Integrated Signaling Pathways Mediate Bordetella Immunomodulation, Persistence, and Transmission.

The mammalian immune system includes a sophisticated array of antimicrobial mechanisms. However, successful pathogens have developed subversive strategies to detect, modulate, and/or evade immune control and clearance. Independent disciplines study host immunology and bacterial pathogenesis, but interkingdom signaling between bacteria and host during natural infection remains poorly understood. An efficient natural host infection system has revealed complex communication between Bordetella spp. and mice, identified novel regulatory mechanisms, and demonstrated that bordetellae can respond to microenvironment and inflammatory status cues. Understanding these bacterial signaling pathways and their complex network that allows precisely timed expression of numerous immunomodulatory factors will serve as a paradigm for other organisms lacking such a powerful experimental infection system. VIDEO ABSTRACT.

Seasonal breeding drives the incidence of a chronic bacterial infection in a free-living herbivore population.

Understanding seasonal changes in age-related incidence of infections can be revealing for disentangling how host heterogeneities affect transmission and how to control the spread of infections between social groups. Seasonal forcing has been well documented in human childhood diseases but the mechanisms responsible for age-related transmission in free-living and socially structured animal populations are still poorly known. Here we studied the seasonal dynamics of Bordetella bronchiseptica in a free-living rabbit population over 5 years and discuss the possible mechanisms of infection. This bacterium has been isolated in livestock and wildlife where it causes respiratory infections that rapidly spread between individuals and persist as subclinical infections. Sera were collected from rabbits sampled monthly and examined using an ELISA. Findings revealed that B. bronchiseptica circulates in the rabbit population with annual prevalence ranging between 88% and 97%. Both seroprevalence and antibody optical density index exhibited 1-year cycles, indicating that disease outbreaks were seasonal and suggesting that long-lasting antibody protection was transient. Intra-annual dynamics showed a strong seasonal signature associated with the recruitment of naive offspring during the breeding period. Infection appeared to be mainly driven by mother-to-litter contacts rather than by interactions with other members of the community. By age 2 months, 65% of the kittens were seropositive.

Of mice and men: asymmetric interactions between Bordetella pathogen species.

In a recent experiment, we found that mice previously infected with Bordetella pertussis were not protected against a later infection with Bordetella parapertussis, while primary infection with B. parapertussis conferred cross-protection. This challenges the common assumption made in most mathematical models for pathogenic strain dynamics that cross-immunity between strains is symmetric. Here we investigate the potential consequences of this pattern on the circulation of the two pathogens in human populations. To match the empirical dominance of B. pertussis, we made the additional assumption that B. parapertussis pays a cost in terms of reduced fitness. We begin by exploring the range of parameter values that allow the coexistence of the two pathogens, with or without vaccination. We then track the dynamics of the system following the introduction of anti-pertussis vaccination. Our results suggest that (1) in order for B. pertussis to be more prevalent than B. parapertussis, the former must have a strong competitive advantage, possibly in the form of higher infectivity, and (2) because of asymmetric cross-immunity, the introduction of anti-pertussis vaccination should have little effect on the absolute prevalence of B. parapertussis. We discuss the evidence supporting these predictions, and the potential relevance of this model for other pathogens.

Mice lacking the orphan G protein-coupled receptor G2A develop a late-onset autoimmune syndrome.

Mice with a targeted disruption of the gene encoding a lymphoid-expressed orphan G protein-coupled receptor, G2A, demonstrate a normal pattern of T and B lineage differentiation through young adulthood. As G2A-deficient animals age, they develop secondary lymphoid organ enlargement associated with abnormal expansion of both T and B lymphocytes. Older G2A-deficient mice (>1 year) develop a slowly progressive wasting syndrome, characterized by lymphocytic infiltration into various tissues, glomerular immune complex deposition, and anti-nuclear autoantibodies. G2A-deficient T cells are hyperresponsive to TCR stimulation, exhibiting enhanced proliferation and a lower threshold for activation. Our findings demonstrate that G2A plays a critical role in controlling peripheral lymphocyte homeostasis and that its ablation results in the development of a novel, late-onset autoimmune syndrome.

