Immunoglobulin A-Mediated Protection against Bordetella pertussis Infection

Infection with Bordetella pertussis, the causative agent of pertussis (whooping cough) in humans, is followed by the production of antibodies of several isotypes, including immunoglobulin A (IgA). Little is known, however, about the role of IgA in immunity against pertussis. Therefore, we studied targeting of B. pertussis to the myeloid receptor for IgA, FcalphaRI (CD89), using either IgA purified from immune sera of pertussis patients or bispecific antibodies directed against B. pertussis and FcalphaRI (CD89 BsAb). Both IgA and CD89 BsAb facilitated FcalphaRI-mediated binding, phagocytosis, and bacterial killing by human polymorphonuclear leukocytes (PMNL) and PMNL originating from human FcalphaRI-transgenic mice. Importantly, FcalphaRI targeting resulted in enhanced bacterial clearance in lungs of transgenic mice. These data support the capacity of IgA to induce anti-B. pertussis effector functions via the myeloid IgA receptor, FcalphaRI. Increasing the amount of IgA antibodies induced by pertussis vaccines may result in higher vaccine efficacy.     

Role of antibodies against Bordetella pertussis virulence factors in adherence of Bordetella pertussis and Bordetella parapertussis to human bronchial epithelial cells

Immunization with whole-cell pertussis vaccines (WCV) containing heat-killed Bordetella pertussis cells and with acellular vaccines containing genetically or chemically detoxified pertussis toxin (PT) in combination with filamentous hemagglutinin (FHA), pertactin (Prn), or fimbriae confers protection in humans and animals against B. pertussis infection. In an earlier study we demonstrated that FHA is involved in the adherence of these bacteria to human bronchial epithelial cells. In the present study we investigated whether mouse antibodies directed against B. pertussis FHA, PTg, Prn, and fimbriae, or against two other surface molecules, lipopolysaccharide (LPS) and the 40-kDa outer membrane porin protein (OMP), that are not involved in bacterial adherence, were able to block adherence of B. pertussis and B. parapertussis to human bronchial epithelial cells. All antibodies studied inhibited the adherence of B. pertussis to these epithelial cells and were equally effective in this respect. Only antibodies against LPS and 40-kDa OMP affected the adherence of B. parapertussis to epithelial cells. We conclude that antibodies which recognize surface structures on B. pertussis or on B. parapertussis can inhibit adherence of the bacteria to bronchial epithelial cells, irrespective whether these structures play a role in adherence of the bacteria to these cells.     

