CTB 2013 PDF
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Commercial CTB was used as a positive control. Plates were blocked and then washed five times. After washing, plates were revealed by the addition of 10 mg o-phenylenediamine OPD in 10 mL of a 0. The reaction was stopped by the addition of 4 M H2SO4. The absorbance was measured at nm in all cases. An incision was made in the neck, so as to expose the trachea.
Using a 5 mL syringe, 1 mL of PBS was slowly introduced into the lungs via the trachea and then syringed out. This was repeated twice. At least two independent experiments were performed with consistent results. The plates were washed three times with PBS-T.
Protection Assay in a Mouse Respiratory Model The challenge assay was performed using the mouse respiratory model of B. Inoculation was performed by pipetting the inoculum into the nostrils. Animals were euthanized by CO2 overdose 8 days postinfection PI , and the lungs were excised. Left lung was used for bacterial recovery as described previously [ 30 ], and the right lung was split in two samples, one used for cytokine level and the other for routine histological examination.
A melting curve analysis was performed immediately after amplification at a linear temperature transition rate of 0. The size of all PCR products was confirmed by agarose gel electrophoresis. When necessary, data were analyzed by one-way analysis of variance followed by the Bonferroni multiple comparison test to determine significant differences between groups. Results 3. The fusion protein was expressed and purified as described previously [ 28 ].
The results are shown in Figures 1 b and 1 c , respectively.
These data demonstrated that the recombinant E. Figure 1: Immunogen design, expression, purification, and immunoblot analysis of the CTB-Fim2 fusion protein. NI and I stand for noninduced and induced bacterial extract, respectively.
In contrast, when protein was solubilized in nondenaturing Laemmli sample buffer without boiling lane 2 it migrated with a MW of kDa. These results suggest that native CTB has a pentameric structure after the refolding process.
Lane 1 is the sample boiled in reducing conditions while lane 2 is the same protein sample not subjected to boiling and in nonreducing conditions.
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For detection, anti-CTB serum was used Figure 2 b. These results strongly suggest that CTB-Fim2 has a pentameric structure and indicate that the presence of Fim2 does not abrogate either the formation of the pentamer or binding to its GM1 receptor.
The results obtained at 8 days after the challenge showed a significant reduction in bacterial counts in mice immunized either by the intraperitoneal or the intranasal route with CTB-Fim2 compared to the controls treated with PBS or CTB IP 3. As expected, a significant decrease was seen between BpFim2, isolated from B. At 8 days postinfection, animals were killed and lung samples were collected for UFC counting.
These results indicate that intraperitoneal or intranasal administration of CTB-Fim2 was effective in protecting mice against intranasal challenge with B. Moreover, CTB improved the protective capacity of rFim2 when it was genetically fused to the antigen. As expected, the specific humoral response increased after each immunization in all groups.
To simplify the analysis, only the values of serum IgG at day 35, one day before challenge with B. Figure 4: Specific IgG antibodies in serum. Anti-Fim2 IgG titers of individual serum collected immediately before challenge on day 36 are shown.
These results mainly indicate that rFim2, when administered intranasally, was not capable of generating a strong humoral response in BAL, but when CTB was genetically fused to the antigen, specific antibody levels significantly increased in BAL, giving high titers of IgG and IgA with the fusion protein. Analysis of Th Cell Response To gain partial insight into the nature of immune responses induced by intranasal immunization with CTB-Fim2, we first quantified serum IgG subtypes G1 and G2a specific to Fim2 at day 36 postimmunization and before challenge Figure 6.
In addition, the histopathology analysis Figure 8 showed a more intense inflammation in the lungs of mice immunized with CTB-Fim2 compared with the rFim2 and negative control groups.
Figure 6: IgG1 and IgG2a antibody isotype response. Sera were collected from mice on day Figure 7: Cytokine mRNA expression levels in lung. Moderate lung inflammation in Fimb2 immunized animals c. More severe lung inflammation with extensive cell exudate and some consolidation pneumonia was observed in lungs of CTB-Fimb2 immunized animals d. Hematoxilin-eosin staining in all cases.
