2.1 IM Immunization With MF59/preF Vaccine Induces Systemic Humoral and B-Cell Immune Responses

A robust antibody response has always been a hallmark of an ideal vaccine [46]. Therefore, we first evaluated the antibody response of the MF59/preF vaccine following IM immunization. BALB/c mice in the low- and high-dose groups received IM vaccinations with 5/10 µg of the adjuvanted RSV preF protein vaccine, respectively. The control group was given PBS, MF59, or 10 µg of RSV preF protein alone. The immunization schedule was designed following a prime-boost regimen, wherein the intervals between administrations were set at 21 days (Figure 1A). An initial injection was carried out on Day 0, and two booster inoculations were subsequently performed on Days 21 and 42, respectively (Figure 1A). Serum was obtained 14 days post each immunization (Days 14, 35, and 56), while BALF was obtained on Day 72 to assess preF- and postF-specific IgG and IgA. ELISA data revealed that IM immunization with preF protein alone induced low preF- and postF-specific IgG levels in Day 56 serum, with IgG titers reaching 10⁴ for postF and 10⁵ for preF, whereas the MF59-like adjuvanted vaccine significantly augmented the production of IgG antibodies, with titers > 10⁶ (Figure 1B). Furthermore, serum anti-preF and anti-postF IgG levels showed no significant differences between the low- and high-dose MF59/preF groups (Figure 1B).

Neutralizing antibodies can effectively neutralize pathogens, diminish pathogen titers, and safeguard tissues or cells from being infected [47]. Subsequently, we conducted live virus neutralization assays on Day 56 serum to evaluate neutralizing capacities triggered by the MF59/preF vaccine. To be more specific, the high-dose group exhibited GMTs of 49,315 and 20,482 against RSV A2 and RSV B, respectively, while the low-dose group showed GMTs of 19,000 and 11,394; these differences were not significant (Figure 1C). Compared to preF protein alone, the GMTs were elevated by 32.0- and 20.5-fold in the high-dose group and by 12.3- and 11.4-fold in the low-dose group for RSV A2 and RSV B, respectively (Figure 1C). These findings demonstrate that the MF59 adjuvant is essential and that 5 µg of preF protein suffices to generate a robust serum humoral response.

Moreover, local antibody responses, including antigen-specific IgG and IgA, were assessed. The outcomes manifested that the intramuscularly immunized MF59/preF vaccine was only capable of eliciting IgG antibodies instead of IgA in BALF (Figure 1D), which implied that this vaccine failed to stimulate a localized immune response after IM administration. The detectable IgG antibodies presumably reached the bronchi through the bloodstream.

Antibodies produced by effector B cells upon vaccination are essential for providing protection [48]. These antibodies can neutralize pathogens, enabling other immune cells to recognize and eliminate them. Thus, we verified the activation of the systemic antigen-specific B cell response following vaccination. Four weeks after the last vaccination, bone marrow, spleen, and lung samples were subjected to ELISpot analysis. The results showed that irrespective of dose, IM administration of MF59/preF induced abundant preF-specific IgG⁺ antibody-secreting cells (ASCs) in bone marrow (Figure 1E) and spleen (Figure 1F) but relatively few in the lung (Figure 1G), whereas preF alone generated fewer ASCs in all tissues. Noticeably, IM immunization failed to trigger the generation of preF-specific IgA⁺ ASCs within any of these tissues (Figure S1A–C). This finding further corroborated that the MF59/preF vaccine, when administered through IM immunization, drives systemic IgG⁺ B cell immunity but is ineffective in stimulating IgA⁺ B cells to synthesize mucosal antibodies.