Role of Neutrophils in Response to Bordetella pertussis Infection in Mice

Pertussis is an acute respiratory disease caused by the bacterium Bordetella pertussis, for which humans are the only known reservoir. During infection, B. pertussis releases several toxins, including pertussis toxin (PT) and adenylate cyclase toxin (ACT), which have both been shown to play roles in promoting bacterial growth during early infection in a mouse model. Furthermore, in vitro and in vivo studies suggest that PT and ACT affect neutrophil chemotaxis and/or function, thereby altering the innate immune response. In this study we depleted animals of neutrophils to investigate whether neutrophils play a protective role during B. pertussis infection in mice. In addition, by infection with toxin-deficient strains, we investigated whether neutrophils are the main targets for PT and/or ACT activity in promoting bacterial growth. Surprisingly, we found no role for neutrophils during B. pertussis infection in naïve mice. However, in previously infected (immune) mice or in mice receiving immune serum, we observed a significant role for neutrophils during infection. Furthermore, in this immune mouse model our evidence indicates that neutrophils appear to be the main target cells for ACT, but not for PT.Pertussis, also known as whooping cough, is an acute respiratory disease caused by the bacterium Bordetella pertussis. B. pertussis infects the human respiratory tract and binds to ciliated cells in the upper and lower airways (8, 15, 27, 38). B. pertussis releases several toxins, including pertussis toxin (PT) and adenylate cyclase toxin (ACT), which have both been shown to play a role in promoting bacterial growth in the airways in a mouse model (4, 6). Furthermore, evidence suggests that both toxins have effects on cells of the immune system, including neutrophils and macrophages, which are important effectors of the innate immune response (2, 7, 28, 34-36, 40).Neutrophils play a protective role in response to infection by several different respiratory pathogens (23), including Pseudomonas aeruginosa (33), Legionella pneumophila (29), and Klebsiella pneumoniae (39). A study by Tsai et al. showed that inhibition of neutrophil recruitment in response to P. aeruginosa lung infection in mice resulted in increased bacterial loads and a decreased survival rate of the mice (33), and data reported by Ye et al. suggest that induction of neutrophil recruitment through a Th17 response is essential for protection against K. pneumoniae (39). Furthermore, neutrophils have been shown to be essential for effective clearance of lower respiratory tract Haemophilus influenzae infection in a mouse model (31).PT, which is produced exclusively by B. pertussis, has been shown by us and others to suppress several aspects of the immune response, including macrophage function, serum antibody production, and neutrophil chemotaxis (5-7, 18, 30). Previous data from our lab demonstrated a role for PT in establishing B. pertussis infection in the mouse respiratory tract (6), and we recently showed that PT inhibits neutrophil recruitment to the airways in response to infection by suppressing the release of neutrophil-attracting chemokines by resident airway cells (2).ACT, another toxin released by B. pertussis, has been described to have inhibitory effects on neutrophil function by decreasing phagocytosis and superoxide generation and inhibiting chemotaxis (10). Mobberley-Schuman et al. demonstrated that mutant strains of B. pertussis that do not produce ACT (ΔCYA) are readily ingested by human neutrophils, while wild-type (WT) strains are able to resist phagocytosis (25). ACT has been shown to inhibit both CD11b/CD18-mediated phagocytosis (25) as well as antibody-mediated phagocytosis via the Fc receptor by human neutrophils (24, 34). Opsonizing antibodies generally increase phagocytosis of pathogens by phagocytes (16), and a study by Weingart et al. showed that a ΔCYA strain of B. pertussis was efficiently phagocytosed by human neutrophils in the presence of immune serum (35). However, in the presence of ACT, opsonization of B. pertussis did not increase phagocytosis when compared to unopsonized controls (34). Evidence suggests that the increased levels of cyclic AMP in these cells lead to a decrease in phagocytosis and superoxide generation as well as inhibition of chemotaxis, thereby affecting a major part of the innate immune response (10).Based on these observations, we hypothesized that neutrophils play a role in controlling and/or clearing B. pertussis infection in the airways and that PT and ACT play roles by inhibiting neutrophil chemotaxis and function. Furthermore, we hypothesized that the absence of neutrophils would increase bacterial loads as well as the duration of the infection and that the mutant strains, ΔPT and ΔCYA, would be able to grow to WT levels in a neutropenic mouse model. Surprisingly, we found that neutrophils apparently did not play a role in controlling or clearing B. pertussis infection in naïve mice. Furthermore, neutrophils did not appear to be the main targets of PT and ACT in this model. However, in previously infected (immune) mice, the absence of neutrophils led to a significant increase in bacterial loads, and the ΔCYA strain, but not the ΔPT strain, infected neutropenic mice at WT levels, suggesting that ACT targets neutrophils in this model and that neutrophils play a role in controlling the infection.     

Crystallization of pertussigen from Bordetella pertussis.

A method is described for crystallizing pertussigen from Bordetella pertussis. The crystalline material induced histamine hypersensitivity in mice at a dose of 0.5 ng of protein and leukocytosis at a dose of 100 ng and was toxic at a dose of 429 microgram. The histamine-sensitizing activity and the toxicity were as high as ever reported.     

