Rectal and Intranasal Immunizations with Recombinant Urease Induce Distinct Local and Serum Immune Responses in Mice and Protect against Helicobacter pylori Infection

To determine the optimal inductive sites for immunization against Helicobacter pylori infection, the protective efficacy of recombinant urease (rUre) was assessed for mice given the vaccine by either the oral (p.o.), intranasal (i.n.), or rectal route. When mice were immunized with rUre (25 μg p.o. or rectally or 10 μg i.n.) plus heat-labile toxin from Escherichia coli as the mucosal adjuvant, all routes afforded protection against challenge with H. pylori, as indicated by a significant reduction in gastric urease activity (P < 0.0005) compared to that of sham-immunized controls. Quantitative H. pylori culture of stomach tissue demonstrated a >97% reduction in bacterial burden in mice immunized by all routes (P < 0.05). Induction of antiurease immunoglobulin A (IgA) levels in gastric luminal secretions after p.o. immunization was greater than after i.n. administration (means, 6.0 and 1.02 ng/ml, respectively) and was dependent upon challenge with H. pylori. However, immunization by the rectal route resulted in the generation of the highest levels of gastric antiurease IgA (mean, 40.89 ng/ml), which was detectable prior to challenge with H. pylori. Immunohistochemical staining of stomach tissue for cells secreting urease-specific antibody and CD4⁺ T cells showed levels of recruitment to be dependent upon challenge with H. pylori and equivalent for all routes. These results identify both the rectum and nasal passages as suitable inductive sites for urease immunization.     

A Recombinant Live Attenuated Strain of Vibrio cholerae Induces Immunity against Tetanus Toxin and Bordetella pertussis Tracheal Colonization Factor

An attenuated strain of Vibrio cholerae was used as a carrier for the expression of heterologous antigens such as fragment C from tetanus toxin (TetC) and tracheal colonization factor from Bordetella pertussis (Tcf). In vitro, high levels of protein were obtained when the Escherichia coli nirB promoter was used and the bacteria were grown with low aeration. Intranasal immunization of mice with IEM101 expressing TetC elicited serum vibriocidal activity and induced antibodies against tetanus toxin which were protective against lethal challenge with 10 times the 50% lethal dose of tetanus toxin. Bacterial viability was essential for the induction of anti-TetC antibodies. Intranasal administration of IEM101 expressing Tcf induced a significant reduction in bacterial colonization of the tracheas of mice challenged with wild-type B. pertussis. These data are in agreement with the putative role of Tcf in Bordetella tracheal colonization. In conclusion, we have demonstrated that V. cholerae may be used as a live vector to deliver heterologous antigens in vivo and that protection to both systemic and local challenge may be achieved.     

Contribution of Bordetella bronchiseptica Filamentous Hemagglutinin and Pertactin to Respiratory Disease in Swine