2.2 IM Immunization With MF59/preF Vaccine Induces Systemic T-Cell Immune Responses

Vaccination-induced T-cell immunity is crucial for combating infections [49]. Effective responses from both CD4⁺ and CD8⁺ T-cells are essential in preventing pathogen invasions [50]. Consequently, we further investigated T cell activation following IM vaccination. Four weeks after the last vaccination, splenic lymphocytes were isolated and stimulated in vitro with RSV preF protein. Flow cytometry (FCM) analysis revealed that, compared to the preF protein alone, the MF59-adjuvanted RSV preF formulation markedly increased the proportions of antigen-experienced (CD44⁺) and activated (CD69⁺) CD4⁺ and CD8⁺ T cells in the spleen, whereas the naked preF induced only a slight rise in CD4⁺ CD69⁺ T cell frequency (Figure 2A). Subsequently, ELISpot further demonstrated that the MF59/preF vaccine induced significantly more IFN-γ-producing cells than the naked preF protein (Figure 2B). Moreover, splenic lymphocyte culture supernatants showed significantly elevated IFN-γ levels in the low/high-dose group (Figure 2C). Intracellular cytokine staining (ICS) was utilized to further characterize the cell types responsible for IFN-γ production. We observed increased frequencies of IFN-γ-producing CD8⁺ (Figure 2D) and CD4⁺ (Figure 2E) T cells within the spleens of mice administered MF59/preF intramuscularly, whereas preF alone showed no such increase in cytokine-secreting cells (Figure 2B,D,E) or cytokines (Figure 2C). These findings demonstrate that IM delivery of the MF59/preF vaccine induces robust systemic antigen-specific T cell activation, unlike the unadjuvanted preF protein.

Germinal centers (GC) are sites of antibody diversification and maturation, generating long-lasting plasma cells and memory B cells that provide immunity against subsequent infections [51]. Follicular helper T (Tfh) cells are pivotal in initiating GC development and producing antibodies with high affinity and memory B cells [52]. Subsequently, we evaluated the Tfh (CD4⁺CXCR5⁺PD-1⁺) and preF-specific GC B (B220⁺GL7⁺CD95⁺) cell responses in ILN. As expected, high-dose MF59/preF vaccination generated markedly higher levels of both Tfh (Figure 2F) and antigen-specific GC B cells (Figure 2G) in ILN compared to the preF protein alone group. This augmentation is crucial for the induction of a powerful and enduring humoral immunity against RSV [53]. These findings demonstrated that IM administration of the MF59/preF vaccine can confer an excellent systemic cellular immune reaction.

2.3 IN Immunization With MF59/preF Vaccine Induces Local Humoral and B-Cell Immune Responses

The MF59 adjuvant, commonly administered intramuscularly, boosts immunity in influenza vaccines [54, 55]. Recent studies indicate that IN administration of MF59 also elicits robust local IgA responses, making it a promising strategy against respiratory viruses by promoting IgA and T_RM cells critical for mucosal immunity [56-58]. Accordingly, we evaluated the immune response elicited by MF59/preF, delivered intranasally, using the same schedule as in the IM study (Figure 3A). We first examined serum IgG production on Days 14, 35, and 56. As depicted in Figure 3B, a single IN immunization with the high-dose vaccine elicited an elevation in antigen-specific IgG within the serum (especially preF-specific), whereas preF protein alone had little effect (Figure 3B). On the 56th day, the serum anti-preF and anti-postF IgG titers in the high-dose group exceeded 10⁵, being > 4-fold and > 10-fold greater than those in the low-dose and naked protein groups, respectively (Figure 3B). Significant differences were observed between the high-dose group and the remaining two groups (Figure 3B). Additionally, naked preF protein booster immunization elicited modest serum antigen-specific IgG responses (Figure 3B). Notably, IN delivery of the MF59/preF vaccine-induced antigen-specific IgG titers approximately 10-fold lower than IM immunization (Figure 1B).

