 1 h. Cells were washed twice with PBS and fresh medium was added to the incubator for continued incubation. Cells and supernatants were collected at 1, 6, 12, 20, 24, 36 and 48 h.

Flow Cytometry: The rrRSV-infected cells were collected, washed twice with PBS, and detected by flow cytometry for infection rates.

qRT-PCR: Total cellular RNA was extracted using an RNA rapid extraction kit (BioFlux, China). RNA was transferred to cDNA using Evo M-MLV Mix Kit with gDNA Clean for qPCR (Accurate Biology, AG11728, China). Real-time PCR was performed using the ABI PRISM 7900 HT (Applied Biosystems, Foster City, CA). The number of RSV copies was quantified with a TaqMan probe, and a standard curve of plasmid DNA was used to estimate the exact copy number of RSV N gene. Specific primers and probes for RSV A2 N gene and plasmid sequences are shown in Table 1 .

TEM: This was performed as described above.

Plaque assay: The supernatants were 10-fold serially diluted to a 10–⁷ dilution and added into wells of 24-well plates grown with HEp-2 cells in monolayer. After 1 h of infection, the viral solutions were removed and 1 mL of a 1:1 mixture of 0.6% agarose and 2X DMEM with 5% FBS was added. Five days post incubation, 4% paraformaldehyde was added to fix cells for 1 h. The agarose overlay was removed, the cells were stained with 0.05% neutral red, and the number of plaques was counted.

Flow Cytometry: The rrRSV supernatants (200 μL) were added into 24-wells plates grown to 80% fusion and incubated at 37°C for 2 h. Subsequently, the viral solutions were removed, fresh medium was added, and the incubation was continued for 24 h. Cells were collected for infection rates via flow cytometry.

RNA was extracted from THP-1 cells from T0, T2 and T5 groups using the RNA extraction kit (Genstone Biotech, Beijing, China). Then RNA sequencing (RNA-seq) was done by BGI Genomics Co., Ltd (Shenzhen, China). Differential expression analysis was performed using the R packag2 ‘DESEQ-2’, and genes with p < 0.05, and |log2FC| > 1 were selected as differentially expressed genes. The clusterProfiler package was used for the KEGG enrichment analysis using P < 0.05 as the statistical criteria.

The levels of IL-1β, IL-6, IL-10, and TNF-α in culture supernatants were measured using commercial ELISA kits (NEOBIOSCIENCE, Shenzhen, China) according to the manufacturer’s instructions.

THP-1 cells were pre-incubated for 1 h with 20 µg/mL neutralization antibodies against TLR4 (clone HTA125) (Invitrogen,14-9917-82), or blocking antibodies (10 µg/mL) against FcγRI (10.1, BD), or FcγRIIa (BioXcell, BE0224-1MG). Then, the cells were infected to assess RSV entry rate and 12 h-infection rates.

Cells were collected 24 h after infection and subjected to real-time PCR using the previously described method. Human FcγRI, FcγRIIa and FcγRIIb levels were normalized against GAPDH for relative quantification. Specific primers for human FcγRI, FcγRIIa, FcγRIIb and GAPDH are shown in Table 1 .

Firstly, to investigate the conditions under which ADE occurs, we used nirsevimab and palivizumab for study. Nirsevimab is an IgG1 antibody targeting the site Ø of RSV pre-F protein, while palivizumab is an IgG1 antibody targets site II of pre-F and post-F protein. Additionally, nirsevimab has a 50-fold greater neutralizing capacity than palivizumab (26).

We mixed different dilutions of nirsevimab, nirsevimab Fab, or palivizumab with RSV, then infected HEp-2 cells and THP-1 cells. After 24 h, the percentage of RSV-positive cells was detected by flow cytometry and normalized by the infection rate in the absence of antibodies. The results showed that in FcγR-negative HEp-2 cells, both palivizumab and nirsevimab demonstrated concentration-dependent neutralization against RSV infection ( Figure 1A ). However, in FcγR-bearing THP-1 cells, enhanced RSV infection was observed at dilutions ranging from 10–³ to 10–⁵ for palivizumab and 10–⁴ to 10–⁶ for nirsevimab, and ADE occurred; ADE disappeared when we used nirsevimab Fab with the Fc fragment removed ( Figure 1B ). These findings suggest that FcγRs are essential for the development of ADE during RSV infection when anti-F antibodies at sub-neutralizing levels.

