Bordetella parapertussis Survives inside Human Macrophages in Lipid Raft-Enriched Phagosomes

Bordetella parapertussis is a human pathogen that causes whooping cough. The increasing incidence of B. parapertussis has been attributed to the lack of cross protection induced by pertussis vaccines. It was previously shown that B. parapertussis is able to avoid bacterial killing by polymorphonuclear leukocytes (PMN) if specific opsonic antibodies are not present at the site of interaction. Here, we evaluated the outcome of B. parapertussis innate interaction with human macrophages, a less aggressive type of cell and a known reservoir of many persistent pathogens. The results showed that in the absence of opsonins, O antigen allows B. parapertussis to inhibit phagolysosomal fusion and to remain alive inside macrophages. The O antigen targets B. parapertussis to lipid rafts that are retained in the membrane of phagosomes that do not undergo lysosomal maturation. Forty-eight hours after infection, wild-type B. parapertussis bacteria but not the O antigen-deficient mutants were found colocalizing with lipid rafts and alive in nonacidic compartments. Taken together, our data suggest that in the absence of opsonic antibodies, B. parapertussis survives inside macrophages by preventing phagolysosomal maturation in a lipid raft- and O antigen-dependent manner. Two days after infection, about 15% of macrophages were found loaded with live bacteria inside flotillin-enriched phagosomes that had access to nutrients provided by the host cell recycling pathway, suggesting the development of an intracellular infection. IgG opsonization drastically changed this interaction, inducing efficient bacterial killing. These results highlight the need for B. parapertussis opsonic antibodies to induce bacterial clearance and prevent the eventual establishment of cellular reservoirs of this pathogen.     

The O Antigen Is a Critical Antigen for the Development of a Protective Immune Response to Bordetella parapertussis

Despite excellent vaccine coverage in developed countries, whooping cough is a reemerging disease that can be caused by two closely related pathogens, Bordetella pertussis and B. parapertussis. The two are antigenically distinct, and current vaccines, containing only B. pertussis-derived antigens, confer efficient protection against B. pertussis but not against B. parapertussis. B. pertussis does not express the O antigen, while B. parapertussis retains it as a dominant surface antigen. Since the O antigen is a protective antigen for many pathogenic bacteria, we examined whether this factor is a potential protective antigen for B. parapertussis. In a mouse model of infection, immunization with wild-type B. parapertussis elicited a strong antibody response to the O antigen and conferred efficient protection against a subsequent B. parapertussis challenge. However, immunization with an isogenic mutant lacking the O antigen, B. parapertussis Δwbm, induced antibodies that recognized other antigens but did not efficiently mediate opsonophagocytosis of B. parapertussis. The passive transfer of sera raised against B. parapertussis, but not B. parapertussis Δwbm, reduced B. parapertussis loads in the lower respiratory tracts of mice. The addition of 10 μg of purified B. parapertussis lipopolysaccharide (LPS), which contains the O antigen, but not B. parapertussis Δwbm LPS drastically improved the efficacy of the acellular vaccine Adacel against B. parapertussis. These data suggest that the O antigen is a critical protective antigen of B. parapertussis and its inclusion can substantially improve whooping cough vaccine efficacy against this pathogen.Bordetella pertussis and B. parapertussis are the causative agents of whooping cough, resulting in approximately 50 million cases and 300,000 deaths annually worldwide (28). While whooping cough is considered by the CDC to be a reemerging disease (5), the relative incidences of B. pertussis and B. parapertussis are not clear (50). It is known, however, that the resurgence of whooping cough roughly correlates with the introduction of acellular pertussis vaccines (5). These vaccines contain only B. pertussis-derived antigens and confer little to no protection against B. parapertussis (9, 14, 15, 23, 27, 28). Current acellular pertussis vaccines contain some combination of filamentous hemagglutinin, pertactin, and fimbriae 2 and 3, all of which are expressed by both B. pertussis and B. parapertussis, and pertussis toxin, which is B. pertussis specific (33, 34). Based on genome sequences, the levels of amino acid sequence identity between B. pertussis and B. parapertussis filamentous hemagglutinin, pertactin, and fimbria 2 and 3 proteins are about 98, 91, 71, and 92% (35), and antibodies raised against these antigens from B. pertussis cross-react with B. parapertussis (17, 31). However, immunization with purified B. pertussis filamentous hemagglutinin or pertactin does not confer protection against B. parapertussis (17). B. pertussis fimbriae confer some protection against B. parapertussis, but at much lower levels than they protect against B. pertussis (52). Based on these observations and the fact that B. parapertussis infection induces protective immunity to itself (56, 58), we hypothesized that the lack of protective antigens from B. parapertussis may be part of the reason why current whooping cough vaccines are ineffective against this bacterium.Although B. pertussis and B. parapertussis are very closely related (8, 35, 48), they differ in the structure of their lipopolysaccharides (LPS) (1, 2, 39, 40, 47). B. pertussis produces a lipooligosaccharide containing lipid A and a branched-chain core oligosaccharide with a complex trisaccharide modification but lacks the O antigen due to a natural deletion of the wbm locus responsible for its synthesis (39, 47). B. parapertussis LPS is similar to B. pertussis LPS but lacks the trisaccharide modification and includes an O antigen (39, 40). In addition to conferring serum resistance by inhibiting C3 deposition onto the surfaces of bacteria (11), the O antigen enables B. parapertussis to avoid B. pertussis-induced immunity by preventing antibody binding to cross-reactive antigens on the surfaces of B. parapertussis cells (56, 59). Since the O antigen is one dominant surface antigen recognized by B. parapertussis immune sera (56) and has been shown previously to be a protective antigen of various pathogenic bacteria (22, 36), we hypothesized that the O antigen is a protective antigen of B. parapertussis.To assess the role of the O antigen in the generation of an adaptive immune response to B. parapertussis, the immunity and protection generated by B. parapertussis infection or vaccination were compared to those generated by an isogenic mutant of B. parapertussis lacking the O antigen (Δwbm) (39). Animals immunized with B. parapertussis, but not B. parapertussis Δwbm, were protected against subsequent challenge with B. parapertussis. Mice immunized with B. parapertussis Δwbm were also deficient in the production of B. parapertussis-specific antibodies, and sera collected from these mice were less effective at reducing B. parapertussis colonization upon passive transfer than sera raised against B. parapertussis. The inclusion of LPS from B. parapertussis, but not from B. parapertussis Δwbm, rendered the acellular B. pertussis vaccine Adacel efficacious against B. parapertussis challenge. Together, these data indicate that the O antigen is an important protective antigen of B. parapertussis.     