Subsequently, we performed the live virus neutralization assays using the serum on Day 56 to evaluate the neutralizing capacities elicited by IN immunization with the MF59/preF vaccine. IN administration of 10 µg RSV preF protein alone merely elicited a negligible neutralization response, with GMTs of 52 and 48 against RSV A2 and RSV B, respectively (Figure 3C). Conversely, the high-dose MF59/preF vaccine generated GMTs that were 101.3- and 53.4-fold higher than the naked preF group against RSV A2 and RSV B, respectively, and 5.6- and 3.4-fold higher than the low-dose group (Figure 3C). In addition, unlike IM injection (Figure 1B,C), only the high-dose MF59/preF vaccine administered intranasally can induce a sufficiently strong serum humoral immune response. Consistent with the IgG antibody, the neutralizing antibodies induced by IN inoculation of the same dose of the MF59/preF vaccine were 10-fold lower than those by IM inoculation (Figure 1C).

Mucosal antibodies, especially IgA, are vital for frontline defense against localized viral infections [59]. Thus, we further assessed antigen-specific IgG and IgA in BALF from mice immunized intranasally. In line with serum antibody data, high-dose MF59/preF vaccination elicited elevated IgG and IgA titers in BALF (Figure 3D), demonstrating that IN administration of MF59/preF induces a stronger local antibody response (particularly IgA) compared to IM injection.

To evaluate how IN vaccination influences B cell activation in immunized mice, we employed the ELISpot assay to examine the generation of antigen-specific IgG⁺ and IgA⁺ ASCs in bone marrow, spleen, and the lung. As shown in Figure 3E–J, IN administration of naked protein and low-dose vaccine induced negligible IgG⁺ and IgA⁺ ASCs. High-dose MF59/preF with IN immunization triggered substantial preF-specific IgG⁺ ASCs in the lung (Figure 3G) and bone marrow (Figure 3E), with lower levels in the spleen (Figure 3F). Compared to IM immunization (Figure S1A–C), the high-dose group via intranasal administration elicited preF-specific IgA⁺ ASCs in bone marrow (Figure 3H), spleen (Figure 3I), and lung (Figure 3J), peaking in the lung (Figure 3J), suggesting that the MF59/preF vaccine administered intranasally provoked local and systemic IgA⁺ ASCs, whereas the quantity of IgG⁺/IgA⁺ ASCs in the spleen was relatively weaker.

2.4 IN Immunization With MF59/preF Vaccine Induces Local T-Cell Immune Responses

Apart from IgA antibodies, T_RM cells constitute a crucial element of mucosal immunity, furnishing a prompt response to impede viral infection [60, 61]. Therefore, we assessed T_RM cell frequencies in BALF and lung tissues of mice receiving IN vaccination. As expected, IN immunization with the high dose of MF59/preF vaccine notably elevated the frequencies of antigen-experienced CD4⁺/CD8⁺ T_RM (characterized by CD44⁺CD69⁺CD103⁺) cells in the lung (Figure 4A,B) and elevated their numbers in BALF (Figure 4C,D). This presented a striking contrast to the naked preF protein and the low-dose vaccine, neither of which induced the generation of CD4/CD8 T_RMs.

In the ensuing experiments, we delved deeper into the role of IN immunization with the MF59/preF vaccine in activating T cell responses. Four weeks after the last vaccination, we harvested lung tissues, prepared lung single-cell suspensions, and then conducted in vitro stimulation using RSV preF protein. FCM analysis indicated that IN administration with the high-dose of MF59/preF vaccine markedly elevated the percentage of CD4⁺CD69⁺ and CD8⁺CD69⁺ T cells in the lung compared to the naked preF protein and the low-dose groups (Figure 4E). Furthermore, the high-dose group showed a significant rise in CD4⁺CD44⁺ T cell proportions relative to the naked protein group, while neither dose affected CD8⁺CD44⁺ T cell levels (Figure S2A). Consistently, the low-dose vaccine was insufficient to enhance CD4/CD8 expression in the lung (Figure 4E). ELISpot analysis revealed elevated numbers of IFN-γ-producing cells in both low- and high-dose MF59/preF groups compared to the naked preF group, with a significant difference observed between the two vaccine doses (Figure 4F). Subsequently, ELISA analysis then detected IFN-γ in lung cell culture supernatants, finding it present solely in the high-dose group (Figure 4G). Additionally, ICS analysis revealed comparable outcomes, with the high-dose group showing enhanced levels of IFN-γ⁺ CD8⁺ T cells (Figure 4H) but not IFN-γ⁺ CD4⁺ T cells (Figure S2B). Overall, the data suggest that the low-dose vaccine elicits only limited IFN-γ production, whereas robust antigen-specific T cell induction and activation in the lung occur exclusively with the high-dose vaccination.

