Replacing water-repelling parts improves stability and immune response of respiratory syncytial virus fusion protein

To construct prefusion-stabilized RSV F with high expression levels in cultured cells, full-length RSV F variants with various amino acid mutations were cloned into the pcDNA3.1 vector and expressed in HEK293T cells (Table S1). As expected, RSV F variants with the 10-amino acid (FLGFLLGVGS) deletion in FP maintained a postfusion conformation (Fig. 1A and B). No detectable prefusion F was observed in F_ΔFP-transfected cells when stained with D25, a monoclonal antibody specific for prefusion RSV F protein. Compared to the wild-type (WT) F, the DS mutations (S155C, S290C) increased the conformational stability of prefusion F but resulted in decreased protein expression levels (Fig. 1A through E). At 37°C, RSV F constructs with cavity-filling (Cav1) mutations (S190F, V207L) exhibited a 2.6, 1.5, and 1.3-fold higher ratio of prefusion F (D25/motavizumab) on the cell surface than WT F, DS and DS-Cav1 variants, respectively (Fig. 1C through E). Moreover, the total F protein expression level of the Cav1 variant, as stained by motavizumab, is higher than that of WT F, DS, and DS-Cav1 variants. These results suggest that Cav1 mutations are important for stabilizing the prefusion conformation of DS-Cav1 and enhancing its protein expression level in cells.

Subsequently, we observed that SC mutation, which introduced a GS linker between F1 and F2 subunits, did not enhance the protein expression level or improve its stability on the cell membrane (Fig. 1C through E). Combinations of cavity-filling mutation with DM (N67I, S215P) or TM (N67I, S215P, E487Q) resulted in two prefusion-stabilized full-length RSV F variants, pre-F-IFLP_ct and pre-F-IFLPQ_ct. At both 37°C and 55°C, the pre-F-IFLP_ct variant, which includes four hydrophobic residue mutations (N67I, S190F, V207L, S215P), substantially increased RSV F expression levels and maintained a more stable prefusion F conformation compared to the other F variants tested in this study (Fig. 1C through E). Compared with wild-type RSV F, all the mutated F variants produced fewer and smaller syncytia in cells. Notably, the pre-F-IFLP_ct and pre-F-IFLPQ_ct variants exhibited lower membrane fusion activity than the other tested RSV F variants (Fig. 1F).

To generate soluble prefusion-stabilized RSV F proteins, the ectodomains of RSV F were engineered with various mutations and fused with T4 fibritin trimerization motif and His tag at C-terminus, then expressed in HEK293T cells. All soluble RSV F constructs were subsequently prepared for physical stability evaluation. Hardly any prefusion F was detected in the ΔFP solution (Fig. 2A and B), consistent with previous reports that soluble RSV F proteins lacking the FP adopted a postfusion conformation (20). At 37°C or 55°C, the prefusion F fraction of pre-F-IFLP was higher than that of Fwt, DM, TM, and DS-Cav1 (Fig. 2A and B). After incubation at pH 3.5 or 10, the prefusion F of DM, TM, Cav1, and DS-Cav1 was largely rearranged to a postfusion conformation (Fig. 2C and D). In contrast, both pre-F-IFLP and pre-F-IFLPQ remained stable at these pH levels. Additionally, most pre-F-IFLP retained in its prefusion conformation after 10 days of storage at 25°C (Fig. 2E through G). Although some prefusion F structures in the pre-F-IFLP solution transitioned to the postfusion conformation after 30 days of storage at 25°C, pre-F-IFLP maintained a higher prefusion fraction compared to other tested F variants (Fig. 2H). In contrast, nearly all F_wt changed to postfusion structure after 10 days of storage at 25°C (Fig. 2C and D). Overall, these results suggest that the four hydrophobic residue mutations in pre-F-IFLP substantially enhance its stability in the prefusion conformation.

