A murine model in which protection correlates with pertussis vaccine efficacy in children reveals complementary roles for humoral and cell-mediated immunity in protection against Bordetella pertussis

Abstract

The results of phase 3 efficacy trials have shown that acellular and whole-cell pertussis vaccines can confer protection against whooping cough. However, despite the advances in vaccine development, clinical trials have not provided significant new information on the mechanism of protective immunity against Bordetella pertussis. Classical approaches based on measurement of antibody responses to individual antigens failed to define an immunological correlate of protection. A reliable animal model, predictive of acellular and whole-cell pertussis vaccine potency in children, would facilitate an elucidation of the mechanism of immune protection against B. pertussis and would assist in the regulatory control and future development of pertussis vaccines. In this study, we have shown that the rate of B. pertussis clearance following respiratory challenge of immunized mice correlated with vaccine efficacy in children. Using this model together with mice with targeted disruptions of the gamma interferon (IFN-gamma) receptor, interleukin-4 or immunoglobulin heavy-chain genes, we have demonstrated an absolute requirement for B cells or their products in bacterial clearance and a role for IFN-gamma in immunity generated by previous infection or immunization with the whole-cell pertussis vaccine. The results of passive immunization experiments suggested that protection early after immunization with acellular pertussis vaccines is mediated by antibody against multiple protective antigens. In contrast, more complete protection conferred by previous infection or immunization with whole-cell pertussis vaccines reflected the induction of Th1 cells. Our findings suggest that the mechanism of immunity against B. pertussis involves humoral and cellular immune responses which are not directed against a single protective antigen and thus provide an explanation for previous failures to define an immunological correlate of protection.

FIG. 1

Kinetics of B. pertussis clearance from the lungs after respiratory challenge of mice immunized with Pw or Pa. Groups of 20 BALB/c mice were immunized intraperitoneally at 0 and 4 weeks with 0.2 human dose of either W Pw, PM Pw, CLI Pw, SB Pa2, SB Pa3, PM Pa2, CLL Pa5, or CB Pa3 (a, lot used in Italian trial manufactured in 1992; b, clinical lot manufactured in 1996) or with alum alone (control). Two weeks after the second immunization, mice were challenged with B. pertussis W28 and CFU counts were performed on individual lung homogenate at intervals after challenge. Results are mean (±standard error) viable B. pertussis counts from four mice per group at each time point.

FIG. 2

Correlation between bacterial clearance from the lungs following aerosol challenge of immunized mice and vaccine efficacy in children. The areas under the curves of bacterial clearance 0 to 14 days after challenge of mice immunized with a vaccine, expressed as a ratio of area under the curves for control unimmunized mice in the same experiment (as shown in Fig. 1, except area was calculated up to 14 days in all cases), were plotted against the estimated efficacy of the vaccine shown in Table 1. The efficacies of the CLI Pw and W Pw were taken as the means of the values given in Table 1.

FIG. 3

Bacterial clearance following B. pertussis respiratory challenge of immunized or convalescent gene-disrupted mice. IL-4^−/− (A) and IFN-γR^−/− (B) and the wild-type C57BL/6 and 129Sv/Ev mice were infected with B. pertussis, allowed to clear the bacteria (12 to 14 weeks), and challenged 2 weeks later. Ig^−/−, IL-4^−/− (C and E), and IFN-γR^−/− (D and F) mice and the wild-type C57BL/6 and 129Sv/Ev mice were immunized twice (weeks 0 and 4) with the W Pw (C and D) or Pa prepared from rPT, FHA, and PRN adsorbed to alum (E and F) and were challenged 2 weeks after the second immunization. Results from four experiments are mean (±standard error) CFU per lung for four to nine mice per time point.

FIG. 4

T-cell responses in immune wild-type and gene-disrupted mice. BALB/c (B/c), 129Sv/Ev (129), IFN-γR^−/−, C57BL/6 (C57BL), IL-4^−/−, or Ig^−/− mice were immunized or infected as described in the legend to Fig. 3, and responses were assessed on the day of challenge. Spleen cells were stimulated in vitro with killed B. pertussis, inactivated PT, FHA, or PRN, and supernatants were assessed for IL-5 and IFN-γ by immunoassay. Results are mean responses for four mice assessed individually in triplicate.

FIG. 5

Anti-PT IgG subclasses in immune wild-type and gene-disrupted mice. Mice were immunized or infected as described in the legends to Fig. 3 and 4, and antibody responses were tested by enzyme-linked immunosorbent assay on the day of challenge. Results are reciprocal endpoint titers.

FIG. 6

Clearance of B. pertussis following active or passive immunization with Pw or Pa. (A) Active immunization. BALB/c mice were immunized twice (0 and 4 weeks) with W Pw or Pa (rPT, FHA, and PRN). (B) Passive immunization. Serum was prepared from BALB/c mice immunized three times (0, 3, and 6 weeks) with Pa or Pw, and recipient mice were injected with 0.1 ml 4 h before respiratory challenge. Results are mean (±standard error) CFU per lung for four mice per time point.

FIG. 7

Multiple protective antigens of B. pertussis. (A) Active immunization. Mice were immunized twice (0 and 4 weeks) with 5 μg of either FHA, PRN, rPT, rPT and FHA, or rPT, FHA, and PRN adsorbed to alum or with alum only (control) and were challenged 2 weeks later. (B) Passive immunization. Sera were recovered from mice 2 weeks after three immunizations (0, 3, and 6 weeks) with rPT, FHA, or PRN only and 0.1 ml of each antiserum, in the combinations described for active immunization, injected (intravenously) into naive BALB/c mice, which were challenged 4 h later. Results are mean (±standard error) CFU per lung for four mice per time point.