Then, we evaluated the Tfh and preF-specific GC B cell responses in mLN from intranasally vaccinated mice. As anticipated, IN administration of the high-dose MF59/preF vaccine markedly elevated Tfh cell (Figure 4I) and preF-specific GC B cell (Figure 4J) frequencies in mLN, showing significant differences versus both the naked preF and low-dose groups. These results support the high dose as the preferred option for IN vaccination of the MF59/preF vaccine.

2.5 Combination of IM and IN Immunization Using MF59/preF Vaccine Elicits Both Local and Systemic Humoral and B-Cell Immune Responses

The above results indicate that IM vaccination alone does not elicit mucosal IgA, while IN vaccination alone yields markedly lower systemic IgG than the IM route. Therefore, to overcome the limitations of single-route administration, we implemented a combined IM and IN immunization strategy to enhance the comprehensiveness of the immune response generated by the MF59/preF vaccine. Mice were immunized with a high-dose prime-boost strategy at 21-day intervals (Figure 5A). Mice in the IM-IM-IN group were given the initial two doses intramuscularly, followed by an IN booster. In the IM-IN-IN group, the primary immunization was IM, and the two booster immunizations were IN (Figure 5B). ELISA results showed that both the IM-IM-IN and IM-IN-IN regimens elicited robust preF- and postF-specific IgG titers in mouse serum, demonstrating no significant differences between them (Figure 5C).

Live virus neutralization assays were assessed on Day 56 serum to evaluate the neutralizing capacities induced by the MF59/preF vaccine administered via IM-IM-IN and IM-IN-IN regimens. The IM-IN-IN group exhibited GMTs of 19,377 and 11,316 against RSV A2 and RSV B, respectively, representing 2.1- and 2.6-fold increases over the IM-IM-IN group (Figure 5D), demonstrating superior neutralizing capacity with the IM-IN-IN schedule.

Then, we further assessed antigen-specific IgG and IgA in BALF following IM-IM-IN and IM-IN-IN vaccination. Both regimens elevated IgG titers in BALF (Figure 5E), whereas IM-IN-IN induced significantly greater IgA levels, indicating that two IN doses are required to achieve robust local IgA responses.

To assess the impact of IM-IM-IN and IM-IN-IN immunization on local and systemic B cell activation, ELISpot assays measured antigen-specific IgG⁺ and IgA⁺ ASCs in bone marrow, spleen, and lung. The IM-IN-IN regimen significantly induced higher levels of IgG⁺ and IgA⁺ ASCs across all tissues (Figure 5F–K) compared to IM-IM-IN, demonstrating that two IN doses following one IM injection elicit stronger B cell responses.

2.6 Combination of IM and IN Immunization Using MF59/preF Vaccine Elicits both Local and Systemic T-Cell Immune Responses

To evaluate the impact of IM-IM-IN and IM-IN-IN vaccination schedules on local T cell responses in immunized mice. The IM-IN-IN regimen markedly elevated the levels of CD4⁺CD44⁺, CD8⁺CD44⁺, CD4⁺CD69⁺, and CD8⁺CD69⁺ T cells in the lung relative to the PBS control (Figure 6A). Conversely, the IM-IM-IN regimen elevated CD4⁺/CD8⁺CD69⁺ and CD4⁺CD44⁺ T cell levels in comparison to the PBS control without affecting CD8⁺CD44⁺ T cells (Figure 6A). Although the overall count of CD4⁺/CD8⁺ CD44⁺ T cells remained comparable, the relative frequency of these T cells in the IM-IN-IN regimen was significantly (at least with a tendency) higher than that in the IM-IM-IN regimen (Figure 6A). Then, ELISpot results showed more IFN-γ-producing cells in the IM-IN-IN group, with a statistically significant difference compared to the IM-IM-IN group (Figure 6B). The ICS assay showed that the IM-IN-IN regimen elicited a higher proportion of pulmonary IFN-γ⁺CD8⁺ T cells compared to the IM-IM-IN regimen (Figure 6C). Similar to the results obtained with three IN immunizations (Figure S2B), neither regimen effectively increased pulmonary IFN-γ⁺CD4⁺ T cell frequencies (Figure S3A).

