Construction, purification, and characterization of the chimeric protein, KFD2-rPAc

The expression plasmid pET28a-KFD2-rPAc was constructed by substituting hyper-variable region domains D2 and D3 of flagellin KF with rPAc (Fig. 1a and b) and the recombinant protein was prepared as described in the Materials and Methods section. In the present study, KF-rPAc, KFD2-rPAc, and rPAc in the soluble fraction of cell lysates were purified in parallel. The purified recombinant proteins were tested by SDS-PAGE (Fig. 1c) and Western blotting assay (Fig. 1d). Mice splenocytes from C57BL/6 WT or TLR5 KO mice were used as an in vitro model to test the TLR5 agonist efficacy of the recombinant proteins. As shown in Fig. 1e, compared to rPAc or medium alone, both 10 nM of KF-rPAc and KFD2-rPAc induced significantly higher production of IL-6 and IFN-γ from wild type splenocytes but not from TLR5 KO ones. Surprisingly, KFD2-rPAc was less efficient in inducing IL-6 and IFN-γ than KF-rPAc at 1 nM concentration. This demonstrated that KFD2-rPAc has TLR5 agonist activity, but less efficient than its first generation counterpart, KF-rPAc.

Figure 1 Construction and verification of the recombinant protein KFD2-rPAc. (a) Construction process of plasmid pET28a-KFD2-rPAc. (b) Diagram of KF, KF-rPAc, and KFD2-rPAc. (c) The purity of the recombinant proteins verified by SDS-PAGE. (d) Western blotting analysis of the recombinant proteins probed with anti-His tag monoclonal antibody. (e) IL-6 and IFN-γ secreted into the culture supernatant from splenocytes of C57BL/6 background WT or TLR5 KO mice stimulated with 10 nM rPAc, 1 or 10 nM KF-rPAc or KFD2-rPAc for 20 hours. Dose-dependent (f) and time-dependent (g) IL-8 and MCP-1 secreted into the culture supernatant from Caco-2 cells stimulated with indicated concentrations of rPAc, KF-rPAc or KFD2-rPAc. Data are represented as mean ± SEM from triplicate samples of one representative experiment. *p < 0.05; **p < 0.01; and ***p < 0.001; n.s., non-significant. Full size image

To further compare the TLR5 agonist efficiency of KF-rPAc and KFD2-rPAc, extensive assays were carried out on Caco-2 cells, which constitutively express TLR5. At first, the dose-dependent effects of KFD2-rPAc on TLR5 agonist were compared with that of KF-rPAc at 6 hours post stimulation. The results showed that KFD2-rPAc induced IL-8 and MCP-1 in a dose-dependent manner similar to that for KF-rPAc, but with less activity in the concentrations lower than 10 nM (Fig. 1f). To verify this, we further compare the TLR5 activating efficiency between these two recombinants in a wider range of time points during 24 hours post stimulation. Similar to above results, 10 nM of KFD2-rPAc induced comparable IL-8 and MCP-1 production as 10 nM of KF-rPAc, but 1 nM of KFD2-rPAc induced significant less of these soluble mediators than 1 nM of KF-rPAc, at all times points (Fig. 1f).

Besides, the NLRC4 activating efficiency of KF-rPAc, KFD2-rPAc was measured on bone marrow derived macrophages (BMMs). Results showed that similar to KF, KF-rPAc and KFD2-rPAc induced minor production of IL-1β, cell death and caspase-1 p10. Therefore, both KF-rPAc and KFD2-rPAc have poor efficacy in activating the cytosolic NAIP/NLRC4 inflammasome pathway (see Supplementary Fig. S1b–d).

In brief, the second-generation flagellin-rPAc fusion protein, KFD2-rPAc, retained the TLR5 agonist activity but less efficient than KF-rPAc. Moreover, KFD2-rPAc has poor NLRC4 pathway activating efficacy as KF-rPAc and KF.