Multiple roles for Bordetella lipopolysaccharide molecules during respiratory tract infection.

Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica are closely related subspecies that cause respiratory tract infections in humans and other mammals and express many similar virulence factors. Their lipopolysaccharide (LPS) molecules differ, containing either a complex trisaccharide (B. pertussis), a trisaccharide plus an O-antigen-like repeat (B. bronchiseptica), or an altered trisaccharide plus an O-antigen-like repeat (B. parapertussis). Deletion of the wlb locus results in the loss of membrane-distal polysaccharide domains in the three subspecies of bordetellae, leaving LPS molecules consisting of lipid A and core oligosaccharide. We have used wlb deletion (Deltawlb) mutants to investigate the roles of distal LPS structures in respiratory tract infection by bordetellae. Each mutant was defective compared to its parent strain in colonization of the respiratory tracts of BALB/c mice, but the location in the respiratory tract and the time point at which defects were observed differed significantly. Although the Deltawlb mutants were much more sensitive to complement-mediated killing in vitro, they displayed similar defects in respiratory tract colonization in C5(-/-) mice compared with wild-type (wt) mice, indicating that increased sensitivity to complement-mediated lysis is not sufficient to explain the in vivo defects. B. pertussis and B. parapertussis Deltawlb mutants were also defective compared to wt strains in colonization of SCID-beige mice, indicating that the defects were not limited to interactions with adaptive immunity. Interestingly, the B. bronchiseptica Deltawlb strain was defective, compared to the wt strain, in colonization of the respiratory tracts of BALB/c mice beginning 1 week postinoculation but did not differ from the wt strain in its ability to colonize the respiratory tracts of B-cell- and T-cell-deficient mice, suggesting that wlb-dependent LPS modifications in B. bronchiseptica modulate interactions with adaptive immunity. These data show that biosynthesis of a full-length LPS molecule by these three bordetellae is essential for the expression of full virulence for mice. In addition, the data indicate that the different distal structures modifying the LPS molecules on these three closely related subspecies serve different purposes in respiratory tract infection, highlighting the diversity of functions attributable to LPS of gram-negative bacteria.

Modulation of host immune responses, induction of apoptosis and inhibition of NF-kappaB activation by the Bordetella type III secretion system.

Bordetella bronchiseptica establishes respiratory tract infections in laboratory animals with high efficiency. Colonization persists for the life of the animal and infection is usually asymptomatic in immunocompetent hosts. We hypothesize that this reflects a balance between immunostimulatory events associated with infection and immunomodulatory events mediated by the bacteria. We have identified 15 loci that are part of a type III secretion apparatus in B. bronchiseptica and three secreted proteins. The functions of the type III secretion system were investigated by comparing the phenotypes of wild-type bacteria with two strains that are defective in type III secretion using in vivo and in vitro infection models. Type III secretion mutants were defective in long-term colonization of the trachea in immunocompetent mice. The mutants also elicited higher titres of anti-Bordetella antibodies upon infection compared with wild-type bacteria. Type III secretion mutants also showed increased lethal virulence in immunodeficient SCID-beige mice. These observations suggest that type III-secreted products of B. bronchiseptica interact with components of both innate and adaptive immune systems of the host. B. bronchiseptica induced apoptosis in macrophages in vitro and inflammatory cells in vivo and type III secretion was required for this process. Infection of an epithelial cell line with high numbers of wild type, but not type III deficient B. bronchiseptica resulted in rapid aggregation of NF-kappaB into large complexes in the cytoplasm. NF-kappaB aggregation was dependent on type III secretion and aggregated NF-kappaB did not respond to TNFalpha activation, suggesting B. bronchiseptica may modulate host immunity by inactivating NF-kappaB. Based on these in vivo and in vitro results, we hypothesize that the Bordetella type III secretion system functions to modulate host immune responses during infection.

Manipulating the host to study bacterial virulence.

The ability to manipulate animal hosts as well as bacterial pathogens greatly expands the utility of in vivo models of infection. For example, the construction of mice that harbor human tissues or express specific transgenes can provide ligand-receptor interactions that are essential for pathogenesis. Interactions between virulence factors and specific host defenses can sometimes be resolved by challenging selectively immuno deficient mice with bacteria containing virulence gene mutations. Transgenic animals expressing inducible reporters can be used to conveniently identify cells in which specific response pathways have been activated during infection. These and other approaches promise to improve the quality of information obtainable from in vivo assessments of pathogenesis.