Purified pre-F-IFLP and DS-Cav1 maintain a trimeric conformation, as demonstrated by native-PAGE and native-western blot (WB) analysis (Fig. S1A). Next, the trimeric RSV F was collected via size exclusion chromatography (Fig. S1B). Although some pre-IFLP and DS-Cav1 trimers were dissociated into monomers after SDS-PAGE and SDS-WB under non-reduced conditions, the trimeric forms of pre-F-IFLP and DS-Cav1 still displayed a higher band intensity compared to their monomers (Fig. S1C). No monomers and trimers were detected in pre-F-IFLP and DS-Cav1 after denaturing SDS-WB with dithiothreitol, suggesting that both disulfide bonds in pre-F-IFLP and DS-Cav1 were completely reduced (Fig. S1C). Surface plasmon resonance (SPR) assays were subsequently conducted to detect the binding affinity of pre-F-IFLP and DS-Cav1 to neutralizing antibodies. Pre-F-IFLP showed a strong binding affinity to the D25 antibody. The dissociation equilibrium constant (KD) for D25 binding to pre-F-IFLP and DS-Cav1 was <0.024 and 0.282 nM, respectively (Fig. 3A and B), suggesting a stronger affinity of D25 with pre-F-IFLP than with DS-Cav1. In addition, the KD values for pre-IFLP binding to AM14 or motavizumab were at the nanomolar levels comparable to those observed for DS-Cav1 with the two neutralizing antibodies (Fig. 3C through F). Taken together, these results suggest that the purified pre-F-IFLP maintains a trimeric prefusion conformation with highly potent antigenic epitopes recognized by prefusion F-specific neutralizing antibodies.

To evaluate the in vivo immunogenicity of pre-F-IFLP and its protective efficacy to prevent RSV infection, BALB/c mice were vaccinated with the pre-F-IFLP or DS-Cav1 formulated with AddaVax adjuvant. On days 14 and 28, the mice received first and second boost immunizations, respectively (Fig. 4A). Seven days after the prime immunization, immunoregulatory factors in the mouse serum were detected using enzyme-linked immunosorbent assay (ELISA). Mice immunized with pre-F-IFLP exhibited a slight increase in the secretion level of interferon γ (IFN-γ) (Fig. S2A), while other proinflammatory or immunoregulatory factors remained comparable to those in the AddaVax control group (Fig. S2B through D). Furthermore, flow cytometry analysis revealed that pre-F-IFLP immunization led to an increased proportion of CD4⁺CD44⁺IL4⁺cells in spleen lymphocytes without altering the proportion of CD8⁺CD44⁺IL4⁺cells (Fig. S2F and G). These results suggest that pre-F-IFLP presumably activates the Th1-associated immune responses.

Following two immunizations with either pre-F-IFLP or DS-Cav1, neutralizing antibodies were generated in the mouse serum and showed strong binding affinity to their respective immunogens. Sera from both pre-F-IFLP- and DS-Cav1-immunized mice significantly neutralized RSV infection. Notably, the neutralizing antibodies induced by pre-F-IFLP after the first boost immunization had a 26-fold higher potency in preventing RSV infection compared to those induced by DS-Cav1 (Fig. 4B). Compared with DS-Cav1 immunization, the neutralizing antibodies in the serum of pre-F-IFLP-immunized mice exhibited lower binding titers to soluble prefusion RSV F constructs (Fig. 4C). Furthermore, the ratio of the binding titer to the neutralizing titer in pre-F-IFLP-immunized mice was significantly lower than that observed in DS-Cav1-immunized mice (Fig. 4D). These results suggest that the neutralizing antibodies induced by pre-F-IFLP immunization are more effective in reducing RSV infection than those generated by DS-Cav1 immunization.

In comparison to the first boost immunization, the second boost immunization substantially enhanced the neutralizing capacity of mouse serum against RSV infection. Following two boost doses of pre-F-IFLP, the average serum neutralizing titers in vaccinated mice reached levels of 64,000, nearly 32-fold higher than those in mice that received only the first boost immunization (Fig. 5A and B). Furthermore, the serum binding titers to prefusion F immunogens in pre-F-IFLP-immunized mice markedly increased following the second boost immunization (Fig. 5B and C), indicating that the second boost immunization elicited a greater abundance of neutralizing antibodies. The serum neutralizing titers in pre-IFLP-immunized mice were 72-fold higher than those in mice immunized with DS-Cav1 (Fig. 5A). Importantly, neutralizing antibodies induced by pre-F-IFLP demonstrated a stronger efficacy in inhibiting RSV infection, as evidenced by significantly lower ratios of binding titer to neutralizing titers in sera from pre-F-IFLP-immunized mice compared to those from DS-Cav1-immunized mice (Fig. 5C).

