HEp-2 cells (ATCC, CCL-23™) were cultured in Minimum Essential Medium (MEM; Thermo Fisher Scientific, Waltham, MA, USA, Cat. #42360099) supplemented with 10% inactivated fetal bovine serum (Thermo Fisher Scientific, Cat. #10099141) and 100 U/ml penicillin–streptomycin (Thermo Fisher Scientific, Cat. #15140-122). The RSV A2 strain (ATCC), recombinant virus, A2-mKate and RSV clinical isolates (7445 and 7127) were produced in HEp-2 cells as previously described (Rameix-Welti et al., 2014) and stored at −80 °C to ensure the stability. Viral titers were determined via plaque assay as previously mentioned (Zhang et al., 2019).

Expression and purification of soluble RSV-F proteins and antibodies against the F protein were performed according to previous methods (Mclellan et al., 2013). In brief, plasmids encoding the pre-F protein (DS-Cav1), post-F protein and antibodies against RSV F protein were transiently transfected into Expi293F™ cells (Thermo Fisher Scientific, Cat. #A14528). After 7 days, the cell culture supernatants were collected and centrifuged to remove cell debris. Ni Sepharose fast-flow 6 resin (GE Healthcare, USA) was used for RSV-F proteins, followed by further purification on HiLoad 16/600 Superdex 200 pg size exclusion column (GE Healthcare) according to the manufacturer's instructions. For antibodies against the F protein, the protein A agarose column (GenScript, Cat. #L00210) was used. Purified trimeric F proteins and antibodies were dialyzed against 1 × PBS at pH 7.4, and stored at −80 °C until further use.

At 24 h post-infection, HEp-2 cells infected with RSV A2-mKate were detached and prepared as single-cell suspensions. Cells were resuspended in staining buffer and incubated on ice with primary antibodies targeting distinct epitopes of the F protein (10 μg/ml) for 30 min. After two washes with staining buffer, samples were incubated on ice in the dark with the appropriate BV421-conjugated secondary antibodies (A85-1, R2-40 or G18-145, BD Bioscience https://www.bdbiosciences.com/zh-cn) for 30 min. Following final washes, cells were resuspended in staining buffer and immediately acquired on the BD LSRFortessa X-20. Data were analyzed using FlowJo software (version 10.10.0, BD Bioscience https://www.flowjo.com/company).

HEp-2 cells infected with RSV A2-mKate at a multiplicity of infection (MOI) of 1 were seeded in 96-well cell culture plates at 3 × 10⁴ cells per well and cultured for 24 h at 37 °C, 5% CO₂. After gently washing twice with phosphate-buffered saline containing 0.05% Tween-20 (PBST), cells were fixed with 4% paraformaldehyde for 15 min, and then blocked for 2 h with 5% non-fat dry milk in PBS at room temperature. Hundred microliter of 0.1 mg/ml RSV F protein antibody was added to each well, incubated for 1 h at room temperature, followed by a 2,000-fold diluted anti-mouse Alexa Fluor 488 secondary antibody (Thermo Fisher Scientific, Cat. #A32723) for 1 h. Nuclei were stained with a 2,000-fold dilution of DAPI for 5 min. Cells were washed three times with PBST after each step.

HEp-2 cells were infected with RSV A2 or A2-mKate at an MOI of 2, incubated at 37 °C, 5% CO₂ for 14 h. Then, the cells were trypsinized and reseeded into a 96-well plate at 3.5 × 10⁴ cells per well, followed by an additional 8 h of incubation. The cells were gently washed twice with PBST and centrifuged to remove residual liquid. Subsequently, cells were fixed with various concentrations of formaldehyde or methanol solution at room temperature for 30 min. Following extensive washing, plates were blocked with 200 μl of blocking buffer per well for 2 h at 37 °C. Serially diluted antibodies or heat-inactivated serum were added and incubated for 1 h at 37 °C. After washing five times with PBST, plates were incubated for 1 h with HRP-conjugated goat anti-human/mouse IgG Fc secondary antibody (Abcam https://www.abcam.com/en-us, Cat. # ab97225/ab97265) at 1:5,000 dilution. Finally, the plates were developed by addition of the HRP substrate, TMB for 15 min at 37 °C, and the reaction was stopped with sulfuric acid before measuring absorbance at 450 nm/630 nm [OD_{(450nm/630nm)}]. Binding titers were calculated as the antibody concentration or the serum dilution that resulted in a 50% reduction in absorbance by three-parameter logistic regression in GraphPad Prism (version 10.4.1, GraphPad Software https://www.graphpad.com) 10, expressed as EC₅₀ titers or ED₅₀ titers.