Pregenomic comparative analysis between bordetella bronchiseptica RB50 and Bordetella pertussis tohama I in murine models of respiratory tract infection.

We describe here a side-by-side comparison of murine respiratory infection by Bordetella pertussis and Bordetella bronchiseptica strains whose genomes are currently being sequenced (Tohama I and RB50, respectively). B. pertussis and B. bronchiseptica are most appropriately classified as subspecies. Their high degree of genotypic and phenotypic relatedness facilitates comparative studies of pathogenesis. RB50 and Tohama I differ in their abilities to grow in the nose, trachea, and lungs of BALB/c mice and to induce apoptosis, lung pathology, and an antibody response. To focus on the interactions between the bacteria and particular aspects of the host immune response, we used mice with specific immune defects. Mice lacking B cells and T cells were highly susceptible to B. bronchiseptica and were killed by intranasal inoculation with doses as low as 500 CFU. These mice were not killed by B. pertussis, even when doses as high as 10(5) CFU were delivered to the lungs. B. bronchiseptica, which was highly resistant to naive serum in vitro, caused bacteremia in these immunodeficient mice, while B. pertussis, which was highly sensitive to naive serum, did not cause bacteremia. B. bronchiseptica was, however, killed by immune serum in vitro, and adoptive transfer of anti-Bordetella antibodies protected SCID-beige mice from B. bronchiseptica lethal infection. Neutropenic mice were similarly killed by B. bronchiseptica but not B. pertussis infection, suggesting neutrophils are critical to the early inflammatory response to the former but not the latter. B. bronchiseptica was dramatically more active than B. pertussis in mediating the lysis of J774 cells in vitro and in inducing apoptosis of inflammatory cells in mouse lungs. This side-by-side comparison describes phenotypic differences that may be correlated with genetic differences in the comparative analysis of the genomes of these two highly related organisms.

Probing the function of Bordetella bronchiseptica adenylate cyclase toxin by manipulating host immunity.

We have examined the role of adenylate cyclase-hemolysin (CyaA) by constructing an in-frame deletion in the Bordetella bronchiseptica cyaA structural gene and comparing wild-type and cyaA deletion strains in natural host infection models. Both the wild-type strain RB50 and its adenylate cyclase toxin deletion (DeltacyaA) derivative efficiently establish persistent infections in rabbits, rats, and mice following low-dose inoculation. In contrast, an inoculation protocol that seeds the lower respiratory tract revealed significant differences in bacterial numbers and in polymorphonuclear neutrophil recruitment in the lungs from days 5 to 12 postinoculation. We next explored the effects of disarming specific aspects of the immune system on the relative phenotypes of wild-type and DeltacyaA bacteria. SCID, SCID-beige, or RAG-1(-/-) mice succumbed to lethal systemic infection following high- or low-dose intranasal inoculation with the wild-type strain but not the DeltacyaA mutant. Mice rendered neutropenic by treatment with cyclophosphamide or by knockout mutation in the granulocyte colony-stimulating factor locus were highly susceptible to lethal infection by either wild-type or DeltacyaA strains. These results reveal the significant role played by neutrophils early in B. bronchiseptica infection and by acquired immunity at later time points and suggest that phagocytic cells are a primary in vivo target of the Bordetella adenylate cyclase toxin.

An IgG3-IL-2 fusion protein recognizing a murine B cell lymphoma exhibits effective tumor imaging and antitumor activity.