Mixed outbreak ofBordetella pertussis andBordetella parapertussis infection in Finland

The epidemiology of whooping cough in a vaccinated population was studied during an outbreak of paroxysmal cough in an elementary school with 258 pupils in Turku, Finland. Nasopharyngeal specimens for isolation ofBordetella pertussis and/or sera for ELISA detection of antipertussis immunoglobulin M, A and G antibodies were taken from 94 % of children who were prospectively followed for two months.Bordetella pertussis was isolated in six patients, and 17 culture-positive cases withBordetella parapertussis were identified. Patients withBordetella pertussis orBordetella parapertussis were found simultaneously in the same classrooms. Comparison of immunoglobulin M responses toBordetella pertussis andBordetella parapertussis was used for differential diagnosis of these two infections. Twenty-six cases with pertussis and 27 cases with parapertussis were diagnosed. The results of this prospective study suggest thatBordetella parapertussis is a more common etiologic agent of mild respiratory tract infection among vaccinated school-aged children than is generally recognised. The possibility thatBordetella pertussis was converted toBordetella parapertussis during this outbreak is discussed.     

Monitoring of Bordetella isolates circulating in Saint Petersburg, Russia between 2001 and 2009

Bordetella isolates in the Saint Petersburg region have been monitored since 1998. Over the past ten years, concomitant with the increase in pertussis whole-cell vaccine coverage, the incidence of whooping cough has decreased. However, this decrease exists only for Bordetella pertussis infections, as the incidence of Bordetella parapertussis confirmed cases has remained stable, suggesting that pertussis-vaccine-induced immunity is not protective against parapertussis, as expected. B. pertussis and B. parapertussis clinical isolates were analyzed using serotyping, immunoblotting, pulsed-field gel electrophoresis of chromosomal DNA (after digestion with XbaI) and sequencing of virulence genes. The bacterial population is now similar to that observed in other European countries.     