To investigate the mechanism of Fc-FcγR-mediated enhancement of RSV infection, we dissected the different steps of RSV replication in cells during ADE, which mainly include viral attachment, endocytosis, replication, and release. Experimental groups were established using HEp-2 and THP-1 cells under three conditions: RSV infection without antibodies (H0/T0), with fully protective nirsevimab concentrations (10–² dilution, H2/T2), and ADE-inducing concentrations (10–⁵ dilution, H5/T5).

To delineate Fc-FcγR interactions in early RSV infection, we analyzed viral attachment and endocytosis across experimental groups. Key findings revealed cell-type-specific differences. Flow cytometry showed no significant differences in RSV attachment or entry rates between antibody-treated (H2/H5) and untreated (H0) groups in HEp-2 cells ( Figures 2A, C ). However, in THP-1 cells, both T2 and T5 groups exhibited significantly elevated RSV attachment and entry rates compared to the T0 control ( Figures 2B, D ). Increase of attachment and entry disappeared when using nirsevimab Fab, which confirmed FcγRs dependence ( Figures 2B, D ). TEM allows direct observation of viral particles endocytosed into cells, and the results are consistent with flow cytometry data ( Figure 2E ). These findings indicate that F protein antibodies allow RSV attachment and entry into cells even at neutralizing concentrations, while Fc-FcγR interactions mediate additional viral attachment and entry.

To assess antibody effects on viral replication, we quantified intracellular RSV dynamics through three approaches: qPCR for N gene copy number, flow cytometry monitoring rrRSV protein translation (via RFP+ cells), and TEM visualization of viral particles. In HEp-2 cells, neutralizing antibody treatment (H2 group) induced a time-dependent reduction in N gene copy number ( Figure 3A ), near-complete suppression of viral protein synthesis (RFP+ cells ≈0%; Figure 3C ), and TEM-confirmed viral clearance ( Figure 3E ). The above indicators gradually increased over time in the H5 group but remained consistently lower than in H0 group ( Figurse 3A, C, E ), demonstrating partial replication inhibition. However, enhanced RSV replication was observed under sub-neutralizing antibody concentrations; The N gene copy number, RFP+ cell rate, and intracellular viral particles exceeding those of the T0 group ( Figures 3B, D, F ). The results of RSV replication in the T2 group were identical to those of the H2 group. These findings reveal a dual regulation of antibodies to F protein: while neutralizing antibody concentrations universally inhibit endocytosed RSV replication across cell types, sub-neutralizing doses paradoxically enhance viral dissemination in macrophages via FcγR-mediated processes.

Subsequently, we evaluated whether antibody-enhanced infection could lead to the production of infectious viral progeny. Plaque assay revealed that the viral titer in the T5 group was 2 to 3 times higher than that in the T0 group, while no virus was detected in the T2 group ( Figure 4A ). Similarly, the infection of HEp-2 cells with the rrRSV supernatant demonstrated that the proportion of RFP+ cells was nearly zero in the T2 and H2 groups, significantly higher in the T5 group compared to the T0 group, and lower in the H5 group than in the H0 group ( Figures 4B, C ). These results suggest that the release of RSV was also increased when ADE occurs.

Our findings demonstrated that only sub-neutralizing antibody concentrations promote enhanced viral replication following RSV entry. We postulated that this effect might stem from incomplete neutralization, where sub-neutralizing antibodies fail to fully block RSV F protein-mediated membrane fusion despite facilitating increased viral entry. To test this hypothesis, we treated T5-group RSV with nirsevimab, followed by a secondary incubation with high-concentration nirsevimab Fab fragments to achieve completely F protein occlusion. The results showed that RSV entry rates post-Fab treatment remained significantly elevated compared to the T0 group ( Figure 5A ), indicating the blockade of redundant F proteins by Fab did not inhibit the internalization of the virus in THP-1 cells mediated by the interaction of Fc and FcγR. However, RFP+ THP-1 cells were virtually undetectable at 24 h post-infection ( Figure 5B ), and supernatant-reinfected HEp-2 cells similarly exhibited negligible RFP positivity ( Figure 4B ), suggesting the viral replication was inhibited. Collectively, these data demonstrate that ADE is caused by insufficient neutralization of the F protein, and that incomplete blockade of F proteins enables membrane fusion following RSV entry, thereby facilitating viral replication.