Antibody (Ab)-based tumor therapeutics use the tumor-binding specificity of the Ab to target Fc functions or associated molecules to the site of the tumor. We have used an Ab-interleukin-2 (IL-2) fusion protein to deliver IL-2 to a murine B cell lymphoma (38C13). This anti-Id IgG3-CH3-IL-2, which recognizes the idiotype present on the surface of the lymphoma has a half-life in mice approximately 17-fold longer than the half-life reported for IL-2. Gamma camera studies showed that anti-Id IgG3-CH3-IL-2 localizes at the site of a subcutaneous tumor in mice. The anti-Id IgG3-CH3-IL-2 also shows enhanced antitumor activity compared with the combination of Ab and IL-2 administered together. However, the mechanism of antitumor activity appears to depend on the dose and the treatment schedule used. A single dose of fusion protein prevented tumor in only 50% of the animals, although all the survivors showed some evidence of immunologic memory. Although multiple doses are more effective in preventing tumor growth (87% survivors), they are ineffective in generating protective immunologic memory. Our results suggest that Ab-IL-2 fusion proteins will be useful in the diagnosis and treatment of human B cell lymphomas and other related malignancies.

The BvgAS virulence control system regulates type III secretion in Bordetella bronchiseptica.

The BvgAS signal transduction system in Bordetella spp. mediates a transition between infectious (Bvg+) and non-infectious (Bvg-) phases by sensing environmental conditions and regulating gene expression. Using differential display, arbitrary-primed polymerase chain reaction (PCR), we identified a gene expressed in the Bvg+ phase of Bordetella bronchiseptica that shows a high degree of sequence similarity to a locus involved in providing energy for type III secretion in pathogenic gram-negative bacteria (yscN in Yersinia spp.). We determined that the expression of this homologue in B. bronchiseptica (designated bscN) is regulated by bvg. Several open reading frames surrounding the bscN locus also show sequence similarity to loci encoding type III secretion apparatus components in other bacteria. An in-frame deletion of bscN in B. bronchiseptica leads to decreased secretion of several proteins, decreased cytotoxicity towards cultured cell lines and a defect in causing tyrosine dephosphorylation of specific proteins in infected cells in vitro. The deletion strain also revealed that bscN-mediated secretion is required for persistent colonization of the trachea in a rat infection model. Loci encoding type III secretion homologues were identified in four strains of B. pertussis and two strains of B. parapertussis. B. pertussis strain 18323 and an ovine isolate of B. parapertussis show significant transcription of the genes in vitro.

Antibody-IL-2 fusion proteins: a novel strategy for immune protection.

Advances in genetic engineering and expression systems have led to a rapid progress in the development of immunoglobulins fused to other proteins. These 'antibody fusion proteins' have novel properties and include antibodies fused to the cytokine interleukin-2. In the present review we describe strategies for construction of these antibody-interleukin-2 fusion proteins and discuss their in vitro and in vivo properties. Antibody-interleukin-2 fusion proteins retain both antibody associated functions such as antigen binding, complement activation and Fc gamma receptor binding as well as interleukin-2 associated functions such as the stimulation of proliferation of CTLL2 cells. In vivo, they produce strong potentiation of the host immune response against any associated antigen. In addition, these novel molecules are able to target tumor cells and produce a specific and effective T cell response capable of eliminating the tumor. These properties suggest that antibody-interleukin-2 fusion proteins will be useful in the diagnosis and/or treatment of human cancer as well as in the potentiation of human response against any associated antigen.

In vivo properties of an IgG3-IL-2 fusion protein. A general strategy for immune potentiation.

In this report, we describe a strategy for enhancing the immunogenicity of a wide variety of Ags by linking them to IL-2 via an IgG3-IL-2 fusion protein with high affinity for a convenient hapten Ag, dansyl (DNS; N,N-dimethyl-1-aminonaphthalene-5-sulfonyl chloride). This fusion protein, anti-DNS-IgG3-IL-2, combines the functional characteristics of its constituents and has pharmacokinetic properties that are greatly improved over those of IL-2 and a previously described IgG1-IL-2 fusion. The molecule is intact and recoverable from the blood of mice hours after i.p. injection and reaches distant organs throughout the animal. The 7-h in vivo half-life of this molecule is much longer than that of IL-2, addressing a major obstacle in the application of IL-2 to human diseases, including cancer and AIDS. Additionally, the Ab's specificity for the hapten dansyl and the convenient chemistry of dansyl provide a means to link IL-2 to virtually any molecule of interest without the complexities and uncertainties of making IL-2 fusions with each molecule individually. Using hapten-conjugated-BSA (DNS-BSA) as a model Ag we show that the Ab response elicited by anti-DNS-IgG3-IL-2-bound DNS-BSA-Sepharose injected into mice is increased over that of DNS-BSA-Sepharose or anti-DNS-IgG3-bound DNS-BSA-Sepharose. Anti-DNS-IgG3-IL-2 also increased the Ab response to soluble DNS-BSA after a booster injection. This system should be useful in testing the ability of IL-2 to potentiate the immune response to Ag and in screening a large number of potential Ags for use in vaccines. The dramatically improved pharmacokinetics should also overcome one of the major difficulties in applying IL-2 to the treatment of human disease, its short half-life.