Subsequently, we further ascertained the frequencies of T_RM cells in the lung following the IM-IM-IN and IM-IN-IN immunizations. As anticipated, the MF59/preF vaccine delivered by combined IM and IN routes significantly increased antigen-experienced CD4⁺/CD8⁺ T_RM cells in the lung, with the IM-IN-IN regimen inducing notably higher levels than IM-IM-IN (Figure 6D,E). These results show that the IM-IN-IN approach elicits a more robust local T-cell response.

In the following experiments, we examined how the IM-IM-IN and IM-IN-IN regimens affected splenic T-cell responses. Four weeks after the last vaccination, splenic lymphocytes were isolated and stimulated in vitro with RSV preF protein. Both regimens significantly increased the frequencies of CD4⁺CD44⁺, CD8⁺CD44⁺, CD4⁺CD69⁺, and CD8⁺CD69⁺ T cells in the spleen (Figure 6F). Additionally, inoculation with the MF59/preF vaccine via the IM-IM-IN and IM-IN-IN regimens both resulted in a higher abundance of IFN-γ-producing cells in the spleen (Figure 6G) and a greater percentage of splenic IFN-γ⁺CD8⁺ T cells (Figure 6H), while no increase in splenic IFN-γ⁺CD4⁺ T cells was detected (Figure S3B). No notable differences were found between the two regimens, indicating that both vaccination strategies effectively triggered strong splenic T-cell responses.

4.1 Animals

Specific pathogen-free (SPF) female BALB/c mice (6–8 weeks old; Beijing Vital River, China) were kept at 22°C–23°C with 45%–55% humidity under a 12 h light/dark cycle and allowed free access to food and water. After at least one week of acclimation, mice were randomly assigned to experimental groups. All procedures were approved by the Sichuan University Animal Care and Use Committee (Chengdu, China).

4.2 Vaccination of Mice

MF59-like adjuvant was formulated as previously described [80]. The BALB/c mice (6 per group) were intramuscularly or intranasally immunized with PBS, MF59-like adjuvant, preF alone (10 µg, Sino Biological, 11049-VNAS), MF59/preF-low (5 µg preF), or MF59/preF-high (10 µg preF) on Days 0, 21, and 42. Each mouse received 100 µL for IM immunization and 50 µL for IN immunization.

To delve into the synergistic immune impact of a combination of IM and IN vaccines, BALB/c mice were administered three high doses of MF59/preF (10 µg preF) on days 0, 21, and 42. The IM-IM-IN group received two IM injections followed by an IN boost, while the IM-IN-IN group was primed IM and boosted twice intranasally.

Serum was collected 14 days after each immunization. Four weeks after the last vaccination, BALF, lung, spleen, ILN, mLN, and bone marrow were collected for immune analyses.

4.3 Measurement of Antibody by ELISA

Briefly, 96-well ELISA plates (NUNC-MaxiSorp, Thermo Fisher Scientific) were coated overnight at 4°C with either 1 µg/mL of RSV preF or postF protein (Sino Biological, 11049-V49H5-B). Following this, the plates were washed three times with PBST (PBS containing 0.1% Tween-20) and then blocked with 1% bovine serum albumin (BSA) at 37°C for 1 h. Serum or BALF samples were serially diluted, added to the wells, and incubated at 37°C for 1 h. After washing, 100 µL/well of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Thermo Fisher Scientific, 31430) or IgA (Abcam, ab97235) was added and incubated for another hour at 37°C. Following five washes, 100 µL/well of 3,3′,5,5′-tetramethylbenzidine (TMB, Thermo Fisher Scientific, 34029) substrate was added and developed for 10 min in the dark. The reaction was stopped with TMB stop solution (Beyotime, P0215), and absorbance was measured at 450 nm with a 630 nm reference using a microplate reader (Spectramax ABS, Molecular Devices) with SoftMax Pro 7.1 software.

