2.1 IN CCD/preF Vaccine Induced RSV-Specific Mucosal IgA and Serum IgG Responses That Neutralized Virus Infection

Based on our previous studies and dose optimization, we immunized mice with 50/5 µg of CCD/preF intranasally on days 0, 21, and 42 [33, 38]. To explore whether the addition of vaccine dose brings advantages over anti-viral immunity, we vaccinated another group of mice with 100/10 µg of CCD/preF intranasally. Correspondingly, rats were immunized with 200/20 µg or 400/40 µg of CCD/preF via intranasal drops, respectively. Animals receiving PBS, naked preF, or the CCD adjuvant were defined as control groups. Serum from immunized animals was obtained 14, 35, and 56 days after the initial immunization, and bronchoalveolar lavage fluid (BALF) was obtained on day 72 to determine preF- and postF-specific antibodies (Figure 1A). Enzyme-linked immunosorbent assay (ELISA) analysis indicated that immunization with preF protein or CCD adjuvant cannot arouse postF- or preF-specific antibodies in sera (Figure 1B,C). In contrast, antigen-specific IgG antibodies in sera increased significantly on day 14 with titers of >10⁴ in mice (Figure 1B,C) and >10²–10³ in rats immunized with CCD/preF (Figure S1). After one boost, the sera IgG titers ascend to >10⁵ against postF and 10⁶ against preF in mice (Figure 1B,C), which reached >10⁴ specific to postF and 10⁵ against preF protein in rats (Figure S1A,B). The second booster of the intranasal CCD/preF further increased serum antibody titers, which reached over 10⁷ against preF in mice on day 56. No statistically significant differences were observed in the anti-preF and anti-postF serum IgG levels between low- and high-dose groups of CCD/preF except for a slight enhancement of anti-preF IgG antibodies in the high-dose group on day 56, indicating that immunization with 5 µg of preF antigen is sufficient to induce substantial humoral immune responses in the presence of CCD adjuvant. Since the magnitudes of antibody response targeting site Ø is related to neutralizing capacity, we applied D25 competition ELISA to investigate if the tested serum antibodies effectually targeted this preF exclusive site. After the third dose, mice sera from the vaccine groups induced high anti-site Ø IgG titers (Figure 1D).

Subtype analysis revealed that IgG1, IgG2a, IgG2b, and IgG2c antibodies against preF antigen were all enhanced in mice immunized with CCD/preF (Figure 1E). F-specific antibodies can be used to predict virus-neutralization antibodies against RSV [27, 39]. To determine the functions of antibodies, we infected HEp-2 cells with the RSV A2 and B18537 strains and used the collected sera to neutralize viral infection at different dilutions. The results showed that both low and high-dose CCD/preF vaccines induced high levels of neutralizing antibodies against RSV infection with titers of > 2000 (Figure 1F). These results indicated that CCD/preF can induce strong antigen-specific humoral immune responses in the sera of intranasally vaccinated animals.

Next, we analyzed the production of antigen-specific antibodies in BALF, including IgA and IgG. As expected, superior anti-preF IgG and IgA responses were induced in mice inoculated with CCD/preF, but not in mice vaccinated with naked preF protein, CCD, or PBS (Figure 1G,H). Although slightly decreased compared with preF-specific antibodies, IN CCD/preF administration also induced high levels of mucosal IgG and IgA antibodies specific to postF. These results supported that CCD is an excellent mucosal adjuvant for the recombinant subunit vaccine of RSV to induce strong mucosal and systemic antibody responses.

2.2 preF-Specific IgG⁺ and IgA⁺ ASCs Induced in Local and System by IN CCD/preF Vaccine

Apart from humoral immunity, cellular immunity is also an essential part of antiviral immunity [40]. Antibody-secreting cells (ASCs), derived from activated B cells, may differentiate into extracollicular cells or form germinal central responses within secondary lymphoid organs. ASCs are considered the mainstay of humoral immune responses because they can produce a large number of antibodies for host defense continuously [41, 42]. To study the underlying mechanisms, the immunized mice were sacrificed 1 month after the last immunization. Cells from the lungs, spleen, and bone marrow (BM) were isolated and added to preF-coated wells for ASC detection. Enzyme-linked immunospot assay (ELISpot) showed that IN immunization with low- and high-dose CCD/preF induced comparable levels of antigen-specific IgG⁺ and IgA⁺ ASCs in lung, BM, and spleen, but not in PBS and naked protein controls (Figure 2A–C). Meanwhile, preF-specific IgG⁺ and IgA⁺ ASCs in the lung (Figure 2A) were superior to those in the spleen (Figure 2B) and BM (Figure 2C), indicating that the IN CCD/preF vaccine outperformed in activating antigen-specific ASC responses producing mucosal antibodies.

