Mutant Bordetella strains and methods of use thereof
By developing the Bordetella pertussis adhesin-deficient strain BPZE1P, we have solved the challenges of attenuation and immunogenicity of existing vaccines and achieved effective immune responses and protection against respiratory inflammation without causing pathology. The protection significantly reduces the development of airway inflammation.
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example 1
[0064] Example 1 - Construction and characterization of a pertussis adhesin-deficient strain of Bordetella pertussis.
[0065] Escherichia coli DH5α, SM10 and Bordetella pertussis BPZE1, BPSM (Menozzi et al., Infect Immun [Infection and Immunity] 1994;62:769-778) and B1917 (Bart et al. Genome Announc [Genome Bulletin] 2014;2(6)). Bordetella strains were grown on Bordet-Gengou agar (BG) supplemented with 1% glycerol and 10% defibrinated sheep blood at 37°C. After growth, bacteria were harvested by scraping the plate and resuspended in phosphate buffered saline (PBS) at the desired density. For liquid cultures, Bordetella strains were grown at 37°C in a modified Stainer- Grow in Scholte medium (Imaizumi et al. Infect Immun [Infection and Immunity] 1983;41:1138-1143). The E. coli strains used for the cloning procedure were grown in LB broth or LB agar plates. Streptomycin (Sm) at 100 μg / ml, gentamicin (Gm) at 10 μg / ml, and ampicillin (Amp) at 100 μg / ml were used as needed.
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example 2
[0068] Example 2 - BPZE1P colonizes mice like BPZE1.
[0069] As previously described (Mielcarek et al., supra), with 10 6 Groups of 18 6-week-old mice were inoculated intranasally with 20 μl of PBS with live bacteria. At the indicated time points (3 hours, 3 days, 7 days, 14 days, 21 days, and 28 days), 3 mice per group were sacrificed, and lungs were harvested and homogenized to measure the total number of colony forming units (CFUs). . Statistical analysis was performed by two-way ANOVA test using post hoc comparative Bonferroni test (95% confidence interval). see figure 2 , BPZE1 and BPZE1P colonize the animals equally well. Both strains exhibited a proliferation peak 3 days after vaccination, and colonization continued for 4 weeks. No statistically significant differences were observed between these strains in their ability to colonize mouse lungs.
example 3
[0070] Example 3 - BPZE1P is as immunogenic as BPZE1 and protective against virulent Bordetella pertussis challenge.
[0071] BPZE1P-induced immunity compared to BPZE1 was measured by antibody titration of mouse immune sera after nasal vaccination. with 10 5 Groups of 8 mice were intranasally inoculated with live BPZE1 or BPZE1P. After 4 weeks, mice were bled and total IgG titers were measured against total BPSM lysates. Blood was centrifuged at 5,000 x g for 5 minutes to separate serum from cells. Antibody titers against B. pertussis were estimated using an enzyme-linked immunosorbent assay (ELISA) as previously described (Mielcarek et al., supra), lysed using total B. pertussis BPSM at 1 μg total protein per well thing. Statistical analysis was performed using GraphPad Prism software. like image 3 As shown, BPZE1 and BPZE1P vaccinated mice exhibited much higher antibody titers than naive control mice. No significant differences in antibody titers were detected betwee...
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