Dose Response of Attenuated Bordetella pertussis BPZE1-Induced Protection in Mice

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-γ) 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.Pertussis, or whooping cough, caused by the exclusively human pathogen Bordetella pertussis, is a highly contagious acute disease of the respiratory tract and is responsible for approximately 300,000 deaths in children worldwide every year. Despite the availability and intensive use of efficacious vaccines for several decades, pertussis has not been eliminated in any country. In fact, the incidence of the disease is increasing in many countries with high vaccine coverage, and whooping cough remains globally among the top 10 causes of childhood death (4). Although by far most pertussis-linked deaths occur in young infants, adolescent and adult pertussis is also a growing and often underestimated problem in countries with high vaccine coverage (33). In contrast to infant infections, many B. pertussis infections in adolescents and adults are mild or subclinical and are usually not life-threatening (13). However, infected adults and adolescents are now considered an important reservoir for the whooping cough agent, able to transmit the infection to infants before they are sufficiently protected by vaccination. This epidemiological change is likely to be due to progressive waning of vaccine-mediated immunity during adolescence. However, other epidemiological features may also potentially contribute to the increasing pertussis burden in areas of high vaccine coverage, such as adaptation of B. pertussis strains in response to vaccine-induced immunity (3, 9, 10, 26).Several strategies to solve this problem can be envisaged. As one of the potential solutions, it has been proposed to provide regular booster doses to adolescents and adults (7). However, repeated administrations of current pertussis vaccines are sometimes associated with local adverse effects, such as large swellings that may involve the entire limb (15). In addition, compliance of adolescents and adults to receiving booster doses is usually low for any vaccine (32). Maternal immunization has also been described as a potential approach to protect newborns (for a recent review, see reference 25). Nevertheless, the vaccination schedule will have to be carefully defined, both safety and efficacy of such a strategy still need to be assessed in clinical trials, and the acceptance among mothers may constitute an important hurdle. As an alternative, infant vaccination occurring as early as possible, preferably at birth, has been proposed in order to protect children during their most vulnerable period (2, 28). However, early protection by vaccination is hampered by the relative immaturity of the neonatal and infant immune system, especially of the cell-mediated immune arm (31), known to be important for protection against B. pertussis (23). In addition, optimal protection requires at least three doses of the current vaccines (6), usually given at 1- or 2-months intervals. Therefore, acceptable protection would not be achieved before 3 to 4 months, even if vaccination was started at birth.In contrast to vaccination, infection with B. pertussis is able to quickly induce a strong Th1-type immune response in very young children, characterized by the production of high levels of B. pertussis antigen-specific gamma interferon (IFN-γ) (18). Furthermore, studies of nonhuman primates have led to the conclusion that “ultimate protection against whooping cough probably best follows a live B. pertussis inoculation” (14). This has prompted us to construct an attenuated B. pertussis strain as a live vaccine candidate by genetically altering or removing three B. pertussis toxins, pertussis toxin (PTX), tracheal cytotoxin (TCT), and dermonecrotic toxin (DNT). Briefly, this strain, named BPZE1, expresses an enzymatically inactive PTX by altering two key amino acids for the enzymatic activity of the toxin, shows a 100-fold reduction in TCT activity by the replacement of the B. pertussis ampG gene with that of Escherichia coli, and does not produce DNT by the deletion of its structural gene. We showed that BPZE1 still colonizes the mouse respiratory tract and is able to provide protection against B. pertussis challenge after a single nasal administration in a mouse model (22).In this study, we investigated the dose response of a single nasal administration of BPZE1 in mice to identify the protective doses needed against challenge infection with virulent B. pertussis.     

Role of gamma interferon in natural clearance of Bordetella pertussis infection.

Using a mouse model of Bordetella pertussis infection, we have analyzed the role of gamma interferon (IFN-gamma) in bacterial clearance from the respiratory tract. Adult BALB/c mice began to clear a respiratory infection within 3 weeks postinfection, with complete resolution of infection 6 to 8 weeks postinfection. In contrast, neither adult SCID mice (which lack mature B and T lymphocytes) nor adult nude mice (which lack mature T lymphocytes) controlled B. pertussis infection, and both strains died within 3 to 5 weeks postinfection. Short-term T-cell lines generated from the draining lymph nodes of the lungs of infected BALB/c mice were found to be CD4+ and produced IFN-gamma but no detectable interleukin-4. Analyses of IFN-gamma mRNA induction in the lungs of mice following B. pertussis infection showed that in both BALB/c and C57BL/6 mice, IFN-gamma mRNA levels increased sharply by 1 week postinfection and then subsequently declined. Further exploration of a potential role for IFN-gamma demonstrated that infection of adult BALB/c mice depleted of IFN-gamma in vivo with anti-IFN-gamma monoclonal antibodies resulted in greater numbers of bacteria recovered from the lungs than in infected, control BALB/c mice, although IFN-gamma-depleted mice could subsequently clear the infection. Infection of mice which have a disrupted IFN-gamma gene resulted in bacterial clearance with a time course similar to those seen with IFN-gamma-depleted mice. These results indicate that IFN-gamma plays a role in controlling B. pertussis infection.     