2.3 Superior Antigen-Specific B and T Cell Immunity Induced by IN CCD/preF Vaccine at Local Sites

Activation of local and systemic B- and T-cell responses after vaccination was analyzed by flow cytometry (FCM). Germinal centers play a key role in the proliferation of B cells and the generation of memory B cells (MB) [43], whereas follicular helper T cells (Tfh) are important in the regulation of high-quality antibody response [32]. Thus, we detected GCB (CD19⁺GL-7⁺CD95⁺) and Tfh (CD4⁺CXCR5⁺PD-1⁺) cells in mediastinal lymph nodes (mLN) and found that the CCD-adjuvanted RSV IN vaccine significantly increased the frequency of GCB (Figure 3A) and Tfh (Figure 3B) in mLN. preF protein is fluorescently labeled to probe preF-specific B cells. preF-specific B cell (preF⁺CD19⁺) elevation was observed in the mLN of the vaccine group compared with immunization with preF protein (Figure 3A). Plasmablasts induce an early burst of antigen-specific antibodies after vaccination, whilst plasma cells can continuously secrete large numbers of antibodies with high affinity [44]. preF-specific memory B cells (MBCs, CD138⁻CD19⁺CD38⁺), plasma cells (IgD⁻CD19⁻CD138⁺) and plasmablast cells (IgD⁻CD19⁺CD138⁺) in the lung (Figure 3C), BM (Figure 3D), and spleen (Figure 3E) were remarkably elevated in mice inoculated with CCD/preF vaccines regardless of the dosage, further demonstrating the activation of B cell immune responses.

Mucosal tissue-resident memory T cells (T_RMs) have been reported to have the potential for rapid host defense against viral infection in situ [45, 46]. Therefore, we evaluated the production of mucosal T_RMs following IN administration of the CCD/preF vaccine. Mice that received the CCD/preF vaccine, but not pure preF protein, produced significant CD4⁺ and CD8⁺ T_RM cells in BALF (Figure 3F) and the lungs (Figure 3G). The lymphocytes obtained in lung tissues were stimulated by the F-peptide pools. We then used intracellular cytokine staining (ICS) and ELISpot to assess the generation of interferon-γ (IFN-γ). The frequencies of IFN-γ⁺CD4⁺ and IFN-γ⁺CD8⁺ T cells (Figure 3H) and the total number of IFN-γ secreting cells (Figure 3I) were remarkably increased in the lungs of mice vaccinated with CCD/preF. At the same time, ELISA results indicated that lung lymphocytes from the CCD/preF vaccinated mice secreted a large quantity of IFN-γ cytokines into the supernatants after peptide restimulation (Figure 3J). Nevertheless, IN vaccination with CCD/preF failed to activate effective splenic T cells producing IFN-γ against preF antigen (Figure S2). These data suggested that the IN CCD/preF vaccine can induce antigen-specific T cell immune responses at the local sites instead of systemic immune organs.

2.4 IN CCD/preF Vaccine Elicits Long-term Immune Responses for Over One Year

The durability of vaccines’ efficacy is of great importance to avoid repeated vaccination. After immunization with CCD/preF (5 µg) vaccine, the preF- and postF-specific IgG levels in sera did not decline to half of the day 56 levels by day 365, indicating an antibody half-life of over 365 days (Figure 4A). The titers of antigen-specific IgG in BALF showed no or only a slight decreasing trend on day 365 after immunization (Figure 4B). However, the titers of preF- and postF-specific IgA in BALF reduced to less than 1/10 on day 365 compared with day 56 (Figure 4C). These results suggested that the IN CCD/preF vaccine can induce long-lasting humoral immunity for up to 1 year.