Bordetella bronchiseptica is pervasive in swine populations and plays multiple roles in respiratory disease. Most studies addressing virulence factors of B. bronchiseptica are based on isolates derived from hosts other than pigs. Two well-studied virulence factors implicated in the adhesion process are filamentous hemagglutinin (FHA) and pertactin (PRN). We hypothesized that both FHA and PRN would serve critical roles in the adhesion process and be necessary for colonization of the swine respiratory tract. To investigate the role of FHA and PRN in Bordetella pathogenesis in swine, we constructed mutants containing an in-frame deletion of the FHA or the PRN structural gene in a virulent B. bronchiseptica swine isolate. Both mutants were compared to the wild-type swine isolate for their ability to colonize and cause disease in swine. Colonization of the FHA mutant was lower than that of the wild type at all respiratory tract sites and time points examined and caused limited to no disease. In contrast, the PRN mutant caused similar disease severity relative to the wild type; however, colonization of the PRN mutant was reduced relative to the wild type during early and late infection and induced higher anti-Bordetella antibody titers. Together, our results indicate that despite inducing different pathologies and antibody responses, both FHA and PRN are necessary for optimal colonization of the swine respiratory tract.Respiratory disease in pigs is the most important health concern for swine producers today. According to the 2006 NAHMS survey, respiratory disease was the greatest cause of mortality in swine, accounting for 53.7% of nursery deaths and 60.1% of deaths in grower/finisher pigs (68). Bordetella bronchiseptica is widely prevalent in swine populations and contributes to multiple pathologies in respiratory disease. In very young pigs it causes severe bronchopneumonia with high morbidity and, if untreated, mortality. It is a primary etiologic agent of atrophic rhinitis, causing a moderate to mild reversible form, and promotes colonization by toxigenic strains of P. multocida, usually leading to severe, progressive atrophic rhinitis (11, 12). B. bronchiseptica is frequently found in nasal turbinates and lung lesions of fattening pigs who may not exhibit clinical signs of respiratory disease. Nonetheless, field surveys document that subclinical pneumonia can result in substantial economic losses due to slower weight gain, increased days to market, and reduced feed efficiency (4, 22). In addition, B. bronchiseptica infections increase the severity of respiratory disease associated with other bacterial and viral pathogens and is thus a main contributing agent in porcine respiratory disease complex, a multifactorial disease state that is consistently listed as a top research priority by the National Pork Board (3, 6, 7, 10, 11, 72).Infection begins with colonization of the ciliated epithelial cells of the upper respiratory tract. Two well-studied Bordetella virulence factors implicated in the adhesion process are filamentous hemagglutinin (FHA) and pertactin (PRN). Both FHA and PRN are regulated by the BvgAS signal transduction system, which controls the expression of virulence determinants involved in the Bordetella infectious cycle (14). Numerous in vitro studies have demonstrated that FHA functions as an adhesin and contains several different binding domains (2, 16, 25, 27, 28, 40-42, 58, 63, 66-67, 69-71). These domains include a heparin-binding domain that facilitates binding to sulfated polysaccharides (23), a carbohydrate-recognition domain that promotes binding to ciliated epithelial cells of the respiratory tract and macrophages (52), and an Arg-Gly-Asp (RGD) domain. The RGD domain has been shown to play a key role in the upregulation of intercellular adhesion molecule 1 by epithelial cells, through an NF-κβ signaling pathway, by interacting with very late antigen-5 (28, 29). This RGD domain also plays an important role in the upregulation of CR3 binding activity by interacting with the leukocyte response integrin/integrin-associated protein located on monocytes and macrophages (27). In addition, FHA of B. bronchiseptica has been shown to be required for colonization of the rat trachea (16).PRN belongs to the type V autotransporter protein family and, similar to FHA, contains an RGD domain as well (18). Several in vitro studies have demonstrated PRN to function as an adhesin (19, 32, 36, 71); however, an exact host receptor has not been identified. The role of PRN as a protective immunogen is more clearly defined. Active immunization with purified PRN has been shown to provide protection against mortality and reduce pathology and lung colonization in mice and pigs challenged with Bordetella, and passive transfer of a PRN-specific monoclonal antibody has also been shown to provide protection in mice (33, 43, 46, 60).The overwhelming majority of studies addressing virulence factors of B. bronchiseptica are based on isolates derived from hosts other than pigs. Swine isolates of B. bronchiseptica possess unique genetic and phenotypic traits relative to isolates from other host species (20, 44, 55). Recent experiments in rats demonstrate that attachment is a multifactorial process (16, 40), and this is also likely to be true in swine. However, no definitive data exist with respect to swine, either in vivo or with swine tissue or cells in vitro. In this report, we investigate the role of FHA and PRN in Bordetella pathogenesis in swine, by constructing two mutants containing an in-frame deletion of the FHA or the PRN structural gene in KM22, a virulent B. bronchiseptica swine isolate. We compare both of these mutants to KM22 for their ability to mediate adherence in vitro and to colonize and cause disease during respiratory infection in swine.     

Protection against experimental Bordetella bronchiseptica infection in mice by active immunization with killed vaccine.