A comparison of passive protection tests against intranasal and intracerebral challenges with Bordetella pertussis

A survey of a number of rabbit antisera to Bordetella pertussis revealed the existence of two distinct antibodies, one passively protecting mice against lethal infection by the intracerebral route, the other passively protecting mice against lethal infection by the intranasal route. Neither is the antitoxin. Antisera against most, if not all, S forms of B. pertussis contain both types of protective antibody, and so to a lesser extent do B. parapertussis and B. bronchisepticus. Neither of the protective antigens is an agglutinogen, or the haemagglutinin. The two antigens can also be distinguished by active protection tests. Extensive investigations, however, had not led to an in vitro test for either of the protective antigens or their antibodies that would replace the mouse test.

The practical importance of the two distinct antigens and their antibodies is shown by the fact that assay by the intranasal and intracerebral routes of challenge will not arrange vaccines in the same order of potency either in active or in passive protection tests.

     

The pentameric structure of IgM is necessary to enhance opsonization of Bacteroides thetaiotaomicron and Bacteroides fragilis via the alternative complement pathway

Studies were conducted to investigate the mechanisms by which natural IgM antibodies act together with the alternative complement pathway to promote opsonization and adherence of encapsulatedBacteroides thetaiotaomicronandBacteroides fragilisto polymorphonuclear leukocytes (PMN). A model system consisting of the six isolated proteins of the alternative pathway was used. A comparison of the opsonic effects of pentameric and monomeric forms of isolated normal IgM demonstrated that, although the monomeric form bound toBacteroidesas effectively as the pentameric form and promoted complement deposition to the same extent, it was unable to enhance alternative pathway-dependent opsonization and adherence ofBacteroidesto PMN. When opsonization was performed in two steps with pentameric IgM added either before or after alternative pathway components, a marked enhancement of adherence to PMN was observed only in the former case, suggesting IgM must act prior to complement to be effective. Electron microscopic studies demonstrated that, when added with complement, pentameric IgM, but not monomeric IgM, stabilized the bacterial capsule to the dehydration in dimethylformamide used for embedding in Lowicryl K4M. A strong correlation was observed between capsular stability and ability to be bound by PMN. The results suggest that pentameric IgM alters the structure of capsular components, perhaps through crosslinking, and this in turn facilitates interaction of C3bi and C3b with CR3 and CR1, their respective receptors on PMN.     

Evolution of French Bordetella pertussis and Bordetella parapertussis isolates: increase of Bordetellae not expressing pertactin

Bordetella pertussis and Bordetella parapertussis are closely related bacterial agents of whooping cough. Whole-cell pertussis (wP) vaccine was introduced in France in 1959. Acellular pertussis (aP) vaccine was introduced in 1998 as an adolescent booster and was rapidly generalized to the whole population, changing herd immunity by specifically targeting the virulence of the bacteria. We performed a temporal analysis of all French B. pertussis and B. parapertussis isolates collected since 2000 under aP vaccine pressure, using pulsed-field gel electrophoresis (PFGE), genotyping and detection of expression of virulence factors. Particular isolates were selected according to their different phenotype and PFGE type and their characteristics were analysed using the murine model of respiratory infection and in vitro cell cytotoxic assay. Since the introduction of the aP vaccines there has been a steady increase in the number of B. pertussis and B. parapertussis isolates collected that are lacking expression of pertactin. These isolates seem to be as virulent as those expressing all virulence factors according to animal and cellular models of infection. Whereas wP vaccine-induced immunity led to a monomorphic population of B. pertussis, aP vaccine-induced immunity enabled the number of circulating B. pertussis and B. parapertussis isolates not expressing virulence factors to increase, sustaining our previous hypothesis.     

Bordetella parapertussis Infection in Children: Epidemiology, Clinical Symptoms, and Molecular Characteristics of Isolates

The clinical trial conducted in Italy to evaluate the efficacy of acellular pertussis vaccines provided an opportunity to estimate the frequency of clinical infections with Bordetella parapertussis and to compare the clinical characteristics of children suffering from Bordetella pertussis illness with those of children with B. parapertussis illness. This study dealt with 76 B. parapertussis infections diagnosed from a population of 15,601 children participating in the follow-up of suspected cases of pertussis. An overall incidence of 2.1 cases of laboratory-confirmed parapertussis per 1,000 person-years was observed. Children affected by B. parapertussis infections showed a less severe clinical picture both in the duration of symptoms and in the percentage of patients affected, even when compared with vaccinated children with pertussis. To characterize the isolated strains, we performed assays for susceptibility to erythromycin and sulfamethoxazole-trimethoprim, and we examined the genomic DNAs by pulsed-field gel electrophoresis. The results showed a high degree of genetic stability among B. parapertussis strains regardless of time of collection and geographical distribution.     