To delineate the molecular changes underlying RSV ADE, we performed transcriptomic profiling via RNA sequencing (RNA-seq) on THP-1 cells from T0, T2, and T5 groups. Global gene expression analysis revealed that the majority of differentially expressed genes (DEGs) exhibiting inverse trends—genes upregulated in T2 group were downregulated in T5 group, and vice versa (top 50 DEGs visualized; Figure 6A ). Across 16,720 analyzed genes, cells in T2 group displayed 35 significantly upregulated and 472 downregulated genes (fold change >2, P < 0.05 vs. T0; Figure 6B ), whereas cells in T5 group exhibited 137 upregulated and 17 downregulated genes under the same thresholds ( Figure 6C ).

KEGG pathway enrichment analysis of DEGs identified the top 20 enriched signaling pathways, with immune and antiviral response pathways—including NOD-like receptor (NLR), chemokine, TNF, NF-κB, Toll-like receptor, apoptosis, and multiple virus-host interaction pathways—being significant inhibitory/activation of immune and viral response pathways in T2/T5 group ( Figures 7A-D ). Notably, the FcγR signaling pathway—a hallmark of antibody-dependent immune modulation—was prominently enriched in both groups, but was not shown in Figure 7C due to being ranked outside the top 20 pathways in the T5 group. These pathways collectively drive inflammatory cytokine production, prompting us to quantify IL-1β, IL-6, and TNF-α levels in cellular supernatants via ELISA. Mirroring transcriptional findings, supernatants in T5 group showed marked increases in all three pro-inflammatory cytokines, while exhibited significant suppression in T2 group relative to T0 group ( Figures 7E-G ). These results demonstrate that ADE induces a hyperinflammatory state characterized by FcγR-mediated pathway activation and dysregulated cytokine release.

It is reported that FcγR-mediated cytokine responses depend on activating/inhibitory receptor ratios and secondary signals like TLR activation (16, 27–30). Building on evidence implicating FcγRs in RSV ADE, we further investigated their interplay with TLR4, a known receptor of RSV F protein (31). Blockade of TLR4, FcγRI, or FcγRIIa reduced T5-group RSV infection rates, with efficacy hierarchies: FcγRI > TLR4 > FcγRIIa ( Figure 8A ). Notably, residual infection rates in all single-blockade conditions remained significantly elevated over T0 baseline. Dual blockade strategies further attenuated infectivity: concurrent inhibition of FcγRI+TLR4 reduced infection rates to T0-equivalent levels, whereas FcγRIIa+TLR4 blockade retained residual infectivity exceeding T0 ( Figure 8A ). These findings identify FcγRI as the dominant FcγR subtype driving ADE, acting synergistically with TLR4 to potentiate RSV infection.

Given evidence of inflammatory feedback regulating FcγR expression (32), we quantified FcγR transcript dynamics post-infection via qPCR. While T2-group THP-1 cells showed no significant FcγRI/FcγRIIa/FcγRIIb expression changes versus T0, T5-group cells exhibited marked upregulation of all three receptors, with FcγRI induction being most pronounced ( Figure 8B ). This FcγR upregulation, particularly FcγRI, suggests a self-reinforcing cycle: ADE-induced inflammatory signals (e.g., IL-1β/IL-6/TNF-α; Figures 7E-G ) upregulate FcγR expression, which in turn potentiates viral entry and further cytokine dysregulation. Collectively, these data unveil a pathogenic loop wherein FcγRI-TLR4 crosstalk initiates ADE-driven viral internalization, while subsequent inflammatory cascades amplify FcγR availability, creating a feedforward circuit that exacerbates both infection and immunopathology.