We next assessed long-term cellular immunity after IN immunization with the CCD/preF vaccine on day 365. A sharp increase in the frequency of preF-specific MBCs, plasmablast cells, and plasma cells was observed in the lung (Figure 4D). ELISpot assay further supported the activation of B cells in the lung and BM, with elevated levels of ASCs secreting preF-specific IgG and IgA in the vaccine group compared with PBS (Figure 4E,F). Cells isolated from lung tissues in the CCD/preF vaccine group maintained high levels of IFN-γ production after restimulation with F-peptide pools (Figure 4G). Similarly, we discovered that lung CD4⁺ and CD8⁺ T_RM responses remained relatively high in the CCD/preF vaccine group (Figure 4H). Thus, the IN CCD/preF vaccine can induce high and durable humoral and cellular immune responses both locally and systemically for over 1 year.

2.5 Combining IM and IN Immunization of CCD/preF Vaccine Elicits Stronger Mucosal and Systemic Antibody Responses

We next investigated whether combining the conventional IM immunization could enhance the immunogenicity of the IN CCD/preF vaccine. Mice were immunized with different vaccination strategies (Figure 5A,B). NIH mice were intramuscularly primed with CCD/preF on day 0, followed by two-dose boosters via IM and IN delivery (2×IM+1×IN) or only IN delivery (1×IM+2×IN) of the CCD/preF at intervals of 21 days. The mice receiving three IN doses of the CCD/preF vaccine (3×IN) or PBS were used as control (Figure 5B). On day 56, mice sera were collected for the detection of serum humoral immunity. On day 72, lungs, spleens, mLN, spleen, BALF, and BM were collected.

As shown in Figure 5C, the immunizations of 2×IM+1×IN and 3×IN elicited comparable titers of preF-specific and postF-specific IgG titers in serum, whereas 1×IM+2×IN immunization resulted in the highest IgG levels specific to both preF and postF. However, BALF preF- and postF-specific IgG titers were not significantly boosted in the 2×IM+1×IN or 1×IM+2×IN groups, indicating that BALF IgG levels have reached a ceiling for the CCD/preF vaccine after IN administration (Figure 5D). Interestingly, 1×IM+2×IN immunization with CCD/preF vaccine induced comparably high BALF IgA titers compared with 3×IN immunization, whilst a modest reduction of preF-specific and a sharp decrease of postF-specific IgA titers were detected in the 2×IM+1×IN group (Figure 5E). Consistent with the results of antibody titers, 1×IM+2×IN and 3×IN immunization induced the highest percentage of Tfh cells in the mLN. In sharp contrast, 2×IM+1×IN vaccination showed limited activation of Tfh cells (Figure 5F). These results indicated that combining one IM prime and two IN boosters with CCD/preF could provoke comparable mucosal humoral immunity but superior serum IgG titers than pure IN delivery, whilst 2×IM+1×IN delivery compromised the antibody responses both locally and systemically.

2.6 Combining IM and IN Immunization of CCD/preF Vaccine Elicits Strong B and T Cell Immunity both Locally and Systemically

We measured antigen-specific B cells in the lung, spleen, and BM with FCM on day 72 after the prime. We detected a significant increase of preF-specific plasmablasts in the lung, BM, and spleen after administration of the CCD/preF vaccine, irrespective of vaccination route (Figure 6A). In the lung and spleen, 1×IM+2×IN immunization induced the highest proportion of antigen-specific plasmablasts, followed by 3×IN immunization. We also discovered an escalation of preF-specific plasma cells in the lung and spleen after vaccination with the CCD/preF vaccine, especially in the 1×IM+2×IN group (Figure 6B). These results further verified the noninferior capability of the 1×IM+2×IN vaccination strategy in inducing antigen-specific antibodies than 2×IM+1×IN or 3×IN immunization. MB are long-lived and can be activated and differentiate into ASCs rapidly after re-exposure to the same antigen [47]. As expected, 1×IM+2×IN immunization induced a sharp increase in the frequency of MBCs in the lung, BM, and spleen. 2×IM+1×IN and 3×IN immunization also activated MBCs efficiently, but not as effectively as 1×IM+2×IN immunization (Figure 6C).