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Discussion Natural infection with B. The use of the mucosal route of vaccination is an attractive alternative to the use of the parenteral route since it may mimic many aspects of the immune response elicited against the natural infection [ 34 ].
Moreover, a single nasal dose of a live attenuated B. In addition, other pertussis antigens such as pertactin and filamentous hemagglutinin or even the more complex component, the outer membrane vesicles administered via the mucosal route, has proven to be effective against B.
The presence of fimbriae serotypes 2 and 3 in acellular pertussis vaccines has been shown to improve short-term vaccine efficacy in young children [ 38 , 39 ]. Although it has been well established that Fim2 purified from B. The molecular basis of this difference has not been established, but it could be due to differences in the structure of Fim2 obtained from the two sources. Furthermore, the protective capacity of rFim2 delivered mucosally has not been reported previously.
CTB has been widely used as an adjuvant in rodent studies [ 13 , 14 ]. When given via the oral or intranasal route, CTB not only elicits anti-CTB responses at multiple mucosal sites but also induces strong antibody responses to genetically fused administered antigens [ 32 ].
Previous studies have successfully tested the possibility of intranasal immunization with an acellular pertussis vaccine using CTB as an adjuvant, mixing recombinant CTB with pertussis toxoid and formalin-treated filamentous hemagglutinin [ 43 ]. Furthermore, a chimeric protein consisting of a divalent pertussis toxin PTX S1 fragment linked to the cholera toxin A2B fragment elicited protective immunity after three intranasal immunizations, showing the potent effect of CT as a mucosal adjuvant [ 44 ].
The CTB-Fim2 presented here formed a pentameric structure that seemed to be important in relation to its adjuvant capacity [ 13 ]. More importantly, a significant reduction in the number of bacteria recovered from the lungs was observed in mice immunized with CTB-Fim2 compared to those immunized with rFim2 or the control mice groups indicating that some antigen can offer protection when fused to CTB.
The protective capacity of CTB-Fim2 over rFim2 may be due, at least in part, to both the increased uptake of coupled antigen across the mucosal barrier [ 45 ] and more efficient delivery to antigen-presenting cells [ 46 ].
In our conditions and in agreement with other authors [ 42 ], rFim2 alone was not a good immunogen to reduce bacterial burden in lungs when administered via either the intraperitoneal or the intranasal route.
We also observed that intranasal immunization with CTB-Fim2 induced similar levels of anti-Fim2 serum IgG to those of intraperitoneal immunization. This result was not observed with the other treatments performed herein and is consistent with previously published data showing that parenteral administration is less effective than mucosal routes in inducing mucosal responses important for protection [ 53 ].
The enhanced inflammatory response in vaccinated mice, in agreement with other studies [ 57 ], may represent an enhanced host response to clear the bacteria. In addition, open the possibility to be used by other routes, even mucosal routes, as have been done with CTB fused to other antigens in preclinical studies [ 63 ] as well as in humans CTB administered by mucosal way in healthy adult volunteers, trial NCT , NIH, publication pending.
Therefore, the genetic fusion of ctb and fim2 genes provides a new promising antigen against Bordetella pertussis infection. Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper.The CTB-Fim2 presented here formed a pentameric structure that seemed to be important in relation to its adjuvant capacity [ 13 ]. On the edit page that opens, make all the corrections you can.
The most vulnerable to the disease correspond to groups of unvaccinated infants, partially vaccinated children, and persons who have completed the immunization schedule with waning immunity [ 1 ]. Elomaa, A. Please tell us what format you need. The observations from the DLS experiment indicated that only a protein heterodimer of pentamers is formed.
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These results indicate that intraperitoneal or intranasal administration of CTB-Fim2 was effective in protecting mice against intranasal challenge with B.
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