Cholesterol-rich domains are involved in Bordetella pertussis phagocytosis and intracellular survival in neutrophils

Bordetella pertussis-specific antibodies protect against whooping cough by facilitating host defense mechanisms such as phagocytosis. However, the mechanism involved in the phagocytosis of the bacteria under non-opsonic conditions is still poorly characterized. We report here that B. pertussis binding and internalization is cholesterol dependent. Furthermore, we found cholesterol to be implicated in B. pertussis survival upon interaction with human neutrophils. Pre-treatment of PMN with cholesterol sequestering drugs like nystatin or methyl-β-cyclodextrin (MβCD) resulted in a drastic decrease of uptake of non-opsonized B. pertussis. Conversely, phagocytosis of opsonized bacteria was not affected by these drugs, showing that cholesterol depletion affects neither the viability of PMN nor the route of entry of opsonized B. pertussis. Additionally, intracellular survival rate of non-opsonized bacteria was significantly decreased in cholesterol-depleted PMN. Accordingly, confocal laser microscopy studies showed that non-opsonized B. pertussis co-localized with lysosomal markers only in cholesterol-depleted PMN but not in normal PMN. Our results indicate that B. pertussis docks to molecules that eventually prevent cellular bactericidal activity.     

The O antigen enables Bordetella parapertussis to avoid Bordetella pertussis-induced immunity

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 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 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.     

Characterization of Opsonins for Bacteroides fragilis in Immune Sera Collected from Experimentally Infected Mice

Serum collected from mice experimentally infected with Bacteroides fragilis 23745 (immune serum) was analyzed for its ability to opsonize the in vitro ingestion of this organism by mouse peritoneal macrophages. B. fragilis was shown to be phagocytized most efficiently in the presence of immune serum although normal mouse serum demonstrated reduced, but significant, opsonic activity. Phagocytosis was greater in the presence of serum collected from animals inoculated twice with the organism than in the presence of serum from once-inoculated animals. The increased opsonic activity of serum from twice-inoculated animals compared to singly inoculated animals was associated with increases in immunoglobulin G1(IgG1), IgG2a, and IgG2b but not IgM. Adsorption analysis of immune serum with homologous or heterologous bacterial antigens indicated that both antibody and complement act synergistically in opsonizing B. fragilis, although either alone may effectively opsonize this organism. Further evaluation of antibody-mediated opsonization revealed that prior treatment of heat-inactivated immune serum with the reducing agent 2-mercaptoethanol caused a slight, but significant, decrease in opsonic activity, thus indicating that IgM is a minor opsonizing antibody for B. fragilis. When ingestion of a B. fragilis stock strain (23745) was compared to a recent clinical isolate (C-1), it was observed that the stock strain was more easily phagocytized in the presence of normal mouse serum, thus suggesting a possible anti-opsonic-phagocytic property of the clinical strain. In addition, the clinical isolate was phagocytized to a significantly greater degree in an aerobic than an anaerobic environment. Subsequent analysis of in vitro killing of B. fragilis 23745 by peritoneal macrophages reflected the previous results in that optimal killing occurred in the presence of immune serum, although normal serum promoted phagocytic killing to an intermediate degree. Thus, these studies implicate both antibody and complement, either alone or in combination, in the opsonization of B. fragilis. Moreover, the virulence of clinical B. fragilis strains may relate to their refractoriness to opsonization and phagocytosis.     

Phagocytosis of virulent Porphyromonas gingivalis by human polymorphonuclear leukocytes requires specific immunoglobulin G.