Importance of holotoxin assembly in Ptl-mediated secretion of pertussis toxin from Bordetella pertussis

We examined the structural components of pertussis toxin that are required for efficient export from Bordetella pertussis via the Ptl system, a member of the type IV family of macromolecular transporters. First, we constructed a strain of B. pertussis that contains a functional Ptl system but does not produce pertussis toxin. Plasmids which express either the S1 subunit or the B oligomer were then introduced into this strain. We found that the B oligomer of the toxin is not secreted in the absence of the S1 subunit. Conversely, the S1 subunit is also not secreted by a Ptl-mediated mechanism in the absence of the B oligomer. Thus, an assembled holotoxin is required for Ptl-mediated export of pertussis toxin from B. pertussis.     

Role of immune sera in the in-vitro phagocytosis of Bordetella pertussis strains

In this study, phagocytosis of Bordetella pertussis was assessed using a human monocyte-derived macrophage line (THP-1) and immune sera from children who had received primary vaccination during the Italian clinical trial on the efficacy of two acellular three-component (PT-FHA-PRN) and one whole-cell pertussis vaccines. The results demonstrate that phagocytosis of opsonized bacteria with specific immune sera is not significantly enhanced compared with that of non-opsonized bacteria or bacteria opsonized with non-immune sera. A similar result was obtained also using B. pertussis strains showing variants of the pertactin antigen suggesting that those variations do not reduce the capability of the bacterium to invade the monocytes.     

Regulatory Role of Nitric Oxide in Neutrophil Apoptosis

Apoptosis of blood neutrophils from healthy donors was studied under conditions of cell culturing with different concentrations of H₂O₂, selective NO synthase inhibitor, and inductor of NO synthesis (L-arginine). In vitro incubation of neutrophilic leukocytes with 5 mM H₂O₂ led to activation of the apoptotic program in neutrophils, which was seen from increased content of Bax protein in the cells and increased number of apoptotic cells in the culture. Increased content of annexin-positive cells after incubation of neutrophil culture with NO synthase inhibitor suggests involvement of NO in the regulation of neutrophil apoptosis under conditions of oxidative stress, while L-arginine prevented H₂O₂-induced programmed cell death. Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 146, No. 12, pp. 646–650, December, 2008     

Regulatory Role of Nitric Oxide in Cutaneous Inflammation

Inflammation - Nitric oxide (NO), a signaling molecule, regulates biological functions in multiple organs/tissues, including the epidermis, where it impacts permeability barrier homeostasis, wound...     

Role of carbohydrate recognition domains of pertussis toxin in adherence of Bordetella pertussis to human macrophages.

Pertussis toxin (PT) and filamentous hemagglutinin can each mediate the association of Bordetella pertussis with human macrophages. Adherence via filamentous hemagglutinin leads to integrin-mediated entry and survival of the bacteria within the human cell. We determined the contribution of PT to bacterial adherence to human macrophages. Plating macrophages on wells coated with recombinant PT subunit 2 (S2) or S3 decreased PT-dependent bacterial binding by greater than 60%; S1, S4, and S5 were ineffective. S3-dependent adherence was reduced 63% +/- 8% by sialic acid, while S2-dependent adherence was reduced 53% +/- 11% by galactose. Loss of the carbohydrate recognition properties of S2 by deletion of residues 40 to 54 or site-specific mutations at Asn-93, His-47, or Arg-50 eliminated the ability of the subunit protein to competitively inhibit bacterial binding. Peptides corresponding to residues 28 to 45 of S2 and S3 competitively inhibited adherence. Treatment of macrophages with antibodies to Le(a) or Le(x) but not CD14, CD15, CD18, or HLA interfered with PT-mediated binding. Exposure of the macrophages to the B oligomer, S2, or S3 increased binding to the CD11b/CD18 integrin. These results indicate that the carbohydrate recognition domains of both S2 and S3 participate in adherence of B. pertussis to human macrophages. The PT receptor(s), as yet unidentified, appears to carry the Le(a) or Le(x) determinants and is functionally capable of modulating integrin-mediated binding to the macrophage.     