KFD2-rPAc induced a much lower systemic inflammatory response than KF-rPAc

Since the TLR5 activating efficiency of KFD2-rPAc is less than that of KF-rPAc, we hypothesized that the TLR5 pathway-related inflammatory responses induced by KFD2-rPAc could be less than that by KF-rPAc. Therefore, we compared potential inflammatory responses and possible side effects induced by KFD2-rPAc and KF-rPAc in the mouse model. PBS and rPAc were used as the vehicle and irrelevant, non-inflammatory protein control, respectively.

First, we analyzed the body weight changes after intranasal administration of 10 μg or 50 μg of KF-rPAc or KFD2-rPAc in BALB/c mice. At the 10-μg dosage, there were no significant body mass changes induced by KF-rPAc or KFD2-rPAc when compared with PBS. However, when the dose was increased to 50 μg, KF-rPAc induced a significant body weight loss at d 1, d 2, and d 3 post-protein administrations. Interestingly, KFD2-rPAc did not induce a significant body weight change during 6 d after protein administration (Fig. 2a).

Figure 2 KFD2-rPAc induced a much lower systemic inflammatory response after intranasal administration than KF-rPAc. Female BALB/c or C57BL/6 (WT or TLR5 KO) mice were intranasally treated with indicated amount of rPAc, KF-rPAc, or KFD2-rPAc in 30 μl of PBS or PBS alone. (a) Body weight changes of BALB/c mice after 10 μg (left panel) or 50 μg (right panel) recombinant protein administration. IL-6, KC and TNF-α in serum (b) or BALF (c) after 4 h of administration of 50 μg recombinant protein to BALB/c mice were determined by ELISA kits. IL-6, KC and TNF-α in serum (d) or BALF (e) after 4 h of administration of 50 μg recombinant protein to C57BL/6 background WT or TLR5 KO mice were determined by ELISA kits. Data are presented as mean ± SEM from 1 experiment that was repeated 3 times (n = 6 per group). *p < 0.05; **p < 0.01; and ***p < 0.001; n.s., non-significant. Full size image

Next, we analyzed proinflammatory cytokine IL-6, TNF-α and chemokine KC in serum and BALF after intranasal administration of 50 μg of KFD2-rPAc and KF-rPAc33. Compared with PBS or rPAc, KF-rPAc induced about a 2-fold increase of IL-6 in the serum, above a 90-fold increase of IL-6 in BALF, over a 30-fold increase of KC in the serum and about a 15-fold increase of KC in BALF. Meanwhile, KF-rPAc induced about a 1-fold increase of TNF-α in the serum and more than a 100-fold increase of TNF-α in BALF. In line with the body weight changes, KFD2-rPAc induced a much lower systemic inflammatory response, which was indicated as significantly lower IL-6, KC and TNF-α induction in serum. Interestingly, KFD2-rPAc also induced much less TNF-α in local fluid, BALF (Fig. 2b and c).

To assess whether KFD2-rPAc-induced less inflammatory responses depend on the activation of flagellin-TLR5 signaling, we performed parallel experiments in C57BL/6 background WT and TLR5 KO mice. As same as in BALB/c mice, C57BL/6 WT and TLR5 KO mice were administrated intranasally with 50 μg rPAc, KF-rPAc and KFD2-rPAc. As shown in Fig. 2d,e, compared to rPAc, KF-rPAc induced significantly increased production of IL-6, KC and TNF-α in serum and BALF, while KFD2-rPAc induced only local increase of IL-6 and KC. On the contrary, neither KF-rPAc nor KFD2-rPAc induced significant increase of IL-6, KC or TNF-α in serum or BALF. These results demonstrated that the inflammatory effects induced by intranasal administration of KF-rPAc and KFD2-rPAc are mainly dependent on TLR5 pathway. And the decreased systemic inflammatory effect presented by KFD2-rPAc is associated with TLR5 pathway.