Heat-inactivated serum was diluted 1:10 (antibody at 10 μg per well) in cell culture medium and serially diluted in a fourfold series (75 μl final volume). An equal volume of RSV A2 or A2-mKate (1 × 10⁵ PFU/well) was added to each well and incubated at 37 °C, 5% CO₂ for 1 h. HEp-2 cells were then trypsinized, counted, and seeded in a 96-well plate at 3 × 10⁴ cells, 100 μl per well. An equal volume of serum/antibody-virus mixture was added to the cells for incubation at 37 °C, 5% CO₂ for 24–36 h. Cells were fixed with 4% formaldehyde at room temperature for 30 min. After extensive washing, the cell-plates were blocked for 2 h and then incubated with MPE8 (0.5 ng/μl, 100 μl per well) for another 1 h at 37 °C. HRP-conjugated secondary antibody (Abcam, Cat. # ab97225) was then added for a 1 h at 37 °C incubation, respectively. Plates were read for absorbance at 450 nm/630 nm [OD_{(450nm/630nm)}]. Neutralizing titers were calculated as the antibody concentration or the serum dilution that caused a 50% reduction in absorbance by three-parameter logistic regression in GraphPad Prism 10, expressed as IC₅₀ titers or ID₅₀ titers.

Female Balb/C mice (6–8 weeks old) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd., China. All animals were housed in specific pathogen-free animal facilities on a 12 h light/dark cycle and the temperature was kept at a range of 20–24 °C. The mice (n = 5 per group) were intramuscularly primed with 5 μg of F protein with Alum (InvivoGen https://www.invivogen.com, Cat. # vac-alu-50) on day 0, boosted on day 14, and serum was collected on day 21 for neutralization and binding titers assessment against F protein. The animal study protocol was approved by the Laboratory Animal Management Ethics Committee of Xiamen University (approval no. XMULAC20230322).

Serum samples from healthy adult donors used for RSV neutralization assays were obtained with written informed consent, and the study protocol was approved by the Institutional Ethics Committee of the School of Public Health, Xiamen University (approval no. SPHIRB-202101).

Lines present in the graphs represent the mean as indicated. Binding and neutralizing titers were compared using unpaired Student's t test performed using the GraphPad Prism (version 10.4.1). Two-tailed P values of < 0.05 are considered statistically significant.

To establish a cell-based ELISA platform for evaluating the serum binding and neutralizing titers of RSV vaccine candidates targeting the F protein, we first tested whether RSV-infected HEp-2 cells express the F protein on their membranes. Using several reported epitope-specific antibodies (D25, 5C4, hRSV90, pre-F-specific antibodies; 12A12, a post-F-specific antibody; and 1129, MPE8, 101F, antibodies binding both pre-F and post-F) against F protein, flow cytometry analysis revealed that HEp-2 cells infected with RSV A2-mKate (an RSV strain carrying a fluorescent protein gene) were efficiently bound by nearly all antibodies tested, with the notable exception of 12A12. These results confirm the successful expression of F protein on the surface of infected cells and further suggest that the F protein is predominantly presented in the pre-fusion (pre-F) conformation (Figures 1A, B).

Furthermore, we selected three F-specific monoclonal antibodies (5C4, 12A12, and 1129) to investigate the conformational states of F protein on the cell membrane after plating and fixation of infected cells with 4% paraformaldehyde in 96-well plates. Notably, we found that F protein on the membrane exhibited distinct conformational states, with both pre-F and post-F conformations being displayed (Figure 1C).

To evaluate the specificity of post-vaccination sera for binding pre-F or post-F proteins, we explored various cell fixation methods to stabilize F protein on cell membranes in a predominantly single conformational state. First, we determined the optimal viral dose required to achieve near-saturating infection of cells. Using RSV A2-mKate to enable visualization of infection, we observed that an MOI of 2 produced fluorescence intensity that approached saturation relative to higher inocula (Figures 2A, B). Flow-cytometric analysis similarly indicated that infection reached a plateau at MOI = 2, although the mKate-positive fraction remained ~50% (Figure 2C).

Subsequently, we fixed infected cells with different concentrations of formaldehyde or methanol and assessed F protein conformations using 5C4 (pre-F specific) and 12A12 (post-F specific) antibodies. Low formaldehyde concentrations (0.25% and 0.5%) appeared to restrict detectable surface F largely to the pre-F conformation; however, 5C4 signals were comparatively weak under these conditions. Compared with 2% formaldehyde, fixation with 4 or 8% formaldehyde further favored the pre-F conformation, yielding stronger 5C4 reactivity while maintaining minimal 12A12 binding. Although these differences did not reach statistical significance (Figure 2D). In contrast, fixation with 60% methanol shifted surface F predominantly to the post-F conformation, abolishing 5C4 binding and producing robust 12A12 reactivity. Although 40% methanol also increased 12A12 binding, residual 5C4 binding remained detectable (Figure 2E). These results demonstrate that fixation conditions can be tuned to modulate the conformational presentation of RSV F protein on infected cells. Accordingly, we selected 4% formaldehyde and 60% methanol for subsequent experiments to preferentially present pre-F and post-F on RSV-infected cell membranes.