No studies to date clearly define the interactions between Porphyromonas gingivalis and human peripheral blood polymorphonuclear leukocytes (PMN), nor has a protective role for antibody to P. gingivalis been defined. Using a fluorochrome phagocytosis microassay, we investigated PMN phagocytosis and killing of P. gingivalis as a function of P. gingivalis-specific antibody. Sera from a nonimmune rabbit and a healthy human subject were not opsonic for virulent P. gingivalis A7436, W83, and HG405; phagocytosis of these strains (but not 33277) required opsonization with hyperimmune antiserum (RaPg). Diluting RaPg with a constant complement source decreased proportionally the number of P. gingivalis A7436 cells phagocytosed per phagocytic PMN. Enriching for the immunoglobulin G fraction of RAPg A7436 enriched for opsonic activity toward A7436. An opsonic evaluation of 18 serum samples from adult periodontitis patients revealed that only 3 adult periodontitis sera of 17 with elevated immunoglobulin G to P. gingivalis A7436 were opsonic for A7436 and, moreover, that the serum sample with the highest enzyme-linked immunosorbent assay titer was most opsonic (patient 1). However, the opsonic activity of serum from patient 1 was qualitatively and not just quantitatively different from that of the nonopsonic human sera (but was less effective opsonin than RaPg). Strain variability was observed in resistance of P. gingivalis to phagocytosis, and opsonization was strain specific for some, but not all, strains tested. An evaluation of killing of A7436 revealed that serum killing and extracellular killing of P. gingivalis were less effective alone when compared with intracellular PMN killing alone.     

Complex Serology and Immune Response of Mice to Variant High-Molecular-Weight O Polysaccharides Isolated from Pseudomonas aeruginosa Serogroup O2 Strains

The O antigen of the Pseudomonas aeruginosa lipopolysaccharide is the optimal target for protective antibodies, but the unusual and complex nature of their sugar substituents has made it difficult to define the range of these structures needed in an effective vaccine. Most clinical isolates of P. aeruginosa can be classified into 10 O-antigen serogroups, but slight chemical differences among O polysaccharides within a serogroup give rise to subtype epitopes. These epitopes could impact the reactivity of O-antigen-specific antibodies, as well as the susceptibility of a target strain to protective, opsonic antibodies. To define parameters of serogroup and subtype-epitope immunogenicity, antigenicity, and surface expression on P. aeruginosa cells, we prepared high-molecular-weight O-polysaccharide vaccines from strains of P. aeruginosa serogroup O2, for which eight structurally variant O antigens expressing six defined subtype epitopes (O2a to O2f) have been identified. A complex pattern of immune responses to these antigens was observed following vaccination of mice. The high-molecular-weight O polysaccharides were generally more immunogenic at low doses (1 and 10 μg) than at a high dose (50 μg) and usually elicited antibodies that opsonized the homologous strain for phagocytic killing. Some of the individual polysaccharides elicited cross-opsonic antibodies to a variable number of strains that express all of the defined serogroup O2 subtype epitopes. Combination into one vaccine of two antigens that individually elicited cross-reactive opsonic antibodies to most members of the O2 serogroup inhibited, instead of enhanced, the production of antibodies broadly reactive with most serogroup O2 subtype strains. Thus, immune responses to P. aeruginosa O antigens may be restricted to a limited range of epitopes on structurally complex O antigens, and combining multiple related antigens into a single vaccine formulation may inhibit the production of those antibodies best able to protect against most P. aeruginosa strains within a given O-antigen serogroup.It has been established through animal and human experimentation that the lipopolysaccharide (LPS) O antigen of Pseudomonas aeruginosa is a target for protective antibodies (3, 36, 38). The studies of Knirel and colleagues (17, 19) on the chemical composition and structure of the major O-side-chain polysaccharides have provided important insights into the immunochemical properties of these antigens, but our understanding of their antigenic and immunogenic properties is incomplete. This point is highlighted by the inability to date to develop effective, LPS-specific immunotherapies for human P. aeruginosa infection (7).Results obtained with animals by using immunogens and antibodies specific to the O polysaccharides have indicated that slight chemical differences among bacterial strains with otherwise closely related O-side-chain structures can produce a complex pattern of reactions between antibodies and related antigens (13). With standard serologic methods using whole-cell agglutinations, strains of P. aeruginosa can be classified as members of one serogroup (serotype); members of each serogroup share a group-specific antigen. Further subdivision into subtypes, which correlate with structural variants determined by Knirel and colleagues (17), can be accomplished with appropriate antisera (22).To develop safe and effective O-antigen-specific P. aeruginosa vaccines, we have utilized the high-molecular-mass (>100,000-Da) fraction of O polysaccharides. These antigens are safe and immunogenic in humans and animals (13, 27, 37) and elicit protective antibodies to the strains from which they are isolated. However, in recent studies of animals immunized with a heptavalent high-molecular-weight O-polysaccharide vaccine whose individual components were isolated from single strains representative of the major serogroups causing P. aeruginosa infection, opsonic antibody responses to the group-specific antigens were not commonly elicited (13). Thus, in spite of chemical and serologic relatedness among subtype strains within a P. aeruginosa serogroup, single antigens isolated from one subtype strain do not always elicit opsonic antibodies to all of the strains within the serogroup (13). Previous results showed that a particular O antigen from a given serogroup may elicit group-specific immunity, while an O antigen from another serogroup may elicit only immunity specific to the subtype epitopes expressed on that particular O antigen.To explore this situation further and gain additional insight into the serologic diversity among P. aeruginosa LPS O antigens, we prepared high-molecular-weight O-polysaccharide immunogens from five strains of P. aeruginosa serogroup O2 that, together, express all six of the identified subtype antigens (Table ​(Table1).1). These polysaccharides were used to immunize mice, and the resultant sera were assessed by enzyme-linked immunosorbent assay (ELISA) and for opsonic killing activity. The results showed a complex interaction among the strains with regard to high-molecular-weight O-polysaccharide immunogenicity, antigenicity, serogroup and subtype epitope density, and susceptibility to opsonic killing. These findings indicate that the current serogroup classifications of P. aeruginosa are probably inadequate to define the full range of LPS antigens needed to elicit comprehensive immunity to a wide range of clinical isolates.

