Dose response of attenuated Bordetella pertussis BPZE1-induced protection in mice

Abstract

Despite the availability of efficacious vaccines, the incidence of whooping cough is still high in many countries and is even increasing in countries with high vaccine coverage. Most severe and life-threatening pertussis cases occur in infants who are too young to be sufficiently protected by current vaccine regimens. As a potential solution to this problem, we have developed an attenuated live Bordetella pertussis vaccine strain, named BPZE1. Here, we show that after a single administration, BPZE1 induces dose-dependent protection against challenge with virulent B. pertussis in low-dose and in high-dose intranasal mouse lung colonization models. In addition, we observed BPZE1 dose-dependent antibody titers to B. pertussis antigens, as well as cell-mediated immunity, evidenced by the amounts of gamma interferon (IFN-gamma) released from spleen cells upon stimulation with B. pertussis antigens. These two parameters may perhaps be used as readouts in clinical trials in humans that are currently being planned.

Figures

FIG. 1.

In vivo and in vitro safety assessments following BPZE1 inoculation. (a) Weight gain monitoring of 3-week-old BALB/c mice intranasally inoculated with 1 × 106 CFU of B. pertussis BPZE1 (open circles) compared to noninfected mice (closed circles). (b) Histological analysis of lungs from BPZE1- or BPSM-infected mice compared to controls given PBS. Five days after infection, the lungs were aseptically removed and fixed in formaldehyde. Sections were stained with toluidine blue and examined by light microscopy. Original magnification, ×10. (c) Cytotoxicity assessment of BPZE1. Human pulmonary epithelial A549 and monocytic THP-1 cells were exposed for 5 h to live B. pertussis BPZE1 or to E. coli K-12 at the indicated bacterium/cell ratio (MOI). After extracellular bacteria were removed, infected cells were reincubated in fresh medium for 1 (white bars), 3 (gray bars), and 6 (black bars) days before determination of cell viability by trypan blue exclusion.

FIG. 2.

Lung colonization by decreasing the doses of BPZE1. Eight-week-old BALB/c mice were intranasally inoculated with attenuated B. pertussis BPZE1, with doses ranging from 1 × 106 (filled square), 1 × 105 (open diamond), 1 × 104 (filled triangle), 1 × 103 (open circle), or 5 × 102 (filled diamond) bacteria/mouse. The results are expressed as the number of mean CFU (± the number of standard errors of the mean CFU) from three to four mice per group and are representative of two similar experiments. The horizontal dotted line represents the limit for bacterial counts.

FIG. 3.

Serum antibody responses of BPZE1-vaccinated mice. (a) Mice were first intranasally immunized with the highest dose (106 CFU) of BPZE1, and the kinetics of serum antibody responses against total B. pertussis extract was measured during 1 month. (b to d) Mice were then intranasally immunized with the indicated doses of BPZE1, and serum antibody titers against total B. pertussis extract (b), FHA (c), and PTX (d) were measured 2 months later by ELISA. The results are expressed as mean titers (± standard errors of the mean titers) from three to four mice per group and are representative of two similar experiments. P values of <0.05 (*) compared to naïve mice were considered significant.

FIG. 4.

Antibody responses in bronchoalveolar lavage fluids of BPZE1-vaccinated mice to B. pertussis antigens. Mice were immunized intranasally with the highest dose (106 CFU) of BPZE1 or intraperitoneally with one-fifth of acellular pertussis vaccine (aPv) (Tetravac; Aventis-Pasteur, France). Bronchoalveolar lavage fluids were obtained 2 months later, and antibody responses to a total B. pertussis extract were measured by ELISA. Results are expressed as mean titers (± standard errors of the mean titers) from three to four mice per group. P values of <0.05 (*) compared to naïve mice were considered significant.

FIG. 5.

IFN-γ production in culture supernatants of splenocytes from BPZE1-vaccinated mice upon stimulation with FHA, PTX, or total B. pertussis extract. (a) Eight-week-old BALB/c mice were intranasally immunized with 1 × 106 CFU of BPZE1 (filled columns) or with two doses of acellular pertussis vaccine (open columns), and IFN-γ concentrations were measured in the culture supernatant of the splenocytes harvested 6 weeks later and stimulated with B. pertussis antigens. (b, c) Mice were immunized with the indicated doses of BPZE1, and IFN-γ responses were measured after stimulation with FHA (b; open columns), PTX (b; filled columns), or total B. pertussis extract (c). The results are expressed as mean values (± standard errors of the mean values) for four mice per group tested in triplicate. P values of <0.05 (*) compared to naïve mice were considered significant.

FIG. 6.

Protection against challenge infection with wild-type B. pertussis. Two months after vaccination with the indicated doses of BPZE1, mice were challenged with a low dose (5 × 104 CFU) (a) or a high dose (5 × 106 CFU) (b) of B. pertussis BPSM. Lung CFU counts were determined 3 h (open columns) or 7 days (filled columns) after challenge. The results are expressed as the number of mean CFU (± the number of standard errors of the mean CFU) from three to four mice per group and are representative of two similar experiments. The horizontal dotted line represents the detection limit for bacterial counts. P values of <0.05 (*) compared to naïve mice were considered significant.

FIG. 7.

Anamnestic responses to FHA and PTX in BPZE1-vaccinated mice. Eight-week-old BALB/c mice were immunized with indicated doses of BPZE1 and challenged 2 months later with 5 × 106 CFU of B. pertussis BPSM. Antibody titers against FHA (a) and PTX (b) were measured at the time of challenge infection and 7 days later, with three to four mice per group. The results are expressed as the difference between the mean titers at day 0 and those at day 7 after challenge.