To evaluate the antigen-specific B-cell immunity, lymphocytes from the lung, BM, and spleen were isolated and subjected to an ELISpot assay with preF protein. As shown in Figure 6D–F, the CCD/preF vaccine induced preF-specific ASCs producing IgG antibodies in the lung, BM, and spleen, regardless of different immunization routes. 2×IM+1×IN immunization is weaker in inducing IgG ASCs in the lung whereas 3×IN immunization is weaker in activating IgG ASCs in the spleen, making 1×IM+2×IN immunization the most stable and effective method to induce preF-specific IgG-producing B cell responses both locally and systemically. We also assessed the production of IgA-producing ASCs after combining IM and IN immunization. We found that 1×IM+2×IN and 3×IN immunization induced comparatively higher levels of ASCs producing preF-specific IgA antibodies in the lung, BM, and spleen compared with 2×IM+1×IN immunization. These results indicated that the combination of one IM prime and two IN booster immunizations can induce significantly higher B cell immune responses both at local sites and systemically.

We next analyzed whether the combination of IM and IN immunization of CCD/preF vaccine can induce superior T cell responses. In BALF, we found that 1×IM+2×IN immunization induced comparable numbers of CD8⁺ and CD4⁺ T_RM cells as 3×IN immunizations, in contrast to the decreased tendency of 2×IM+1×IN immunization (Figure 7A). Similar results were observed in the frequency of preF-specific T cells in the lung. T cells in the lung tissues of the vaccinated mice were harvested and restimulated with F-peptide pools before being analyzed with ICS. We found that both 1×IM+2×IN and 3×IN immunization induced high levels of CD8⁺IFN-γ⁺ and CD4⁺IFN-γ⁺ T cells in the lung, whereas 2×IM+1×IN immunization did not (Figure 7B). We found that 3×IN immunization with the CCD/preF vaccine is insufficient to elicit T-cell responses in the spleen (Figure S2). However, combining one or two IM immunizations can overcome this obstacle by increasing the frequencies of preF-specific CD4⁺ and CD8⁺ T cells producing IFN-γ and/or IL-4 cytokines in the spleen (Figure 7C). Therefore, combining IM and IN immunization can induce comparably strong antigen-specific local immunity compared with IN immunization while maintaining the potential of CCD/preF in activating strong systemic immune responses.

4.1 Materials

preF (cat: 11049-VNAS), Biotinylated-preF (cat: 11049-VNAS-B), and postF (cat: 11049-V08B) were obtained from SinoBiological. Horseradish peroxidase (HRP)-conjugated anti-mouse IgG (cat: 0107-05) and IgA (cat: 1040-05) antibodies and Horseradish peroxidase (HRP)-conjugated goat anti-rat IgG (Cat: 3030-05) were provided by southern biotech. RSV A2 (cat: VR-1540) and B18537 (cat: VR-1580) were obtained from an American-type culture collection (ATCC). D25 antibody (cat: PABL-322) was purchased from Creative Biolabs.

4.2 CCD Preparation

The CCDs were synthesized according to our previous work [33]. Briefly, pre-prepared CD-1.8k and linkers were dissolved in ethanol followed by a reaction at 80°C for three days with N₂ protection. Ethanol was adequately removed from the reaction mixture and mixed solvent (TFA and CH₂Cl₂) was added and stirred for 16 h at room temperature (RT). The solvent was then fully removed using vacuum evaporation. Dissolve the residue in deionized water and purify it using dialysis with 0.1N HCl solution (MWCO 3500) for 48 h and deionized water for 24 h. The final product was freeze-dried to obtain CCD.

4.3 Vaccine Formulations and Vaccination of Animals

For vaccine preparation, 100 µg CCD was incubated with 5/10 µg of preF protein for 30 min at RT. Then, the volume was refilled to 50 µL with PBS. Six- to eight-week-old female NIH mice (SPF) were purchased from Beijing Vital River Laboratory Animal Technologies Co., Ltd (China) and raised in the animal facility (SPF) of the Animal Center of the State Key Laboratory of Biotherapy.

To identify the immune response induced by IN immunization, the mice were randomly divided into five groups: mice in the vaccine group were immunized intranasally with 50/5 µg or 100/10 µg doses of vaccine (CCD/preF, 50 µL/one mouse). Mice receiving 100 µg of CCD or 5 µg of the naked protein were used as the control. In the PBS group, mice were inoculated with an equal volume of PBS.

To investigate the efficacy of integrating IM and IN immunization, NIH mice (5 mice/group) were immunized with 5 µg preF antigen plus CCD adjuvant. One group of mice was inoculated with two doses of vaccine via the IM pathway and then received the third dose of vaccine via the IN pathway (2×IM+1×IN). Mice in the 1×IM+2×IN group were first immunized intramuscularly and then boosted twice via the IN route.