TABLE 1

Strains used for immunogen production, their serologic classification by subtype epitope, and chemical structures of the associated O antigens Open in a separate window^aBoldface type indicates a feature of a structure that distinguishes it from a related structure of the same serogroup. Abbreviations: FucNAc, 2-acetamido-2,6-dideoxygalactose (N-acetylfucosamine); Man(NAc)₂A, 2,3-diacetamido-2,3-dideoxymannuronic acid; Man(2NAc3N)A, 2-acetamido-3-acetamidino-2,3-dideoxymannuronic acid; Gul(NAc)₂A, 2,3-diacetamido-2,3-dideoxyguluronic acid. ^bThe lower structure is also part of the O antigen of strain 170007; there is about a 2:1 ratio of the upper and lower structures.      

Interactions between Bordetella pertussis and the complement inhibitor factor H

Bordetella pertussis causes whooping cough in humans, a highly contagious disease of the upper respiratory tract. An increase in cases of whooping cough in adolescents and adults in many countries has been reported, despite high immunization rates in children. To efficiently colonize the host the bacteria have to resist complement, the first defence line of innate immunity. B. pertussis has previously been shown to bind the classical pathway inhibitors C4b-binding protein and C1-inhibitor being thereby able to escape the classical pathway of complement. In this study recent clinical isolates of B. pertussis and B. parapertussis were found to survive alternative pathway attack in fresh non-immune serum better than the reference B. pertussis strain, Tohama I. By using adsorption assays, flow cytometry and a radioligand binding assay we observed that both B. pertussis and B. parapertussis bound the alternative pathway inhibitor factor H (FH) from normal human serum. The surface attached FH maintained its complement regulatory activity and promoted factor I-mediated cleavage of C3b. The main binding region was located to the C-terminal part of FH, into short consensus repeat domains 19-20. In contrast, the avian pathogen B. avium did not bind FH and was sensitive to the alternative pathway of human complement. In conclusion, the human pathogens B. pertussis and B. parapertussis are able to evade the alternative complement pathway by surface acquisition of the host complement regulator FH.     

Evaluation of PCR methods for the diagnosis of pertussis by the European surveillance network for vaccine-preventable diseases (EUVAC.NET)

This study aimed to evaluate the performance of polymerase chain reaction (PCR) methods used for the diagnosis of pertussis in laboratories within Europe in 2011. National reference laboratories in 25 European countries were contacted and a total of 24 laboratories from 19 countries agreed to participate in the study. A panel of seven samples of DNA from Bordetella pertussis, Bordetella parapertussis and Bordetella holmesii plus a negative control were distributed and analysed according to the routine PCR methods in each laboratory. The study took place in 2011. Nineteen laboratories used a real-time PCR approach, four laboratories used block-based PCR and one laboratory used a combination of methods. Six different combinations of amplification targets were used, and ten laboratories tested only for the presence of B. pertussis DNA. All laboratories (24/24) correctly identified a sample with high concentration of B. pertussis DNA, while three misidentified the B. parapertussis DNA as B. pertussis and 15 misidentified the B. holmesii DNA as either B. pertussis or B. parapertussis. There was a wide variation in the methods used for PCR-based diagnosis of pertussis among the European laboratories. Several laboratories were not able to discriminate between DNA samples from different Bordetella species.     