During the initial stage of RSV infection, the viral F protein interacts with the cell membrane and then mediates the virus-cell membrane fusion. The soluble ectodomains of RSV F protein in a high-molarity buffer adopt a pretriggered conformation and are capable of interacting with the cell membrane (10, 21, 22). Upon the pretreatment with diluted serum from AddaVax-immunized mice, a large fraction of soluble F(ecto) adsorbed onto the cell membrane. However, in the presence of the diluted sera from pre-F-IFLP- or DS-Cav1-immunized mice, the amount of F(ecto) on the cell membrane was significantly decreased. Pre-F-IFLP vaccination induced more potent neutralizing antibodies than DS-Cav1 to block the interaction of F(ecto) with the cell membrane (Fig. 5D).

We next determined whether the cell-cell fusion and syncytia formation could be inhibited by the serum from vaccinated mice. In this assay, HEK293T cells were transfected with plasmids encoding full-length RSV F protein and treated with the diluted sera from pre-F-IFLP- or DS-Cav1-immunized mice. Treatment with serum from pre-F-IFLP-immunized mice resulted in a nearly complete inhibition of the formation of large multinucleated syncytia (Fig. 5E). In contrast, cells treated with serum from DS-Cav1-immunized mice displayed numerous multinucleated syncytia, with only a slight decrease in syncytia numbers. Collectively, these results suggest that pre-F-IFLP immunization elicited high titers of RSV-neutralizing antibodies, the neutralizing efficacy of which is more potent than the antibodies induced by DS-Cav1.

To assess the protective effects of prefusion F vaccination against RSV infection, mice were intratracheally inoculated with RSV A2 following the second boost immunization. Lung tissues of mice were collected 5 days postinfection. Compared to AddaVax-immunized mice, viral titers were reduced by 328-fold in DS-Cav1-immunized mice and by 2,068-fold in pre-F-IFLP-immunized mice (Fig. 6A). Severe inflammatory infiltrations, including bronchitis, alveolitis, alveolar damage, and crushed debris accumulation around bronchioles, were observed in RSV-infected mice (Fig. 6B). In pre-F-IFLP-immunized mice, these pathological changes and inflammatory responses, such as bronchitis, hemorrhage, and alveolitis, were apparently alleviated compared with those in AddaVax-immunized mice (Fig. 6C). Further evidence from immunostaining and RT-PCR assays revealed a marked reduction of viral antigens within the lung tissues of pre-F-IFLP-immunized mice (Fig. 6D), in which the transcript levels of viral NS1 and F genes were suppressed to nearly undetectable levels (Fig. 6E). Both DS-Cav1- and pre-F-IFLP-vaccinated mice exhibited a significant decrease in the serum levels of pro-inflammatory cytokine interleukin 6 (IL-6) (Fig. 6F). Moreover, the secretion level of IFN-γ, an antiviral and inflammation mediator, was significantly suppressed by pre-F-IFLP-immunization. In contrast, DS-Cav1 immunization only slightly reduced IFN-γ levels in mouse serum. Consistent with these findings, RT-PCR analysis further confirmed that pre-F-IFLP immunization significantly reduced the transcription levels of proinflammatory cytokines (IFN-γ, TNF-α, IL-6) in the lung tissues compared to AddaVax immunization (Fig. S3).

The human epithelial type 2 cell line (HEp-2) and human embryonic kidney cell line (HEK293T) were purchased from the American Type Culture Collection (ATCC). HEp-2 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS). The RSV A2 strain (VR-1540; ATCC) was propagated in HEp-2 cells, and viral titers were detected using the TCID₅₀ assay.

HEK293T cells were seeded into 24-well plates 1 day before transfection. After incubation for 24 h, the cells were transfected with plasmids encoding various RSV F variants using Lipofectamine 2000 transfection reagent. At 5 h post-transfection, the Opti-MEM medium was replaced with fresh cell growth medium. At 48 h post-transfection, the cell culture supernatant was discarded, and the cells were washed twice with PBS. The RSV F proteins in the cells were then detected using humanized monoclonal antibody D25 (PABL-322; Creative Biolabs) or motavizumab (TAB-709; Creative Biolabs) for 2 h at room temperature (RT), followed by staining with Alexa Fluor 488-conjugated anti-human secondary antibody (A11013; Thermo Fisher Scientific) for 1 h at RT. After washing with PBS, the cells were visualized and photographed under a fluorescent microscope.

