Use of a genetically defined double mutant strain of Bordetella bronchiseptica lacking adenylate cyclase and type III secretion as a live vaccine

Abstract

While most vaccines consisting of killed bacteria induce high serum antibody titers, they do not always confer protection as effective as that induced by infection, particularly against mucosal pathogens. Bordetella bronchiseptica is a gram-negative respiratory pathogen that is endemic in many nonhuman mammalian populations and causes substantial disease in a variety of animals. At least 14 different live attenuated vaccines against this pathogen are available for use in a variety of livestock and companion animals. However, there are few published data on the makeup or efficacy of these vaccines. Here we report the use of a genetically engineered double mutant of B. bronchiseptica, which lacks adenylate cyclase and type III secretion, as a vaccine candidate. This strain is safe at high doses, even for highly immunocompromised animals, and induces immune responses that are protective against highly divergent B. bronchiseptica strains, preventing colonization in the lower respiratory tract and decreasing the bacterial burden in the upper respiratory tract. This novel B. bronchiseptica vaccine candidate induces strong local immunity while eliminating damage caused by the two predominant cytotoxic mechanisms.

FIG. 1.

Lethality of B. bronchiseptica strains in susceptible mice. Groups of 5 to 10 (A) TLR4^def or (B) TNF-α^−/− mice were inoculated intranasally with approximately 5 × 10³, 5 × 10⁴, or 5 × 10⁵ CFU of RB50 or 5 × 10⁵ CFU of AVS in a 50-μl volume, as indicated.

FIG. 2.

Lung pathology in susceptible mouse strains. Groups of four to six WT, TLR4^def, or TNF-α^−/− mice were inoculated intranasally with approximately 5 × 10⁵ CFU of either RB50 or AVS in a 50-μl volume, as indicated. On day 3 postinoculation, the trachea and lungs were excised, inflated with 10% formaldehyde, sectioned, stained, and examined by a veterinary pathologist blinded to experimental treatment. (A) Pathology scores. (B) Lung histology pictures. *, P value of <0.05.

FIG. 3.

AVS protection in susceptible mouse strains. Groups of four (A) TLR4^def or (B) TNF-α^−/− mice were inoculated intranasally with approximately 5 × 10⁵ CFU of AVS in a 50-μl volume. On day 49 postinoculation, the mice were then challenged with approximately 5 × 10⁵ CFU of RB50 in a 50-μl volume. On day 52, the mice were sacrificed, and the numbers of RB50 CFU in the nasal cavity, trachea, and lungs were measured. The dashed line indicates the limit of detection. *, P value of <0.05. Error bars indicate standard errors.

FIG. 4.

AVS colonization in WT mice. Groups of four to six WT mice were inoculated intranasally with approximately 5 × 10⁵ CFU of either RB50 or AVS in a 50-μl volume, as indicated. Bacterial numbers were measured in the nasal cavity, trachea, and lungs on days 0, 3, 7, 10, 28, and 56 postinoculation. The dashed line indicates the limit of detection. *, P value of <0.05. Error bars indicate standard errors.

FIG. 5.

AVS-induced antibody response and its effect on RB50 colonization. Groups of three or four WT mice were intranasally inoculated with approximately 5 × 10⁵ CFU of either WT RB50 or AVS in a 50-μl volume, as indicated. A group of four WT mice were rechallenged with AVS on day 49 postinoculation. Sera (IS) (A) and lung homogenates (LH) (B) were collected on day 49 postinoculation or day 3 post-secondary challenge. B. bronchiseptica-specific antibody titers were measured by ELISA. (C) Groups of four WT mice were injected intraperitoneally with 200 μl of either naïve serum or immune serum (IS) raised against RB50 or AVS, as indicated, and then intranasally inoculated with approximately 5 × 10⁵ CFU of RB50 in a 50-μl volume. Bacterial numbers in the lungs, trachea, and nasal cavity were measured on day 3 postinoculation and -transfer. The dashed line indicates the limit of detection. *, P value of <0.05. Error bars indicate standard errors.

FIG. 6.

AVS-induced splenocyte production of IFN-γ, IL-10, and IL-4. Spleens from groups of three or four naïve, AVS-inoculated, or RB50-inoculated WT mice were restimulated in triplicate with heat-killed RB50 (MOI of 5) for 3 days. IFN-γ, IL-10, and IL-4 production was determined by sandwich ELISA. *, P value of <0.05. Error bars indicate standard errors.

FIG. 7.

Intranasal vaccination with AVS protects WT mice. Groups of four WT mice were vaccinated intranasally with AVS. On day 49 postvaccination, the mice were inoculated intranasally with approximately 5 × 10⁵ CFU of AVS, RB50, 1127, or 253, as indicated, in a 50-μl volume, and bacterial numbers were measured at 3 days postinoculation. The dashed line indicates the limit of detection. *, P value of <0.05. Error bars show standard errors.

FIG. 8.

Intranasal vaccination with AVS induces protective immunity against Bordetella pertussis and Bordetella parapertussis in the lower respiratory tract. WT mice were vaccinated intranasally with approximately 100 CFU of AVS in a 5-μl volume. On day 49 postvaccination, the mice were inoculated intranasally with approximately 5 × 10⁵ CFU of either (A) B. pertussis or (B) B. parapertussis in a 50-μl volume. Bacterial numbers were measured at 3 days postinoculation. The dashed line indicates the limit of detection. *, P value of <0.05. Error bars indicate standard errors.