RSV F protein contains six antigenic epitopes, including five neutralizing epitopes: site Ø and V (pre-F-specific), and sites II, III, IV (shared between pre-F and post-F). To evaluate epitope preservation post-fixation, we tested the reactivity of neutralizing epitope-specific mAbs (D25 for site Ø, 1129 for site II, MPE8 for site III, 101F for site IV, hRSV90 for site V). To assess the generalizability of the fixation conditions, HEp-2 cells were infected with multiple RSV strains, including RSV A2-mKate and the clinical isolates 7445 and 7127. Under 4% formaldehyde fixation, all five antibodies showed strong reactivity, although 1129 exhibited slightly reduced binding; the post-F–specific antibody 12A12 also retained weak binding, except in cells infected with clinical isolate 7445 (Figure 3B). These results indicates that 4% formaldehyde preferentially preserves F protein in a pre-F–enriched state while retaining a minor post-F. In contrast, with 60% methanol fixation, binding by antibodies targeting sites 0 and V was largely abolished, whereas antibodies against sites II–IV, together with 12A12, retained robust reactivity (Figures 3A–C), confirming that methanol fixation locks F protein in post-F conformation. Collectively, these data indicate that both fixation methods preserve critical neutralizing epitopes corresponding to their respective conformational states.

Neutralizing antibody titers are a key metric for evaluating RSV vaccine efficacy. To evaluate the cell-based ELISA platform as a potential neutralization assay, fourfold serial dilutions of monoclonal antibodies 5C4 and 12A12 were preincubated with RSV A2 mKate and then applied to HEp-2 cells. Neutralization potency was determined by three independent assays. In the classical plaque-reduction neutralization assay, 5C4 showed an IC₅₀ of 12.46 ng/ml against A2 mKate, while 12A12 exhibited no detectable neutralizing activity (Figure 4A). In the fluorescence-based neutralization assay (Sun et al., 2018), 5C4 effectively neutralized A2-mKate with an IC₅₀ of 14.7 ng/ml, whereas 12A12 showed no activity (Figure 4B). For the cell-based ELISA method, infected cells fixed with 4% formaldehyde or 60% methanol were probed with 0.5 ng/μl MPE8 (a pre/post-F cross-reactive antibody), followed by an HRP-conjugated secondary antibody. Both fixation methods allowed determination of neutralizing titers of 5C4 and 12A12, but cells fixed with 4% formaldehyde produced substantially stronger signals and a 5C4 IC₅₀ value closer to those obtained with the other two assays (4% formaldehyde: IC₅₀ = 13.56 ng/ml; 60% methanol: IC₅₀ = 18.35 ng/ml), indicating higher sensitivity with 4% formaldehyde fixation (Figures 4C, D). Using the 4% formaldehyde cell-based ELISA, we were also able to effectively measure neutralizing titers of 5C4 and 12A12 against RSV clinical isolates (Figures 4E, F). These results indicate that the cell-based ELISA is a viable method for assessing RSV neutralizing activity.

To further assess the ability of the cell-based ELISA platform to reflect vaccine serum neutralization potency against RSV wild strains, Balb/c mice were immunized with 5 μg of RSV pre-F protein (DS-Cav1) or post-F protein using a two-dose regimen. Serum was collected 1 week post-boost. Treated cells infected with RSV A2 (4% formaldehyde for pre-F stabilization, 60% methanol for post-F stabilization) were used to measure binding specificity with serially diluted heat-inactivated sera. DS-Cav1-immunized sera showed significantly reduced binding to 60% methanol-fixed cells (post-F conformation) compared with 4% formaldehyde-fixed cells, indicating that the induced antibodies preferentially recognize pre-F-specific epitopes. In contrast, the post-F immunized sera showed comparable binding to both conformations (Figure 5A). Importantly, Neutralization assays against RSV A2 using the cell-based ELISA platform revealed superior activity in DS-Cav1 sera compared to post-F sera, with a difference of 100 times (Figure 5B). These data indicate that DS-Cav1 immunization induces potently neutralizing antibodies, whereas post-F immunization elicits antibodies that bind strongly but display limited neutralizing activity.

To further validate the cell-based ELISA platform for quantifying serum antibody binding titers and neutralization, we tested sera from healthy adult donors. Binding titers to HEp-2 cells fixed with either 4% formaldehyde or 60% methanol were comparable across the cohort, indicating that adult sera contain antibodies capable of recognizing both pre-F and post-F conformations (Figure 5C). We next compared the neutralizing titers against RSV A2-mKate measured by the fluorescence-based assay and the cell-ELISA platform. Although the cell-ELISA yielded modestly lower neutralizing titers than the fluorescence assay, it reliably discriminated differences in neutralizing capacity among individual sera (Figure 5D). These results collectively validate the platform's capacity to simultaneously evaluate conformational binding specificity and functional neutralization of RSV vaccine candidates.