KFD2-rPAc induced less flagellin-specific but comparable rPAc-specific antibody responses in mice

As the main antigenicity region of flagellin KF, D2 and D3 were replaced with rPAc, and the immunogenicity of the flagellin part in fusion protein KFD2-rPAc would be significantly reduced in our prospects. To comparatively analyze the immunogenicity of KF in KFD2-rPAc and KF-rPAc, mice were intranasally (i.n.) immunized with an equivalent molar amount of protein thrice. Consistent with our hypothesis, KF-specific serum IgG, serum IgA, and salivary IgA induced by KFD2-rPAc were 10 times less than that induced by KF-rPAc (Fig. 3a).

Figure 3 Flagellin- and rPAc-specific antibody responses induced by i.n. immunization of recombinant proteins in mice. Mice were i.n. immunized 3 times at 4-week intervals with PBS, 1 μg of rPAc, 1.7 μg of KF-rPAc, or 1.4 μg of KFD2-rPAc in a 10-μl aliquot. The serum and saliva collected on d 70 were detected by ELISA for antibody response against KF (a) or rPAc (b). (a) KF-specific serum IgG, serum IgA, and salivary IgA. (b) rPAc-specific serum IgG, serum IgA, and salivary IgA. (c) Biofilm formation inhibition of immunized mice serum or saliva was determined by biofilm assay. Data are represented as mean ± SEM for 6 mice of 1 representative experiment. *p < 0.05; **p < 0.01; and ***p < 0.001; n.s., non-significant. Full size image

Since the TLR5 activating efficiency of KFD2-rPAc was lower than that of KF-rPAc, we wondered whether the target antigen and rPAc-specific antibody response would be affected. Therefore, the quantity of rPAc-specific antibodies in the serum and saliva was tested and determined by ELISA. Surprisingly, the rPAc-specific serum IgG, serum IgA, and salivary IgA induced by KFD2-rPAc were all on a similar level as that induced by KF-rPAc (Fig. 3b). The antibodies induced by either KFD2-rPAc or KF-rPAc were 100-fold higher than that induced by rPAc alone (Fig. 3b). Then, the quality of the specific antibody responses in the serum and the saliva were tested by an in vitro biofilm formation model, which could indicate the inhibitory efficacy of rPAc-specific antibody responses in samples34. As shown in Fig. 3c, sera of KFD2-rPAc-immunized mice efficiently inhibited biofilm formation when compared with sera from PBS- or rPAc-immunized mice. More importantly, the inhibiting efficiency of sera from KFD2-rPAc-immunized mice was the same as that from KF-rPAc-immunized mice. In parallel, the saliva of the KFD2-rPAc-immunized group also showed similar efficacy in inhibiting biofilm formation as the saliva of the KF-rPAc-immunized group.

All in all, these results indicated that KFD2-rPAc induces significantly lowered flagellin-specific antibody responses while retaining comparable robust rPAc-specific antibody responses compared with KF-rPAc in mice.

KFD2-rPAc exhibits high prophylactic efficacy against caries as does KF-rPAc

Since KFD2-rPAc induces lowered flagellin-specific but comparable rPAc-specific antibody responses in mice, we wondered whether the changed antibody responses by KFD2-rPAc also exist in rats and provide efficient protection against caries or not. First, we analyzed the prophylactic efficacy against caries in the S. mutans-challenged rat model based on our previous study12, which is briefly depicted in Fig. 4a. Rats were intranasally (i.n.) immunized with an equivalent molar amount of protein thrice. The antibody levels and caries scores were evaluated at the end of the experiments. Similar as observed in the mice, i.n. immunization of KFD2-rPAc induced significantly lower KF-specific serum IgG, serum IgA, and salivary IgA than that of KF-rPAc in the rats (Fig. 4b). Meanwhile, i.n. immunization of KFD2-rPAc induced robust rPAc-specific serum IgG and IgA responses similar to KF-rPAc in the rats, in which both induced about 1000 times higher than that by rPAc alone. Interestingly, it should be noted that i.n. immunization of KFD2-rPAc induced higher rPAc-specific salivary IgA than KF-rPAc (Fig. 4c). Moreover, in KFD2-rPAc-immunized rats, the titer of KF-specific antibody response was over 20 times less than that of the rPAc-specific antibody response (Fig. 4b and c). These results suggested that the replacement of the D2/D3 region of KF with rPAc not only reduces flagellin-specific antibody responses, but it tends to induce more secretory rPAc-specific IgA in saliva.