Mice immunized with a killed vaccine of phase I Bordetella bronchiseptica were challenged with various numbers of virulent B. bronchiseptica by intraperitoneal, intracerebral, or intranasal routes. The course of infection was compared among these routes, and the protective effect of vaccination was quantitatively analyzed. In ddN mice infected intraperitoneally with 1.8 X 10(8) cells (ca. 80 times the 50% lethal dose [LD50]) the organisms rapidly increased in the intraperitoneal fluid, spleen, and liver within few days and caused splenic atrophy, septicemia, and death. However, immunizations with 5 X 10(9) cells gave the mice a high agglutinin titer and suppressed the increase in the number of organisms. With four immunizations, the lungs and livers were clear within 3 days, and with one or two immunizations, they were clear within 7 days. These immunizations effectively protected the mice from death but did not protect them from splenic atrophy. In the intracerebral infection with 1.4 X 10(6) cells (ca. 1.2 X 10(5) LD50), the number of organisms rapidly increased in the brain and caused encephalitis, splenic atrophy, and death. However, four or five immunizations completely suppressed the increase in the brain and protected the mice from death and splenic atrophy. After intranasal infection with 4 X 10(6) cells (ca. 25 LD50), the organisms rapidly increased in the nasal cavity and lungs and caused pneumonia and death. Immunization with 5 X 10(9) cells was effective in clearing the organisms from the lungs and in protecting against death and splenic atrophy. However, the organisms were not cleared from the nasal cavity for 60 to 150 days after the challenge with as little as 10(2) cells, even in the mice with an agglutinin titer as high as 1:10,000.     

Respiratory Disease in Kennelled Dogs: Serological Responses to Bordetella bronchiseptica Lipopolysaccharide Do Not Correlate with Bacterial Isolation or Clinical Respiratory Symptoms

The role of Bordetella bronchiseptica in a natural outbreak of canine infectious respiratory disease was investigated both by culture and serological analysis. B. bronchiseptica was found in the lungs of a large proportion of clinically healthy dogs and in a greater proportion of dogs with respiratory disease. Using a lipopolysaccharide (LPS) antigen-based enzyme-linked immunosorbent assay, we analyzed the serological responses of a large number of dogs. Dogs with high antibody levels showed no protection from disease, and there was no correlation between the development of disease and rising antibody titer. Similarly, there was no difference in antibody levels in dogs with and without B. bronchiseptica in the lungs. Antibodies to LPS have no predictive value in determining which animals will contract respiratory disease, how severe the disease will be, or which dogs will have B. bronchiseptica colonizing the lungs.     

Maternal Vaccination with a Fimbrial Tip Adhesin and Passive Protection of Neonatal Mice against Lethal Human Enterotoxigenic Escherichia coli Challenge

Globally, enterotoxigenic Escherichia coli (ETEC) is a leading cause of childhood and travelers'' diarrhea, for which an effective vaccine is needed. Prevalent intestinal colonization factors (CFs) such as CFA/I fimbriae and heat-labile enterotoxin (LT) are important virulence factors and protective antigens. We tested the hypothesis that donor strand-complemented CfaE (dscCfaE), a stabilized form of the CFA/I fimbrial tip adhesin, is a protective antigen, using a lethal neonatal mouse ETEC challenge model and passive dam vaccination. For CFA/I-ETEC strain {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407, which has been extensively studied in volunteers, an inoculum of 2 × 10⁷ bacteria resulted in 50% lethal doses (LD₅₀) in neonatal DBA/2 mice. Vaccination of female DBA/2 mice with CFA/I fimbriae or dscCfaE, each given with a genetically attenuated LT adjuvant (LTK63) by intranasal or orogastric delivery, induced high antigen-specific serum IgG and fecal IgA titers and detectable milk IgA responses. Neonates born to and suckled by dams antenatally vaccinated with each of these four regimens showed 78 to 93% survival after a 20× LD₅₀ challenge with {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407, compared to 100% mortality in pups from dams vaccinated with sham vaccine or LTK63 only. Crossover experiments showed that high pup survival rates after ETEC challenge were associated with suckling but not birthing from vaccinated dams, suggesting that vaccine-specific milk antibodies are protective. In corroboration, preincubation of the ETEC inoculum with antiadhesin and antifimbrial bovine colostral antibodies conferred a dose-dependent increase in pup survival after challenge. These findings indicate that the dscCfaE fimbrial tip adhesin serves as a protective passive vaccine antigen in this small animal model and merits further evaluation.     

