A live attenuated Bordetella pertussis candidate vaccine does not cause disseminating infection in gamma interferon receptor knockout mice

Abstract

Bordetella pertussis is the cause of whooping cough and responsible for 300,000 infant deaths per annum. Current vaccines require 6 months to confer optimal immunity on infants, the population at highest risk. Recently, an attenuated strain of B. pertussis (BPZE1) has been developed to be used as a low-cost, live, intranasal, single-dose vaccine for newborns. Preclinical proof of concept has been established; however, it is necessary to evaluate the safety of BPZE1, especially in immunodeficient models, prior to human clinical trials. Here, the preclinical safety of BPZE1 was examined in well-characterized murine models. Immunocompetent and gamma interferon (IFN-gamma) receptor knockout mice were challenged by aerosol with either virulent B. pertussis or BPZE1. The two strains colonized the lung at equal levels, but inflammation was associated with carriage of only virulent bacteria. Virulent bacteria disseminated to the liver of IFN-gamma receptor-deficient mice, resulting in atypical pathology. In contrast, attenuated BPZE1 did not disseminate in either immunocompetent or immunodeficient mice and did not induce atypical pathology. In neonatal challenge models, virulent B. pertussis infection resulted in significant mortality of both immunodeficient and immunocompetent mice, whereas no mortality was observed for any neonatal mice challenged with BPZE1. BPZE1 was shown to elicit strong IFN-gamma responses in mice, equivalent to those elicited by the virulent streptomycin-resistant B. pertussis Tohama I derivative BPSM, also inducing immunoglobulin G2a, a process requiring TH1 cytokines in mice. These data indicate that a live attenuated whooping cough vaccine candidate shows no signs of disseminating infection in preclinical models but rather evokes an immunological profile associated with optimal protection against disease.

Figures

FIG. 1.

Attenuated BPZE1 and virulent BPSM colonize lungs at similar levels. 129/Sv (closed symbols) and IFN-γR KO (open symbols) mice were challenged by aerosol with either BPSM (squares) or BPZE1 (circles) such that colonization of between 4 × 105 and 7 × 105 CFU/lung was achieved. Groups of mice were sacrificed at intervals after challenge, and the number of viable bacteria was estimated by performing colony counts on individual lung homogenates. Results are presented as mean (±SEM) CFU in the lungs, detected in triplicate from three mice at each time point and for each group. SEMs are present for all points but may be masked by the symbol at some data points.

FIG. 2.

Virulent B. pertussis BPSM (A, C, E, G, I, and K) induces pathology not seen with attenuated BPZE1 (B, D, F, H, J, and L) in the lungs of 129/Sv (A, B, E, F, I, and J) or IFN-γR KO (C, D, G, H, K, and L) mice, at day 10, 14, or 21 postchallenge. Periairway/perivascular lymphoid cuffing was noted to various degrees in the lungs of 129/Sv (E and F) or IFN-γR KO (G and H) mice on day 14 after infection with either BPSM (E and G) or BPZE1 (F and H), indicative of the formation of bronchus-associated lymphoid tissue hyperplasia (b). BPZE1-challenged mice showed reduced levels of pulmonary inflammation (PI) and inflammatory exudate (e) compared to those of mice challenged with BPSM. High levels of pathology (+++) are noted at day 21 postinfection in 129/Sv (I and J) and IFN-γR KO (K and L) mice challenged with BPSM (I and K), whereas no airway damage was observed (−/+) in BPZE1-challenged mice (J and L). Original magnification, ×200 (A to D) or ×100 (E to L). Lung tissue was fixed, embedded, and stained using hematoxylin and eosin. Histology was scored using a previously described semiquantitative system (14).

FIG. 3.

Priming with either virulent B. pertussis BPSM or attenuated BPZE1 induced a TH1-polarized response. Cell-mediated immune responses were examined from spleen cell cultures after sham inoculation, virulent BPSM infection, or attenuated BPZE1 challenge. IFN-γ (closed bars) and IL-5 (open bars) recall responses were measured in spleen cell culture supernatant after restimulation with inactivated BPSM at 1 × 104 CFU/ml. Determinations were made from four mice, and each was carried out in triplicate; results are expressed as means (±SEM).

FIG. 4.

(A) BPZE1 does not cause lethal infection in neonatal KO mice. Neonate IFN-γR KO (open symbols) or 129/Sv (closed symbols) mice (less than 7 days) were infected with either virulent B. pertussis BPSM (squares) or BPZE1 (circles). Mice were observed over a period of 35 days, and survival was recorded. *, note that BPZE1-challenged 129/Sv mice showed 100% survival but that the curve has been offset from 100% for clarity in this figure. (B) BPZE1 did not disseminate to the liver of neonatal 129/Sv (wild-type) or IFN-γR KO mice. Neonatal 129/Sv and IFN-γR KO mice were challenged with either virulent BPSM or attenuated strain BPZE1, mice were then sacrificed at days 7 and 10, and lungs and livers were removed and examined for B. pertussis colonization. Dissemination was reduced in 129/Sv mice compared to IFN-γR KO mice challenged with BPSM. Results are presented as mean (±SEM) CFU in the liver, detected individually from five mice from each group.