Figure 4 Flagellin- and rPAc-specific antibody responses and prophylactic efficacy of KFD2-rPAc against caries in rats. To analyze the prophylactic efficacy of KFD2-rPAc against caries, rats were immunized before caries were established. The rats were fed antibiotics. After confirming oral bacteria, including Streptococcus, were depleted in the oral cavity of the rats, 30 rats were randomly divided into 5 groups (6 rats per group). Four groups of rats were challenged with 2 × 109 CFU of S. mutans Ingbritt for 3 consecutive days (once daily), while the other group was left untreated and set as a caries baseline and fed the Keyes 2000 diet. After confirming that all of the challenged rats were successfully infected with bacteria, the rats were immunized with 5 μg of rPAc, 5 µg of rPAc equivalent mole of 8.5 μg KF-rPAc, 7ug KFD2-rPAc, or PBS alone in a 10-μl aliquot, and boosted on d 56 and d 84. The day on which the rats completed the first immunization was set as d 0 (d 0). The serum and the saliva were collected on d 70 and analyzed for flagellin- or rPAc-specific antibody responses by enzyme-linked immunosorbent assay. On d 84, all of the rats were killed and caries levels were scored by the Keyes method. (a) Challenge, immunization, and sampling schedule of rats. (b) Flagellin-specific serum IgG, serum IgA, and saliva IgA responses in immunized rats. (c) rPAc-specific serum IgG, serum IgA, and saliva IgA responses in immunized rats. (d) Representative photographs of caries lesion for each group. E, Ds, and Dm lesions are indicated by a green arrow, red arrow, and purple arrow, respectively. (e) Caries scores of enamel (E) and slight dentinal (Ds) and moderate dentinal (Dm) lesions of rats immunized with different immunogens. (f) Total caries scores of different groups (total score = score of E + Ds + Dm). (g) The inhibition ratio of dental caries by immunization with different immunogens. *p < 0.05; **p < 0.01; and ***p < 0.001; n.s., non-significant. n.o., not observed. Full size image

Corresponding to the antibody responses, significantly fewer caries lesions, including enamel lesions (E) and slight dentinal lesions (Ds), were observed in the rats immunized with the fusion protein KF-rPAc or KFD2-rPAc than in the rats immunized with PBS or rPAc alone. No moderate dentinal lesions (Dm) were observed in the rats immunized with the protein KF-rPAc or KFD2-rPAc. Surprisingly, significantly fewer enamel lesions (E) were observed in the rats immunized with KFD2-rPAc than with the KF-rPAc (Fig. 4d,e). Accordingly, significantly lower total caries scores (E + Ds + Dm) were observed in the rats immunized with protein KF-rPAc or KFD2-rPAc than in the rats immunized with PBS or rPAc alone. Lower total caries scores were shown in the rats immunized with the chimeric KFD2-rPAc than in the rats immunized with KF-rPAc, though not significantly (Fig. 4f). Moreover, among the S. mutans-challenged rats, only the KFD2-rPAc-immunized group showed no significant difference in caries lesions from the uninfected rats. Based on the total caries scores of the sham-immunized rats (PBS group, 0%) and the uninfected rats (unchallenged control group, 100%), 84.4% of the mean caries reduction was achieved by i.n. immunization of KFD2-rPAc, 68.5% of the mean caries reduction by KF-rPAc, while only 4.5% was by rPAc alone (Fig. 4g). These results indicated that i.n. immunization of the chimeric KFD2-rPAc greatly prevented teeth against S. mutans-induced dental caries in rats.

Together, compared with KF-rPAc, KFD2-rPAc induces less flagellin-specific but comparable rPAc-specific antibody responses in rats and confers comparable prophylactic protection against caries in rats that were immunized before caries were established.