The 6-week-old rats (female) were immunized intranasally with three doses of 20 µg vaccine (200 µL per rat). The mice and rats were injected on days 0, 21, and 42. Sera were collected on days 14, 35, and 56, and BALF was collected on day 72 to assess the binding and neutralizing antibodies. All animal experiments in this study were approved by the Institutional Animal Care and Use Committee of Sichuan University.

4.4 Enzyme-Linked Immunosorbent Assay

Anti-preF and postF specific IgG and IgA in sera and BALF were identified by ELISA. First, the 96-well plates (NUNC-MaxiSorp; Thermo Fisher Scientific) were coated with preF and postF proteins (1 µg/mL) in the carbonate coating buffer and incubated overnight at 4°C. after the plates were washed three times by PBST (PBS with 0.1% Tween 20), 100 µL blocking buffer (PBST containing 1% BSA) was added and plates were incubated at a 37°C incubator for 1 h. After one washing with PBST, the double gradient diluted serum or BALF was added to the well and reacted for 2 h at 37°C. Then, the plates were washed three times and secondary antibodies (HRP-conjugated anti-mouse IgG, IgA, or anti-rat IgG, 1:10,000, Abcam) were transferred to wells. After reacting for 1 h at 37°C, the plates were added with 100 µL/well of TMB (3,3′,5,5′-tetramethyl biphenyl diamine) and developed fully at RT for 10 min. The reactions were quenched with 1 M H₂SO_4. In the end, the absorbance was detected at 450 nm on a microplate reader (Spectramax ABS; Molecular Devices).

To evaluate the concentration of IFN-γ in the supernatant of lymphocytes, a mouse IFN-γ ELISA Kit (CAT: 88-8314-88, Invitrogen) was used and we completed the whole experiment strictly in accordance with the instructions in the manual.

4.5 Competition ELISA Assay

A competition ELISA was done to measure the amounts of antibodies targeting the PreF exclusive antigenic site Ø in mice sera. Firstly, a SuperBiotin Quick labeling kit (Frdbio, China) was used to conjugate Biotin to the D25 antibody (referred to as a tracer). 96-well ELISA plates were coated with 100 µL/well of PreF (2 µg/mL) and incubated overnight at 4°C. Then, the plates were washed with PBST and then blocked with a blocking buffer for 90 min at RT. The immune serum samples were serially diluted in a blocking buffer and then mixed in equal volumes with a tracer (8 ng/mL). After one washing of the plates, the tracer-sample mixtures were added. Following three times wash, HRP-labeled streptavidin (Cat: A0305, 1:5000, 100 µL/well, Beyotime, China) was added to the plates and incubated for 1 h at RT. Then, plates were washed three times and incubated with 100 µL/well of TMB substrate for 10 min at RT for full reaction. 100 µL/well of 1 M H₂SO₄ was added to stop the reaction. The optical density was measured at 450/620 nm with a microplate reader (Spectramax ABS; Molecular Devices). The serum dilution ratio at 50% inhibition of tracer binding to preF is the antibody titer.

4.6 Enzyme-Linked Immunospot (ELISpot) Assay

The immunized mice were sacrificed on day 72 or 1 year after the prime immunization. preF-specific IgG and IgA antibody-secreting cells (ASCs) in the lung, bone marrow, or spleen were identified using ELISpot. 96-well filtration plates (REF: MSIPS4W10, Millipore) were treated with 35% ethanol for 1 min and then washed five times with sterile water. Each well was added with 100 µL preF (3 µg/mL) and incubated overnight at 4°C. Following that, the plates underwent 5-time washes with PBS. Subsequently, plates were blocked by RPMI-1640 complete medium for at least 1 hat 37°C. Lymphocytes of the immunized mice were obtained with mouse lymphocyte isolation solution and added to the wells. The plates were kept in the incubator overnight and not moved during the process. After three washes with PBS, anti-mouse IgG or IgA Ab (100 µL/well) was introduced to the wells. After being reacted at RT for 2 h, the plates underwent three rounds of washing with PBS and then added to TMB ELISpot substrate solution. After the formation of clear spots, the reaction was stopped by flowing water. Finally, the spates were naturally dried and the spots were counted on the S6 Ultra M2 ELISpot reader (CTL, USA).

