INFECTION AND IMMUNITY, Oct. 2007, p. 4972–4979 Vol. 75, No. 10 0019-9567/07/$08.00ϩ0 doi:10.1128/IAI.00763-07 Copyright © 2007, American Society for Microbiology. All Rights Reserved. The O Antigen Enables Bordetella parapertussis To Avoid Bordetella pertussis-Induced Immunityᰔ Daniel N. Wolfe,1 Elizabeth M. Goebel,2 Ottar N. Bjornstad,3 Olivier Restif,4 and Eric T. Harvill1* Department of Veterinary and Biomedical Sciences, The Pennsylvania State University, 115 Henning Building, University Park, Pennsylvania 168021; Huck Institute of Life Sciences, The Pennsylvania State University, 519 Wartik Lab, University Park, Pennsylvania 168022; Departments of Entomology and Biology, The Pennsylvania State University, 501 Agricultural Sciences and Industry Building, University Park, Pennsylvania 168023; and Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 OES, Great Britain4 Received 5 June 2007/Returned for modification 18 July 2007/Accepted 1 August 2007 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 Downloaded from 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 iai.asm.org 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. at Penn State Univ on September 20, 2007 Bordetella pertussis and Bordetella parapertussis are the caus- progenitor, Bordetella bronchiseptica, and share the majority of ative agents of whooping cough (21, 37), a disease that is their virulence factors (42), cross-immunity could be expected. endemic worldwide despite extensive vaccination efforts (48). Furthermore, genetic comparisons of B. pertussis and B. para- Upon the introduction of B. pertussis vaccines more than 50 pertussis to B. bronchiseptica suggest that B. parapertussis years ago, the incidence of whooping cough greatly declined in emerged as a pathogen more recently than B. pertussis (28, 35, developed countries (9). However, the incidence of this disease 42). Since the mean age at the time of primary B. pertussis has been steadily rising over the past 10 to 20 years in popu- infection for unvaccinated children is less than 5 years (9) and lations despite excellent vaccine coverage (7, 8, 12, 49, 56). infection-induced immunity is protective 5 to 10 years (60), it Although disease caused by B. parapertussis may be less is likely that B. parapertussis successfully emerged in popula- severe than that caused by B. pertussis, infections by these tions that had some level of immunity to B. pertussis (4). bacteria are clinically indistinguishable except for an acute Although adapted to humans, both organisms efficiently col- lymphocytosis that is observed only upon infection of naive onize and rapidly grow throughout the murine respiratory tract hosts by B. pertussis (3, 21, 36, 41). Differentiating between but are ultimately eliminated from the lower respiratory tract these pathogens requires costly laboratory work that does not by B- and T-cell-dependent immunity (24, 25, 27, 62). Wa- affect treatment decisions (37), making differential diagnoses tanabe and Nagai suggested that an immune response induced rare. When specifically investigated, B. parapertussis has been by one species conferred efficient protection against infections found to cause anywhere from less than 1 percent to greater by either species (58). However, bacterial numbers were quan- than 95 percent of whooping cough cases (reviewed in refer- tified only 2 weeks postinoculation, when bacterial numbers ence 59). Thus, it is unclear how many of the estimated 50 were significantly reduced even in the respiratory tracts of million annual cases of whooping cough (10) may be due to B. naive hosts (25, 58). Additionally, the strain used in that study parapertussis and whether or not this pathogen is contributing B. pertussis to the resurgence of this disease. (18-323) is quite distinct from other strains, as The coexistence of these two species in human populations shown by multilocus enzyme electrophoresis and multilocus creates a paradox to the ecological theory that two closely sequence typing (13, 55), and may actually be more closely related immunizing pathogens cannot occupy the same host related to B. bronchiseptica and B. parapertussis strains than population if immunity is cross-protective (1, 14, 