The O antigen enables Bordetella parapertussis to avoid Bordetella pertussis-induced immunity

Abstract

Bordetella pertussis and Bordetella parapertussis are closely related endemic human pathogens which cause whooping cough, a disease that is reemerging in human populations. Despite how closely related these pathogens are, their coexistence and the limited efficacy of B. pertussis vaccines against B. parapertussis suggest a lack of cross-protective immunity between the two. We sought to address the ability of infection-induced immunity against one of these pathogens to protect against subsequent infection by the other using a mouse model of infection. Immunity induced by B. parapertussis infection protected against subsequent infections by either species. However, immunity induced by B. pertussis infection prevented subsequent B. pertussis infections but did not protect against B. parapertussis infections. The O antigen of B. parapertussis inhibited binding of antibodies to the bacterial surface and was required for B. parapertussis to colonize mice convalescent from B. pertussis infection. Thus, the O antigen of B. parapertussis confers asymmetrical cross-immunity between the causative agents of whooping cough. We propose that these findings warrant investigation of the relative role of B. parapertussis in the resurgence of whooping cough.

FIG. 1.

B. pertussis colonization of naive and immunized mice. C57BL/6 mice were inoculated with 5 × 10⁵ CFU of B. pertussis (Bp) or B. parapertussis (Bpp). Immunized and naive mice were challenged 28 days later with 5 × 10⁵ CFU of a nalidixic acid-resistant strain of B. pertussis and sacrificed 3 days after secondary inoculation for the quantification of bacterial numbers in the nasal cavities (A), tracheae (B), and lungs (C). Values are expressed as the log₁₀ means ± standard deviations (SD). For day 3 colonization levels, the difference between each individual value and the mean log₁₀ CFU approximately 10 min after inoculation in the nasal cavities (D), tracheae (E), and lungs (F) was calculated. These data are represented as the mean changes in log₁₀ CFU ± SD. *, P < 0.05; **, P < 0.01 (compared to naive mice).

FIG. 2.

B. parapertussis colonization of naive and immunized mice. C57BL/6 mice were inoculated with 5 × 10⁵ CFU of B. pertussis (Bp) or B. parapertussis (Bpp). Immunized and naive mice were challenged 28 days later with 5 × 10⁵ CFU of a gentamicin-resistant strain of B. parapertussis and sacrificed 3 days after secondary inoculation for the quantification of bacterial numbers in the nasal cavities (A), tracheae (B), and lungs (C). Values are expressed as log₁₀ means ± standard deviations (SD). For day 3 colonization levels, the difference between each individual value and the mean log₁₀ CFU approximately 10 min after inoculation in the nasal cavities (D), tracheae (E), and lungs (F) was calculated. These data are represented as the mean changes in log₁₀ CFU ± SD. *, P < 0.05; **, P < 0.01.

FIG. 3.

IFN-γ production by splenocytes from naive or B. pertussis- or B. parapertussis-immune hosts. C57BL/6 mice were inoculated with 5 × 10⁵ CFU of B. pertussis (Bp) or B. parapertussis (Bpp). Immunized and naive mice were sacrificed 28 days postinoculation, spleens were excised, and splenocytes were exposed to media alone (hatched bars), heat-killed B. pertussis (black bars), or heat-killed B. parapertussis (white bars) for 3 days. The supernatant was collected, and cytokine ELISAs were performed to quantify the levels of IFN-γ produced by splenocytes. Values are expressed as the means ± standard deviations. *, P < 0.05; **, P < 0.01.

FIG. 4.

Recognition of B. pertussis and B. parapertussis by antibodies from convalescent-phase serum. C57BL/6 mice were inoculated with 5 × 10⁵ CFU of B. pertussis (Bp) or B. parapertussis (Bpp), and serum was collected from these mice 28 days later. ELISA was performed on serum from naive (NS), B. pertussis-infected (Bp IS), and B. parapertussis-infected (Bpp IS) mice to quantify titers of antibodies specific for B. pertussis (A) or B. parapertussis (B). The dashed line represents the lower limit of detection. Antibody titers from the ELISA are quantified as the log₁₀ means of the end point titers ± standard deviations. *, P < 0.05; **, P < 0.01. Western blots of pooled B. pertussis-induced (C) and pooled B. parapertussis-induced (D) serum were used to determine if serum raised against one species recognized specific antigens of B. pertussis, B. parapertussis, or the B. parapertussis Δwbm strain (BppΔO-ag).

FIG. 5.

Effect of passive transfer of immune serum to B. pertussis-immune mice on B. parapertussis numbers. C57BL/6 mice were inoculated with 5 × 10⁵ CFU of B. pertussis. Twenty-eight days later, these mice were passively transferred sera from naive (naive serum), B. pertussis-infected (Bp serum), or B. parapertussis-infected (Bpp serum) mice and then challenged with B. parapertussis. B. parapertussis-convalescent mice (Bpp immune) were also challenged with B. parapertussis for comparison. CFU were quantified 3 days after secondary inoculation. Numbers of bacteria are expressed as log₁₀ means ± standard deviations. *, P < 0.05; **, P < 0.01.

FIG. 6.

Antibody recognition of live B. pertussis and B. parapertussis and O-antigen-deficient B. parapertussis by immune serum. C57BL/6 mice were inoculated with 5 × 10⁵ CFU of B. pertussis (Bp) or B. parapertussis (Bpp), and serum was collected 28 days later. Sera from B. pertussis (Bp serum)- or B. parapertussis-infected mice (Bpp serum) or naive sera were examined by ELISA for their ability to bind to live B. pertussis (A) or B. parapertussis (B; white bars) or the O-antigen-deficient B. parapertussis (BppΔO-ag) (B; hatched bars). The dashed line represents the lower limit of detection. Antibody titers are expressed as the end point titers ± standard deviations. **, P < 0.01.

FIG. 7.

Ability of the O-antigen-deficient strain of B. parapertussis to colonize B. pertussis-immune hosts. C57BL/6 mice were inoculated with 5 × 10⁵ CFU of B. pertussis (Bp) or B. parapertussis (Bpp). Immunized and naive mice were challenged 28 days later with 5 × 10⁵ CFU of B. parapertussis (white bars) or the O-antigen-deficient strain of B. parapertussis (BppΔO-ag; hatched bars). Mice were sacrificed 3 days after secondary inoculation for the quantification of bacterial numbers in the nasal cavities (A), tracheae (B), and lungs (C). All values are expressed as log₁₀ means ± standard deviations. *, P < 0.05; **, P < 0.01.