Active and Passive Immunizations with Bordetella Colonization Factor A Protect Mice against Respiratory Challenge with Bordetella bronchiseptica

Bordetella colonization factor A (BcfA) is an outer membrane immunogenic protein, which is critical for efficient colonization of the murine respiratory tract. These properties of BcfA prompted us to examine its utility in inducing a protective immune response against Bordetella bronchiseptica in a mouse model of intranasal infection. Mice vaccinated with BcfA demonstrated reduced pathology in the lungs and harbored lower bacterial burdens in the respiratory tract. Immunization with BcfA led to the generation of BcfA-specific antibodies in both the sera and lungs, and passive immunization led to the reduction of B. bronchiseptica in the tracheas and lungs. These results suggest that protection after immunization with BcfA is mediated in part by antibodies against BcfA. To further investigate the mechanism of BcfA-induced immune clearance, we examined the role of neutrophils and macrophages. Our results demonstrate that neutrophils are critical for anti-BcfA antibody-mediated clearance and that opsonization with anti-BcfA serum enhances phagocytosis of B. bronchiseptica by murine macrophages. We show that immunization with BcfA results in the production of gamma interferon and subclasses of immunoglobulin G antibodies that are consistent with the induction of a Th1-type immune response. In combination, our findings suggest that the mechanism of BcfA-mediated immunity involves humoral and cellular responses. Expression of BcfA is conserved among multiple clinical isolates of B. bronchiseptica. Our results demonstrate the striking protective efficacy of BcfA-mediated immunization, thereby highlighting its utility as a potential vaccine candidate. These results also provide a model for the development of cell-free vaccines against B. bronchiseptica.Respiratory pathogens are a major cause of morbidity and mortality in humans and animals, making the development of efficacious vaccines that protect against these infections a top priority. Bordetellae are small, aerobic, gram-negative coccobacilli that colonize the respiratory tracts of humans and animals (31). Bordetella pertussis infects only humans and causes the acute respiratory disease whooping cough (6). Bordetella parapertussis strains can be divided into two genetically distinct types: those which infect humans, causing a pertussis-like illness, and those which cause respiratory infections in sheep (22, 38). Bordetella avium infects mainly commercially grown turkeys and wild and domesticated birds (43, 45). In contrast, Bordetella bronchiseptica has a broader host range and is considered a cocontributor to a number of respiratory syndromes in agriculturally important and food-producing animals, pets, and nonhuman primates (17). B. bronchiseptica is also a primary etiological agent and/or a predisposing factor that results in porcine reproductive and respiratory disease complex, pneumonia and atrophic rhinitis in swine, infectious tracheobronchitis (i.e., kennel cough) in dogs, and bronchopneumonia in sheep, guinea pigs, rats, mice, rabbits, cats, and nonhuman primates (5, 31). According to the 2000 National Animal Health Monitoring System (NAHMS) survey, respiratory disease was the greatest cause of mortality in swine, accounting for 28.9% of nursery deaths and 39.1% of deaths in grower/finisher pigs. The annual economic impact of atrophic rhinitis and porcine reproductive and respiratory disease complex in the United States alone is estimated to be about $17 million and $40 million, respectively. B. bronchiseptica is also capable of infecting humans, mostly immunocompromised individuals with AIDS or cystic fibrosis (14, 26, 46, 52), although it was recently isolated from an immunocompetent individual (39).Currently available and proposed vaccines against this pathogen include live, attenuated, heat-killed, or genetically modified bacteria (2, 30, 32, 48, 49). Problems associated with these various whole-cell vaccination approaches include the following: persistence of the vaccine strain in animals, poor induction of an antibody response and/or protective immunity, and retention of some of the virulence characteristics by the vaccine strains (2, 30, 32, 48, 49). The genetic mutations that result in the attenuation of many of the commercially available live, attenuated vaccines are unknown, making it likely that these strains may revert to virulent forms because of survival pressures in the host, such as coinfections