16, 30). Be- other B. pertussis strains (2, 40). These problems, along with cause B. pertussis and B. parapertussis evolved from a common clinical studies showing that B. pertussis vaccines poorly protect against B. parapertussis and that these two organisms coexist in the same populations, led us to investigate cross-protective * Corresponding author. Mailing address: Department of Veteri- immunity between these two pathogens. nary and Biomedical Sciences, The Pennsylvania State University, 115 We sought to determine whether or not B. pertussis and B. Henning Building, University Park, PA 16802. Phone: (814) 863-8522. Fax: (814) 863-6140. E-mail: [email protected]. parapertussis induced effective cross-immunity using the se- ᰔ Published ahead of print on 13 August 2007. quenced prototype strains Tohama I (B. pertussis) (42) and 12822 4972 VOL. 75, 2007 B. PARAPERTUSSIS EVADES CROSS-IMMUNITY 4973 (B. parapertussis) (42), which are indistinguishable from current Western blots. Westerns blotting was performed on lysates containing 1 ϫ 108 isolates by multilocus enzyme electrophoresis and multilo- CFU of heat-killed B. pertussis or B. parapertussis. Lysates were run on 7% cus sequence typing (13, 55). B. parapertussis-induced im- sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels in denaturing conditions. Membranes were probed with serum from B. pertussis-orB. para- munity protected against both species; however, B. pertussis- pertussis-infected mice at a 1:100 dilution and goat anti-mouse (immunoglobulin induced immunity protected only against B. pertussis infection. heavy plus light chains) horseradish peroxidase-conjugated antibody (Southern The asymmetrical cross-immunity appeared to be the result of Biotech, Birmingham, AL) (at a dilution of 1:15,000) as the detector antibody. inefficient binding of B. pertussis-induced antibodies to B. para- The membrane was visualized with ECL Western blotting detection reagents (Amersham Biosciences, Piscataway, NJ). pertussis. O antigen prevented B. pertussis-induced antibodies Statistical analysis. The means Ϯ standard deviations (error bars) were de- from binding to B. parapertussis in vitro and from clearing the termined for CFU, IFN-␥ production, and antibody titers. Two-tailed, unpaired bacteria from the respiratory tract in vivo. Together these data Student t tests were used to determine statistical significance between groups. provide a molecular basis for the ability of B. parapertussis to Results were also analyzed by nonparametric Mann-Whitney tests with similar avoid B. pertussis-induced immunity, an ability that may be critical significance. All experiments were performed at least twice with similar results. to the coexistence of these pathogens in human populations. RESULTS MATERIALS AND METHODS Cross-immunity between B. pertussis and B. parapertussis is Bacterial strains and growth. B. pertussis strains 536 and 536NaI (from Duncan asymmetrical. The coexistence of B. pertussis and B. paraper- Maskell, University of Cambridge, United Kingdom) are streptomycin- and Downloaded from nalidixic acid-resistant derivatives of Tohama I, respectively (51). B. parapertussis tussis in humans led us to assess the ability of these pathogens strain 12822 has been described previously (20), and 12822G is a gentamicin- to induce cross-protective immunity. Although considered hu- resistant derivative of the parent strain (62). B. parapertussis strains CN2591 man-adapted pathogens, both species efficiently colonize the (wild type) and CN2591⌬wbm (O antigen deficient) have previously been de- murine respiratory tract (17, 18) and induce sterilizing protec- scribed (44). All were maintained on Bordet-Gengou agar (Difco) containing tive immunity (25), allowing us to test cross-protection in a 10% defibrinated sheep blood (Hema Resources) and appropriate antibiotics. Liquid-culture bacteria were grown at 37°C overnight on a roller drum to mid-log mouse model. Mice were left uninfected or inoculated with B. phase in Stainer-Scholte broth. pertussis or B. parapertussis. Twenty-eight days later, when iai.asm.org Animal experiments. C57BL/6 mice were obtained from Jackson Laboratories strong immune responses had been generated