The pcDNA3.1 plasmids encoding full-length wild-type RSV F or RSV F variants were transfected into HEK293T cells using the Lipofectamine 6000 Kit (C0526, Beyotime). At 48 h post-transfection, the cells were collected and heat-shocked at 55℃ for 10 min and then stained with D25 or motavizumab for 2 h at RT, followed by incubation with Alexa Fluor 488-conjugated goat anti-human secondary antibody for 1 h at RT. After washing three times with PBS, fluorescence intensity was measured using a BD Biosciences flow cytometer. The data were analyzed using FlowJo v.10.

A dual-luciferase assay was performed to quantify intercellular membrane fusion mediated by RSV F proteins. HEK293T cells were seeded into 24-well plates and incubated for 24 h. One group of cells was transiently co-transfected with plasmids expressing RSV F variants and pT7-luc, a plasmid containing the firefly luciferase under the control of T7 RNA polymerase. Another group of cells was transfected with pRL-TK, which encodes Renilla luciferase, and pCAG-T7 Pol, a plasmid expressing T7 RNA polymerase. The two groups were mixed in a 1:1 ratio 24 h post-transfection and then seeded into 96-well plates. In fused cells, the T7 RNA polymerase drives the expression of firefly luciferase, correlating with cell-cell fusion activity. At 72 h post-transfection, cellular luciferase activity was quantified using the Dual-Luciferase Reporter Assay System (Promega, WI, USA).

HEK293T cells were transiently transfected with pcDNA3.1 vectors encoding the ectodomains of RSV F (amino acids 1–513) and its variants with various mutations using Lipofectamine 2000 Transfection Reagent. At 5 days post-transfection, cell supernatants containing soluble RSV F constructs were collected and centrifuged at 2,000 rpm for 10 min to remove cell debris. The supernatants were passed over a Ni-NTA column and eluted with 300 mM imidazole in a buffer containing 50 mM Tris-HCl and 150 mM NaCl (pH 8.0). The eluted fractions were concentrated and further purified by size-exclusion chromatography using a Superdex 200 column (GE Healthcare). The purified proteins were analyzed by SDS-PAGE under reducing or non-reducing conditions, transferred to PVDF membranes, and stained with motavizumab. After incubation at 4°C for 12 h, the membranes were washed three times with PBST (0.05% Tween 20 in PBS) and then incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at RT. The reaction was completed with a chemiluminescent agent, and detection was performed using a chemiluminescence imager (Amersham Imager 600, GE Healthcare).

Soluble RSV F and F variants were blotted onto nitrocellulose membranes, dried under RT, and then blocked with a 5% w/v bovine serum albumin (BSA) solution in PBS for 2 h. To assess the physical stability of the RSV F constructs, the nitrocellulose membranes were subjected to different stress conditions, including low or high pH (3.5 or 10), elevated temperature (55°C), and extended storage periods (1–30 days) at RT. After washing thrice with PBS, the membranes were incubated with D25 or motavizumab, followed by staining with HRP-conjugated secondary antibodies. The membranes were then washed with PBS, incubated with ECL solution, and visualized using a chemiluminescence imager (Amersham Imager 600; GE Healthcare). The integrated density values of each blot were calculated using ImageJ software.

To assess the binding affinity of pre-F-IFLP and DS-Cav1 with RSV F-specific antibodies, surface plasmon resonance assays were conducted at 20°C using a Biacore S200 instrument. RSV F antibodies (D25, motavizumab, and AM14) were diluted in 10 mM sodium acetate buffer and immobilized onto the surface of CM5 sensor chips. A regeneration solution was injected to remove non-covalent bound antibodies from the chips. A blank control with no antibody on the sensor chip surface was used as a reference. Soluble purified RSV F proteins were prepared in serial dilutions with PBS and then flowed over the different sensor channels at a flow rate of 30 µL/min. Binding responses between the antibodies and analyte proteins were recorded by the SPR system, and data were processed using Biacore S200 Evaluation Software.