with other pathogenic organisms. B. bronchiseptica can predispose animals to other infectious agents or exacerbate disease symptoms. For example, B. bronchiseptica colonization leads to increased severity of canine parainfluenza virus 2 infections and predisposition of pigs and rabbits to subsequent Pasteurella multocida colonization (8, 12, 15). Infection of porcine tracheal rings with B. bronchiseptica has also been shown to enhance the adherence of P. multocida bacteria (13).Despite vaccination, animals continue to be carriers, resulting in outbreaks among herds. For laboratory animals like rats, mice, and rabbits, experimental infection with B. bronchiseptica results in a chronic and asymptomatic colonization of the upper respiratory tract. We have been able to isolate B. bronchiseptica from the rat nasopharynx even 85 days after inoculation (our unpublished results), and this bacterium has previously been reported to exist in this site for the life of the infected animals (30). Theoretically, persistent colonization of the upper respiratory tract of the animals vaccinated with live or attenuated strains can create a reservoir of infectious bacteria from which animal-animal and zoonotic transmission can occur. Although transmission of a vaccine strain to humans has not been experimentally proven, a number of such human cases have occurred in individuals exposed to infected, sick, or recently immunized farm and companion animals (20).We propose that an effective acellular vaccination regimen capable of providing long-lasting protective immunity will limit the spread of B. bronchiseptica not only among animals in a herd but also from animals to humans. For B. pertussis, there has been a shift to acellular vaccines because of the high frequency of side effects and multiple adverse reactions associated with the whole-cell vaccines (34). Similarly, development of acellular vaccines capable of protecting against B. bronchiseptica should be given a priority.BcfA (Bordetella colonization factor A) is an outer membrane protein which is positively regulated by the BvgAS signal transduction system (50). We were encouraged to examine the role of BcfA in protective immunity because our previously published research revealed its role in the colonization of the rat trachea. In addition, sera from rats infected with B. bronchiseptica specifically recognized BcfA (50). In the current report, we have evaluated the immunogenicity and protective efficacy of BcfA in an intranasal mouse model of respiratory infection. Both active and passive immunization with BcfA provided protection against subsequent intranasal challenge with B. bronchiseptica, which significantly correlated with the production of subclasses of immunoglobulin G (IgG) antibodies with high opsonic activity. Our results also suggested a role for a Th1-type cellular response in BcfA-mediated protection. Finally, we demonstrated that BcfA is expressed by multiple clinical isolates of B. bronchiseptica. Data presented in the current study underscore the potential utility of an acellular vaccine approach for B. bronchiseptica and highlight the importance of BcfA as a critical protective antigen against B. bronchiseptica infections.     

Biological activities of crystalline pertussigen from Bordetella pertussis.

We studied various biological activities of crystalline pertussigen and found that in mice as little as 0.5 ng of pertussigen induced hypersensitivity to histamine, 8 to 40 ng induced leukocytosis, 2 ng increased production of insulin, 0.1 ng increased production of immunoglobulin E and immunoglobulin G1 antibodies to hen egg albumin, 9.5 ng increased susceptibility to anaphylactic shock, and 0.5 ng increased the vascular permeability of striated muscle. We also found that in Lewis rats 20 ng of pertussigen promoted the induction of hyperacute experimental allergic encephalomyelitis. Pertussigen given intraperitoneally was toxic to mice at a dose of 546 ng. Treatment of pertussigen with glutaraldehyde eliminated this toxicity. Mice immunized with 1,700 ng of detoxified pertussigen were protected against intracerebral challenge with 3 x 10(4) viable Bordetella pertussis cells. When as little as 0.5 ng of pertussigen was given intravenously to mice, the increased susceptibility of the animals to histamine could still be detected 84 days later. The biological properties of crystalline pertussigen indicate its similarity to leukocytosis-promoting factor, Islet-activating protein, late-appearing toxic factor, and mouse-protective antigen of B. pertussis.