Novel Strategy To Protect against Influenza Virus-Induced Pneumococcal Disease without Interfering with Commensal Colonization

Streptococcus pneumoniae commonly inhabits the nasopharynx as a member of the commensal biofilm. Infection with respiratory viruses, such as influenza A virus, induces commensal S. pneumoniae to disseminate beyond the nasopharynx and to elicit severe infections of the middle ears, lungs, and blood that are associated with high rates of morbidity and mortality. Current preventive strategies, including the polysaccharide conjugate vaccines, aim to eliminate asymptomatic carriage with vaccine-type pneumococci. However, this has resulted in serotype replacement with, so far, less fit pneumococcal strains, which has changed the nasopharyngeal flora, opening the niche for entry of other virulent pathogens (e.g., Streptococcus pyogenes, Staphylococcus aureus, and potentially Haemophilus influenzae). The long-term effects of these changes are unknown. Here, we present an attractive, alternative preventive approach where we subvert virus-induced pneumococcal disease without interfering with commensal colonization, thus specifically targeting disease-causing organisms. In that regard, pneumococcal surface protein A (PspA), a major surface protein of pneumococci, is a promising vaccine target. Intradermal (i.d.) immunization of mice with recombinant PspA in combination with LT-IIb(T13I), a novel i.d. adjuvant of the type II heat-labile enterotoxin family, elicited strong systemic PspA-specific IgG responses without inducing mucosal anti-PspA IgA responses. This response protected mice from otitis media, pneumonia, and septicemia and averted the cytokine storm associated with septic infection but had no effect on asymptomatic colonization. Our results firmly demonstrated that this immunization strategy against virally induced pneumococcal disease can be conferred without disturbing the desirable preexisting commensal colonization of the nasopharynx.     

Enzymatic Modification of Lipid A by ArnT Protects Bordetella bronchiseptica against Cationic Peptides and Is Required for Transmission

Pathogen transmission cycles require many steps: initial colonization, growth and persistence, shedding, and transmission to new hosts. Alterations in the membrane components of the bacteria, including lipid A, the membrane anchor of lipopolysaccharide, could affect any of these steps via its structural role protecting bacteria from host innate immune defenses, including antimicrobial peptides and signaling through Toll-like receptor 4 (TLR4). To date, lipid A has been shown to affect only the within-host dynamics of infection, not the between-host dynamics of transmission. Here, we investigate the effects of lipid A modification in a mouse infection and transmission model. Disruption of the Bordetella bronchiseptica locus (BB4268) revealed that ArnT is required for addition of glucosamine (GlcN) to B. bronchiseptica lipid A. ArnT modification of lipid A did not change its TLR4 agonist activity in J774 cells, but deleting arnT decreased resistance to killing by cationic antimicrobial peptides, such as polymyxin B and β-defensins. In the standard infection model, mutation of arnT did not affect B. bronchiseptica colonization, growth, persistence throughout the respiratory tract, recruitment of neutrophils to the nasal cavity, or shedding of the pathogen. However, the number of bacteria necessary to colonize a host (50% infective dose [ID₅₀]) was 5-fold higher for the arnT mutant. Furthermore, the arnT mutant was defective in transmission between hosts. These results reveal novel functions of the ArnT lipid A modification and highlight the sensitivity of low-dose infections and transmission experiments for illuminating aspects of infectious diseases between hosts. Factors such as ArnT can have important effects on the burden of disease and are potential targets for interventions that can interrupt transmission.     

Live Attenuated Mutants of Francisella tularensis Protect Rabbits against Aerosol Challenge with a Virulent Type A Strain

Francisella tularensis, a Gram-negative bacterium, is the causative agent of tularemia. No licensed vaccine is currently available for protection against tularemia, although an attenuated strain, dubbed the live vaccine strain (LVS), is given to at-risk laboratory personnel as an investigational new drug (IND). In an effort to develop a vaccine that offers better protection, recombinant attenuated derivatives of a virulent type A strain, SCHU S4, were evaluated in New Zealand White (NZW) rabbits. Rabbits vaccinated via scarification with the three attenuated derivatives (SCHU S4 ΔguaBA, ΔaroD, and ΔfipB strains) or with LVS developed a mild fever, but no weight loss was detected. Twenty-one days after vaccination, all vaccinated rabbits were seropositive for IgG to F. tularensis lipopolysaccharide (LPS). Thirty days after vaccination, all rabbits were challenged with aerosolized SCHU S4 at doses ranging from 50 to 500 50% lethal doses (LD₅₀). All rabbits developed fevers and weight loss after challenge, but the severity was greater for mock-vaccinated rabbits. The ΔguaBA and ΔaroD SCHU S4 derivatives provided partial protection against death (27 to 36%) and a prolonged time to death compared to results for the mock-vaccinated group. In contrast, LVS and the ΔfipB strain both prolonged the time to death, but there were no survivors from the challenge. This is the first demonstration of vaccine efficacy against aerosol challenge with virulent type A F. tularensis in a species other than a rodent since the original work with LVS in the 1960s. The ΔguaBA and ΔaroD SCHU S4 derivatives warrant further evaluation and consideration as potential vaccines for tularemia and for identification of immunological correlates of protection.     