KFD2-rPAc exhibits a high therapeutic effect against caries as does KF-rPAc

We further analyzed the therapeutic effect against caries in the S. mutans-challenged rat model based on our previous study13, which is briefly depicted in Fig. 5a. Eight weeks after implanting S. mutans, all of the rats developed E and Ds lesions. Rats were then grouped and intranasally immunized with (1) PBS, (2) 5 μg of rPAc, (3) 8.5 μg of KF-rPAc, or (4) 7 μg of KFD2-rPAc, with the equivalent molar dosage of 5 µg rPAc according to the protocol shown in Fig. 5a. After the second boost, KF-specific serum IgG, serum IgA, and salivary IgA induced by KFD2-rPAc were about 10 times less than that elicited by KF-rPAc (p < 0.05) (Fig. 5b). For the rPAc-specific response, both KFD2-rPAc and KF-rPAc induced about 1000-fold higher rPAc-specific serum IgG, 200-fold higher rPAc-specific serum IgA, and 20-fold higher salivary IgA responses than that induced by rPAc alone. Compared with the KF-rPAc immunization, the KFD2-rPAc immunization induced comparable rPAc-specific serum IgG, serum IgA, and salivary IgA (Fig. 5c). Moreover, in the KFD2-rPAc-immunized rats, the titer of KF-specific antibody response was more than 50 times less than that of the rPAc-specific antibody response (Fig. 5b and c).

Figure 5 Flagellin- and rPAc-specific antibody responses and therapeutic effect of KFD2-rPAc against caries in rats. To analyze the therapeutic effect of KFD2-rPAc against caries, the rats were immunized after caries were established. Rats were fed antibiotics and challenged with S. mutans Ingbritt. The day on which the rats completed the challenge was set as d 0 (d 0). Fifty-six days after implanting S. mutans, all of the rats developed E and Ds lesions. Rats were then randomly divided into 5 groups (6 per group): control, PBS, rPAc, KF-rPAc, and KFD2-rPAc. At d 56, the control group was killed and set as the caries baseline, while the other groups were intranasally immunized with PBS, 5 μg of rPAc, 8.5 μg of KF-rPAc, or 7 μg of KFD2-rPAc in a 10-μl aliquot, and boosted on d 84 and d 112, respectively. The serum and the saliva were collected on d 126 and analyzed for flagellin- or rPAc-specific antibody responses by enzyme-linked immunosorbent assay. On d 140, the rats were killed and the caries levels were scored by the Keyes method. (a) Challenge, immunization, and sampling schedule of rats. (b) Flagellin-specific serum IgG, serum IgA, and saliva IgA responses in immunized rats. (c) rPAc-specific serum IgG, serum IgA, and saliva IgA responses in immunized rats. (d) Representative photographs of caries lesion for each group. E, Ds, Dm, and Dx lesions are indicated by a green arrow, red arrow, purple arrow, and black arrow, respectively. (e) Caries scores of enamel (E), slight dentinal (Ds), moderate dentinal (Dm), and extensive dentinal (Dx) lesions of rats immunized with different immunogens. (f) Total caries score of different groups were determined: (total score = score of E + Ds + Dm + Dx). (g) The inhibition ratio of dental caries by immunization of different immunogens. *p < 0.05; **p < 0.01; and ***p < 0.001; n.s., non-significant. n.o., not observed. Full size image

After rats were killed at 4 weeks post-final immunization, the caries scores were detected for each rat subjected to the experiments. As shown in Fig. 5d, significantly fewer E and Ds lesions were observed in the rats immunized with KF-rPAc or KFD2-rPAc than those immunized with PBS or rPAc alone (Fig. 5d,e). Accordingly, significantly lower total caries scores were also observed in the rats immunized with KF-rPAc or KFD2-rPAc than those immunized with PBS and rPAc (Fig. 5f). In comparison with the sham-immunized rats (PBS group, 0%) and the unchallenged control group (killed at 8 weeks post-bacterial challenge, 100%), the rats immunized with KF-rPAc or KFD2-rPAc exhibited similar caries reductions (49.6% vs 51.7%), both of which were significantly higher than that of rPAc groups (Fig. 5g).