4.4 ELISpot Assay

The lungs from immunized mice were harvested, minced with ophthalmic scissors, and digested for 1 h at 37°C in RPMI 1640 medium containing 1% Type I (1 mg/mL) and 1% Type IV (0.5 mg/mL) collagenase (Gibco). The digested tissue was passed through 70 µm cell strainers (Corning, USA) and subjected to red blood cell lysis. After two washes with PBS, the resulting cell suspension was resuspended in RPMI 1640 complete medium containing 10% fetal bovine serum (FBS), 1 mM pyruvate, 100 µg/mL streptomycin, 100 U/mL penicillin (all from Gibco), 20 U/mL IL-2, and 50 µM β-mercaptoethanol (all from Sigma).

The spleens were homogenized with a syringe piston and passed through 70 µm cell strainers to obtain single-cell suspensions. Bone marrow cells were flushed from the tibiae and femora with RPMI 1640 medium. Both spleen and bone marrow suspensions were then isolated with a mouse lymphocyte separation medium (Dakewe, China), followed by red blood cell lysis treatment. After two washes with PBS, the isolated lymphocytes were finally resuspended in RPMI 1640 complete medium.

IgG⁺ and IgA⁺ ASCs in bone marrow, spleen, and lung were quantified by ELISpot. Sterile 96-well plates (Mabtech, Sweden) were first activated with a brief 1 min exposure to 35% ethanol, then washed with PBS. The plates were subsequently coated overnight at 4°C with 3 µg/mL of RSV preF protein. Following a wash step, the plates were blocked with RPMI 1640 complete medium and incubated for 2 h at 37°C. Single-cell suspensions from the spleen, bone marrow, and lung (3 × 10⁵ cells/well) were added and allowed to incubate overnight at 37°C. The next day, the plates were washed and then incubated with HRP-conjugated goat anti-mouse IgG (1:5000) or IgA (1:2000) for 2 h at room temperature. After another wash, the spots were developed using TMB substrate, visualized, and then rinsed to halt the reaction. Finally, the number of spots was recorded with the S6 Ultra M2 ELISpot reader (CTL, USA).

Mouse IFN-γ-spot-forming cells in the lung and spleen were detected following the manufacturer's protocol. Sterile 96-well ELISpot plates (Mabtech) were washed five times with PBS. Next, the plates were incubated with RPMI 1640 complete medium for 2 h at 37°C. Splenic lymphocytes and single-cell suspensions from the lungs (1×10⁵ cells/well) were then added to the plates along with RSV preF protein and left overnight at 37°C. After washing with PBS, the plates were treated with detection antibody (R4-6A2-biotin) for 2 h at room temperature. Following a second wash, streptavidin-alkaline phosphatase (ALP, 1:1000) was added and incubated for 1 h at room temperature. Spots were visualized by adding BCIP/NBT-plus substrate until they appeared, and then the plates were rinsed with water to halt the reaction. The resultant spots were counted using an S6 Ultra M2 ELISpot reader (CTL, USA).

4.5 Measurement of IFN-γ by ELISA

IFN-γ levels in culture supernatants of splenic lymphocytes and single-cell suspensions from the lungs were quantified with a commercial ELISA kit (Thermo Fisher Scientific, 88–7314). Briefly, 96-well plates were coated overnight at 4°C with capture antibody. After washing, the plates were blocked with ELISA diluent for 1 h at room temperature. Next, each sample was added and incubated for 2 h. Following another wash step, the plates were treated with detection antibody for 1 h. Subsequent washes preceded the addition of streptavidin-HRP, which was left to incubate for 30 min. The reaction was developed using TMB substrate for 15 min before being stopped. Absorbance readings were taken at 450 nm using a microplate reader (Spectramax ABS, Molecular Devices) with SoftMax Pro 7.1 software.