Active and Passive Immunity against Borrelia burgdorferi Decorin Binding Protein A (DbpA) Protects against Infection

Borrelia burgdorferi, the spirochete that causes Lyme disease, binds decorin, a collagen-associated extracellular matrix proteoglycan found in the skin (the site of entry for the spirochete) and in many other tissues. Two borrelial adhesins that recognize this proteoglycan, decorin binding proteins A and B (DbpA and DbpB, respectively), have recently been identified. Infection of mice by low-dose B. burgdorferi challenge elicited antibodies against DbpA and DbpB that were sustained at high levels, suggesting that these antigens are expressed in vivo. Scanning immunoelectron microscopy showed that DbpA was surface accessible on intact borreliae. Passive administration of DbpA antiserum protected mice from infection following challenge with heterologous B. burgdorferi sensu stricto isolates, even when serum administration was delayed for up to 4 days after challenge. DbpA is the first antigen target identified that is capable of mediating immune resolution of early, localized B. burgdorferi infections. DbpA immunization also protected mice from B. burgdorferi challenge; DbpB immunization was much less effective. DbpA antiserum inhibited in vitro growth of many B. burgdorferi sensu lato isolates of diverse geographic, phylogenetic, and clinical origins. In combination, these findings support a role for DbpA in the immunoprophylaxis of Lyme disease and suggest that DbpA vaccines have the potential to eliminate early-stage B. burgdorferi infections.     

Targeting Surface Protein SasX by Active and Passive Vaccination To Reduce Staphylococcus aureus Colonization and Infection

SasX is a recently described surface protein of Staphylococcus aureus that is linked to the epidemic success of hospital-associated methicillin-resistant clones, in particular in Asia. It enhances nasal colonization and virulence in skin and lung infection models. Here, we evaluated the potential of SasX as a vaccine component in passive and active immunization efforts using mouse infection models. We found that SasX induced a specific immune response predominantly based on IgG1 antibodies. Active immunization with recombinant SasX or passive immunization with rabbit polyclonal anti-SasX IgG significantly decreased the size of lesions caused by S. aureus in a skin infection model. Furthermore, active immunization reduced acute lung injury in a lung infection model. Moreover, active or passive immunization significantly reduced S. aureus colonization in a nasal colonization model. Finally, anti-SasX IgG enhanced the susceptibility of S. aureus to killing by human neutrophils. We conclude that SasX is a potential target for therapeutics or vaccines designed to moderate colonization and infection by sasX-positive epidemic strains of S. aureus.     

Respiratory disease in kennelled dogs: serological responses to Bordetella bronchiseptica lipopolysaccharide do not correlate with bacterial isolation or clinical respiratory symptoms

The role of Bordetella bronchiseptica in a natural outbreak of canine infectious respiratory disease was investigated both by culture and serological analysis. B. bronchiseptica was found in the lungs of a large proportion of clinically healthy dogs and in a greater proportion of dogs with respiratory disease. Using a lipopolysaccharide (LPS) antigen-based enzyme-linked immunosorbent assay, we analyzed the serological responses of a large number of dogs. Dogs with high antibody levels showed no protection from disease, and there was no correlation between the development of disease and rising antibody titer. Similarly, there was no difference in antibody levels in dogs with and without B. bronchiseptica in the lungs. Antibodies to LPS have no predictive value in determining which animals will contract respiratory disease, how severe the disease will be, or which dogs will have B. bronchiseptica colonizing the lungs.     