To detect IFN-γ-secreting cells in the lungs of immunized mice, a mouse IFN-γ ELISpot kit (CAT:3321-4APT-2) was used. Coated plates were washed with PBS, blocked by 1640 complete medium for 1 h, and then added with cells taken from the lung (1×10⁶/well). Cells in the wells were stimulated with F peptide pools for 16 h at 37°C and then washed with a PBS rinse. Plates were added with diluted detection antibody (100 µL/well) and incubated for 2 h at RT After washing with PBS, each well was added with 100 µL diluted streptavidin-ALP and incubated for 1 h. Following that, plates were washed and BCIP/NBT-plus were transferred into wells for spot formation.

4.7 Virus Neutralization Assay

Virus neutralization assay (VNA) was conducted using collected sera from vaccinated NIH mice. Briefly, sera were heat-inactivated at 56°C for 30 min and then twofold serially diluted ranging from 16 to 32768. Then, the diluted sera were mixed with A2 or B18537 RSV viruses (2 ×10⁴ TCID50/mL) in equal volume. After incubation for 2 h at 37°C, 100 µL mixture was introduced into 96-well plates containing HEp-2 cells (5 ×10⁴/well, ATCC, CCL-23). The HEp-2 cells were incubated at 37°C for 1 week. The presence of cytopathogenic effects (CPE) was observed using a microscope. The lowest dilution resulting in 80% CPE inhibition was determined as the neutralizing antibody titer for the mice serum sample.

4.8 Flow Cytometry

The tissues were obtained on day 30 after the last vaccination. Mediastinal lymph nodes (mLN) were milled, incubated with red blood lysis solution, and filtered to get single cells. Lung tissues isolated from immunized mice were cut into small pieces and digested in digestive buffer (DMEM with type-1 and type-IV collagenases) in advance before treatment like mLN. Lymphocytes in the spleen can be obtained according to the instructions of the lymphocyte separation kit (DAKEWE, CAT: 7211011). Cells in all tissues were analyzed by a NovoCyte Flow Cytometer with NovoExpress 1.4.1 (ACEA Biosciences, Inc.).

Cells in mLN were stained by anti-mouse antibodies including PerCP/Cyanine5.5-CD3 (BioLegend; cat#100218), FITC-CD4 (BioLegend; cat#130308), BV605-CD19 (BioLegend; cat#11540), PE-CD279 (PD-1) (BioLegend; cat#135205), and BV421-CD185 (CXCR5) (BioLegend; cat#145512) at 4°C for half an hour to determine follicular helper T cells (Tfh). To detect germinal center B cells (GCB) and preF-specific B cells in mLN, cells were first treated with the Biotinylated-preF for 30 min at RT. Following that, cells were washed once by PBS and then stained by PerCP/Cyanine5.5-CD3, BV605-CD19, PE-Streptavidin (BioLegend; cat#405204) and BV421-GL-7 (BioLegend; Cat#144614).

To detect T_RMs in BALF or lungs, the following antibodies were used: PerCP/Cyanine5.5-CD3, FITC-CD4, BV421-CD8a (BioLegend; cat#100725), PE-CD69 (BioLegend; cat#164204), PE/Cyanine7-CD44 (BioLegend; cat#103030), and APC-CD103 (BioLegend; cat#110906).

preF-specific plasma cells, plasmablast cells, and memory B cells in the lung, bone marrow, or spleen can be analyzed simultaneously. After being treated with the Biotinylated-preF, cells were stained by BV421-CD4 (BioLegend; cat#100438), PerCP/Cyanine5.5-CD19 (BioLegend; cat#152405), FITC-IgD (BioLegend; cat#405704), APC-GL-7 (BioLegend; cat#144618), BV650-CD138 (BioLegend; cat#142518), PE/Cyanine7-CD38 (BioLegend; cat#165617) and PE-Streptavidin. The immune cells in this study are gated as previously reported [33].

4.9 Statistical Analyses

All statistical analyses were performed using the GraphPad Prism 8.0 program. For comparisons between multiple groups, p-values were calculated using unpaired student's t-tests or one-way ANOVAs followed by Tukey's multiple comparisons and expressed by notation. ****p < 0.0001, ***p < 0.001, **p < 0.01 and *p < 0.05. ns: not significant.