4.6 Live RSV Virus Neutralization Assay

The live RSV virus neutralization assay was performed as described previously [81]. Briefly, A549 cells (5 × 10⁵ cells/mL) were seeded in 96-well plates and cultured for 24 h. Serum samples were heat-inactivated at 56°C for 30 min and then subjected to 3-fold serial dilutions in DMEM containing 2% heat-inactivated FBS. An equal volume of RSV (10⁴ TCID₅₀/mL) was introduced to the samples and incubated at 37°C for 1 h. These mixtures were then transferred onto the A549 cell monolayers and incubated for 24 h at 37°C. Post-incubation, the cells were fixed using a 1:1 acetone/methanol solution at −20°C for 20 min and blocked with PBS containing 1% BSA and 0.1% Tween-20 for 1 h at 37°C. The next step involved staining with an anti-RSV F antibody (CHEMSTAN, CSD00024) for 1 h at 37°C. Following PBS washes, an HRP-conjugated secondary antibody (Abcam, ab6759) was applied for another hour at 37°C. To visualize the results, plates were treated with TrueBlue peroxidase substrate (KPL, 95059–168) for 5 to 15 min at room temperature. The reaction was then halted with PBS, and the resulting spots were counted using an ELISpot Reader (AID, ELR08IFL).

4.7 Flow Cytometry

Four weeks after the last vaccination, BALF and lung tissue were harvested to assess the T_RM cells. Cells were stained with the following antibodies: PerCP/Cyanine5.5-CD3 (BioLegend, 100218), BV421-CD4 (BioLegend, 100412), FITC-CD8a (BioLegend, 100706), BV510-CD44 (BioLegend, 103044), PE-CD69 (BioLegend, 164204), and APC-CD103 (BioLegend, 121414).

Four weeks after the last vaccination, ILN and mLN were collected to analyze Tfh cells and preF-specific GC B cells. For Tfh cells in ILN, the cells were stained with PerCP/Cyanine5.5-CD3, PE-CD19 (BioLegend, 115508), APC-CD4 (BioLegend, 100412), FITC-CD185 (CXCR5) (BioLegend, 145520), and BV421-CD279 (PD-1) (BioLegend, 109121). For Tfh cells in mLN, the cells were stained with PerCP/Cyanine5.5-CD3, PE-CD4 (BioLegend, 100408), FITC-CD19 (BD Pharmingen, 553785), APC-CD185 (CXCR5) (BioLegend, 145506), and BV421-CD279 (PD-1). To analyze the prevalence of preF-specific GC B cells within the ILN and mLN, the cells were first incubated with biotin-tagged preF protein (Sino Biological, HP17OC1701), followed by staining with PerCP/Cyanine5.5-CD3, PE/Cyanine7-B220 (BioLegend, 103222), FITC-CD95 (BioLegend, 152606), APC-GL-7 (BioLegend, 144618), and PE-anti-biotin antibodies (BioLegend, 409004).

For ICS, splenic lymphocytes and lung single-cell suspensions were stimulated with 5 µg/mL RSV preF protein for 12 h. Before cell collection, Brefeldin A (BFA, BD Biosciences) was introduced to inhibit cytokine secretion. Cells were stained with the following antibodies: PerCP/Cyanine5.5-CD3, APC-CD4, FITC-CD8a, PE-CD69, and BV510-CD44 for 30 min at 4°C. After fixation and permeabilization (Biosciences, 554715), cells were stained with PE/Cyanine7-IFN-γ (BioLegend, 505826) antibody for 1 h at room temperature.

4.8 Statistical Analysis

All statistical analyses used GraphPad software Prism 10.1.2, and FCM data analysis was performed with NovoExpress 1.4.1 software (ACEA Biosciences, Inc.). The difference between groups was compared using one-way ANOVA followed by Tukey's multiple comparisons test. ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns, not significant.