An IgG3-IL-2 fusion protein has higher affinity than hrIL-2 for the IL-2R alpha subunit: real time measurement of ligand binding.

The alpha subunit of the interleukin-2 (IL-2) receptor (IL-2R alpha)3 has the highest individual affinity for IL-2 and is the only subunit not known to bind other cytokines. The interactions between IL-2 and IL-2R alpha studied in cell binding assays have revealed a number of factors which may vary significantly in different cell lines used for these assays in different laboratories. In order to avoid the problems associated with cellular assays we used an optical biosensor to examine the interaction between IL-2R alpha and hrIL-2. Real-time measurement of association and dissociation resulted in a calculated KD of 1.9 x 10(-7) M for this interaction. We then examined the IL-2R alpha binding of a potentially bivalent IgG3-IL2 fusion protein previously shown to have a higher affinity than hrIL-2 for the high affinity IL-2R but not the intermediate affinity IL-2R. Biosensor measurements of association and dissociation of IgG3-IL2 to IL-2R alpha yielded a similar association rate but a decreased dissociation rate compared to hrIL-2, resulting in a KD of 5.3 x 10(-8) M. This system is applicable to the numerous IL-2 mutants with different affinities and activities and is generalizable to other cytokine/receptor interactions.

An IgG3-IL2 fusion protein activates complement, binds Fc gamma RI, generates LAK activity and shows enhanced binding to the high affinity IL-2R.

The therapeutic value of Interleukin 2 (IL-2) is limited by its short half life and systemic toxicity. One approach to overcoming these problems is to fuse this protein to an antibody, a protein with a long half life and the ability to target a unique antigen within the body. To examine the biochemical properties of such a molecule a fusion protein was constructed linking the N-terminus of human IL-2 to the C-terminus of IgG3. A similar fusion between IgG1 and IL-2 has previously been shown to bind antigen, generate antibody-dependent cellular cytotoxicity (ADCC) and stimulate T cell proliferation and cytotoxicity. We now extend these studies and show that the fusion protein, termed IgG3-IL2, is appropriately N-glycosylated within the IgG3 CH2 domain, binds the human high affinity Fc receptor (Fc gamma RI) with an affinity slightly lower than that of IgG3, and is able to activate complement via the classical pathway to lyse antigen coated sheep red blood cells (SRBC). When used to stimulate the proliferation of the IL-2 dependent cell line CTLL-2, IgG3-IL2 has a specific activity slightly lower than that of human recombinant IL-2 (hrIL-2). In marked contrast, when comparable unit concentrations, as defined by the standard CTLL-2 proliferation assay, are used to stimulate human peripheral blood lymphocytes (PBL), IgG3-IL2 generates significantly greater lymphokine activated killer (LAK) cell cytotoxicity than does hrIL-2. Competition studies show that IgG3-IL2 binds the intermediate affinity form of the IL-2 receptor (IL-2R), consisting of the beta and gamma subunits, with an affinity slightly less than that of hrIL-2. In contrast, IgG3-IL2 shows a greater affinity than hrIL-2 for the high affinity IL-2R, consisting of alpha, beta and gamma subunits. Our studies show that the IgG3-IL2 fusion protein possesses a combination of the biological properties of IgG3 and IL-2 including antigen binding, complement activation, Fc gamma RI binding, IL-2R binding and stimulation of both proliferation and LAK activity. This combination of activities may allow IgG3-IL2 to target humoral and cell-mediated immune activation to the site of an antigen of interest or target an antigen to IL-2R bearing cells or organs.