All animal experiments were conducted under the ethical approval of the Institutional Animal Care and Use Committee of Jinan University and in compliance with relevant ethical regulations. Six-week-old female BALB/c mice were purchased from Sipeifu, Biotechnology Co., Ltd. (Beijing, China). The mice were randomly divided into different experimental groups, with 24 mice per group. Before immunization, immunogens were gently mixed at a 1:1 vol/vol ratio with AddaVax adjuvant (vac-adx-10; InvivoGen) to reach a final antigen concentration of 0.05 mg/mL. On days 0, 14, and 28, all mice received intramuscular injections of 50 µL AddaVax or immunogens in AddaVax into the gastrocnemius muscle. Blood samples were collected via cardiac puncture on days 7, 24, and 34, and the sera were prepared for the detection of neutralizing and antibody binding titers and inflammatory or immunoregulatory cytokines. The lymphocyte immunophenotype in the spleens of the mice was determined using flow cytometry. On day 27 after the second boost immunization, five mice in each group were intratracheally infected with 10⁵ plaque-forming units (PFU) of RSV in PBS or an equal volume of PBS. The mice were euthanized on day 5 postinfection. Lung tissues and sera of mice were collected and prepared for evaluation of vaccine efficacy.

The neutralizing effect of mouse sera on RSV infection was assessed using a TCID₅₀ assay. Serial dilutions of sera from immunized mice were pre-incubated with virus suspensions for 30 min at 37°C, then added to HEp-2 cells in 96-well plates. Control cells were inoculated with the virus without pre-treatment of sera. At 48 h.p.i., virus suspensions from each well were collected to determine RSV titer using the TCID₅₀ assay.

To determine the binding titers of neutralizing antibodies in the mouse serum to DS-Cav1 or pre-F-IFLP, Maxisorp (Nunc) ELISA plates were coated overnight at 4°C with 0.1 µg/well of antigens (DS-Cav1 or pre-F-IFLP). The plates were then blocked with 4% BSA in PBS at RT for 1 h. Serial dilutions of mouse serum were added to each well and incubated for 2 h at RT. After washing with PBS, an anti-mouse IgG antibody conjugated with alkaline phosphatase (Jackson Immunoresearch) for mouse sera was added. The plates were washed three times and reacted with p-nitrophenyl phosphate substrate for 20 min at RT. The absorbance at 405 nm was measured using a microplate reader. To determine the levels of inflammatory cytokines, mouse serum was collected on day 7 after the prime immunization and 5 days post-RSV infection. Sera and standards were serially diluted and added to ELISA plate wells, followed by incubation for 2 h at RT. The plates were washed and then incubated with the biotin-conjugated antibodies specific for IL-6, IFN-γ, TNF-α, IL-2, or IL-4. After incubation for 1 h at RT, streptavidin-HRP solution was added into each well and incubated for 30 min. The cells were then washed three times with PBS and incubated with the TMB substrate solution. The reaction was stopped after 5–30 min by adding an equal amount of stop solution. Absorbance at 450 nm was detected using a microplate reader.

Spleen lymphocytes were collected from mice at 7 days after prime immunization and cultured in RPMI 1640 medium supplemented with 10% (v/v) FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin for 72 h. The cells were activated by adding DS-Cav1 or pre-F-IFLP at a concentration of 1 µg/mL. Subsequently, the activated splenocytes were treated with Brefeldin A (BD Biosciences) to enhance the intracellular cytokine staining signals. After washing with PBS, the cells were stained with anti-CD8, anti-CD4, and anti-CD44 antibodies (all from BioLegend) for 20 min. The cells were then fixed and permeabilized to allow for intracellular staining with anti-IFN-γ and anti-IL-4 antibodies (all from BioLegend). Flow cytometry data were acquired using a NovoCyte flow cytometer (ACEA Biosciences) and analyzed using FlowJo V10 software.

The lungs of mice were harvested at 5 d.p.i., fixed in 4% paraformaldehyde solution, and subsequently embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). The severity of alveolitis, bronchitis, hemorrhage, and alveolar damage was arbitrarily assessed using a four-tiered scoring system: 0 (negative), 0–1 (mild), 1–2 (moderate), 2–3 (moderately severe), and 3–4 (severe).

Data are presented as mean ± standard deviation (SD) or standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism, version 9.4.1 (GraphPad Software, Inc., La Jolla, CA, USA). Statistical differences between groups were determined using a two-tailed student’s t-test, with a P-value < 0.05 considered statistically significant.

All data needed to evaluate the conclusions in the paper are present in the paper and the supplemental material.