Protective role of immunoglobulin G antibodies to filamentous hemagglutinin and pertactin ofBordetella pertussis inBordetella parapertussis infection

An outbreak of parapertussis was studied prospectively in 38 first and second grade pupils of an elementary school. Eleven (29%) children were confirmed to be culture positive forBordetella parapertussis. Serum samples were collected from 31 children for assay of antibodies to filamentous hemagglutinin (FHA), pertactin (PRN), and pertussis toxin ofBordetella pertussis. At the first sampling, ten children were found to have a cough and 21 were asymptomatic. Of the latter, 12 remained asymptomatic and eight developed cough within 11 to 53 days (mean ± standard deviation, 31±12 days) after sampling. One child was identified as culture positive forBordetella pertussis and, thus, not included in the analysis ofBordetella parapertussis infection. The mean levels of IgC antibodies to FHA and PRN were significantly higher in the 12 asymptomatic children than in the eight children who later developed cough or in 20 healthy control children of the same age (for FHA, p=0.009 and < 0.001, respectively; for PRN, p=0.002 and 0.002, respectively). These preliminary data suggest thatBordetella parapertussis infection is more prevalent than documented, and that children with high levels of IgG antibodies to FHA and PRN can remain asymptomatic.     

Type-Specific Inhibition of Preopsonization Versus Immunoprecipitation by Streptococcal M Proteins

Purified M protein of group A streptococci inhibits in vitro phagocytosis of bacteria nonspecifically when it is mixed directly with the test blood. We devised a method of preopsonization inhibition that avoids direct contact between the test blood and the opsonic inhibitory agent. Each of the M proteins tested by this method inhibited opsonization and phagocytosis of only homologous-type streptococci. Titration of the M antigen in various purified preparations demonstrated a clear dissociation between titers of type-specific precipitating M antigen and type-specific opsonic inhibitory M antigen. Since opsonization reflects protective antibody, assays of M protein based on reaction with opsonic antibody should reflect more accurately the presence of the type-specific M determinant involved in protective immunity against streptococcal infections.     

Role of pertussis toxin in causing symptoms ofBordetella parapertussis infection

Whooping cough can be caused by eitherBordetella pertussis orBordetella parapertussis. Although the two species share an almost complete DNA identity,Bordetella parapertussis does not produce pertussis toxin, which is thought to be the main virulence factor ofBordetella pertussis. In order to elucidate the role of pertussis toxin in causing the typical symptoms of whooping cough, clinical information from 33 patients with culture-positiveBordetella parapertussis infection was collected and compared to that from 331 patients with infection caused byBordetella pertussis. Isolated strains ofBordetella parapertussis lacked pertussis toxin expression, as was demonstrated by negative tests for histamine sensitization. This was further substantiated in vivo by a significantly lower leukocyte count in the parapertussis group as compared to the pertussis group. Frequencies of typical symptoms of whooping cough, such as paroxysmal coughing, whooping and vomiting, were almost identical in the two groups. Nocturnal coughing and contact anamnesis were noted more often in theBordetella pertussis group. Children in the parapertussis group were significantly more often vaccinated with whole-cell pertussis vaccine than children infected withBordetella pertussis. The results indicate that pertussis toxin may not play a decisive role in causing the typical symptoms of whooping cough, such as paroxysmal coughing, whooping and vomiting.     

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

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-γ) 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-γ 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.     

Protective activity of Bordetella adenylate cyclase-hemolysin against bacterial colonization

Bordetella pertussis synthesizes several factors. It has been suggested that one of these factors, the adenylate cyclase-hemolysin (AC-Hly), directly penetrates target cells and impairs their normal functions by elevating intracellular cAMP. In the present study, we show that active immunization with purified B. pertussis AC-Hly or AC (a fragment of the AC-Hly molecule carrying only the adenylate cyclase activity but no toxin activity in vitro) protects mice against B. pertussis intranasal infection. Immunization with AC-Hly or AC significantly shortens the period of bacterial colonization of the mouse respiratory tract. Furthermore, B. parapertussis AC-Hly or AC are also protective antigens against B. parapertussis colonization; their protective activities are equivalent to that of the whole-cell vaccine. These results suggest that AC-Hly may play an important role in Bordetella pathogenesis, in a murine model. If this factor plays a similar role in the human disease, its use as a protective antigen could reduce not only the incidence of the disease, but also the asymptomatic human reservoir by limiting bacterial carriage.