Interactions of pulmonary collectins with Bordetella bronchiseptica and Bordetella pertussis lipopolysaccharide elucidate the structural basis of their antimicrobial activities

Surfactant proteins A (SP-A) and D (SP-D) play an important role in the innate immune defenses of the respiratory tract. SP-A binds to the lipid A region of lipopolysaccharide (LPS), and SP-D binds to the core oligosaccharide region. Both proteins induce aggregation, act as opsonins for neutrophils and macrophages, and have direct antimicrobial activity. Bordetella pertussis LPS has a branched core structure and a nonrepeating terminal trisaccharide. Bordetella bronchiseptica LPS has the same structure, but lipid A is palmitoylated and there is a repeating O-antigen polysaccharide. The ability of SP-A and SP-D to agglutinate and permeabilize wild-type and LPS mutants of B. pertussis and B. bronchiseptica was examined. Previously, wild-type B. pertussis was shown to resist the effects of SP-A; however, LPS mutants lacking the terminal trisaccharide were susceptible to SP-A. In this study, SP-A was found to aggregate and permeabilize a B. bronchiseptica mutant lacking the terminal trisaccharide, while wild-type B. bronchiseptica and mutants lacking only the palmitoyl transferase or O antigen were resistant to SP-A. Wild-type B. pertussis and B. bronchiseptica were both resistant to SP-D; however, LPS mutants of either strain lacking the terminal trisaccharide were aggregated and permeabilized by SP-D. We conclude that the terminal trisaccharide protects Bordetella species from the bactericidal functions of SP-A and SP-D. The O antigen and palmitoylated lipid A of B. bronchiseptica play no role in this resistance.     

Functional and ultrastructural changes in alveolar macrophages from rabbits colonized with Bordetella bronchiseptica.

Alveolar macrophages from rabbits colonized with Bordetella bronchiseptica in their respiratory tract exhibited significant decreases in cell adherence, phagocytic uptake, and bactericidal activity compared with macrophages from uncolonized animals. These dysfunctions were accompanied by ultrastructural changes, including a decrease in overall cell density, a vacuolation of the rough endoplasmic reticulum, and an increase in organelle-poor cell surface projections.     

Anthrax Vaccine Antigen-Adjuvant Formulations Completely Protect New Zealand White Rabbits against Challenge with Bacillus anthracis Ames Strain Spores

In an effort to develop an improved anthrax vaccine that shows high potency, five different anthrax protective antigen (PA)-adjuvant vaccine formulations that were previously found to be efficacious in a nonhuman primate model were evaluated for their efficacy in a rabbit pulmonary challenge model using Bacillus anthracis Ames strain spores. The vaccine formulations include PA adsorbed to Alhydrogel, PA encapsulated in liposomes containing monophosphoryl lipid A, stable liposomal PA oil-in-water emulsion, PA displayed on bacteriophage T4 by the intramuscular route, and PA mixed with Escherichia coli heat-labile enterotoxin administered by the needle-free transcutaneous route. Three of the vaccine formulations administered by the intramuscular or the transcutaneous route as a three-dose regimen induced 100% protection in the rabbit model. One of the formulations, liposomal PA, also induced significantly higher lethal toxin neutralizing antibodies than PA-Alhydrogel. Even 5 months after the second immunization of a two-dose regimen, rabbits vaccinated with liposomal PA were 100% protected from lethal challenge with Ames strain spores. In summary, the needle-free skin delivery and liposomal formulation that were found to be effective in two different animal model systems appear to be promising candidates for next-generation anthrax vaccine development.     

Bordetella bronchiseptica adherence to cilia is mediated by multiple adhesin factors and blocked by surfactant protein A

In the virulent state (Bvg+), Bordetella bronchiseptica expresses adhesins and toxins that mediate adherence to the upper airway epithelium, an essential early step in pathogenesis. In this study, we used a rabbit tracheal epithelial cell binding assay to test how specific host or pathogen factors contribute to ciliary binding. The host antimicrobial agent surfactant protein A (SP-A) effectively reduced ciliary binding by Bvg+ B. bronchiseptica. To evaluate the relative contributions of bacterial adhesins and toxins to ciliary binding, we used mutant strains of B. bronchiseptica in the binding assay. When compared to Bvg+ or Bvg- phase-locked B. bronchiseptica strains, single-knockout strains lacking one of the known adhesins (filamentous hemagglutinin, pertactin, or fimbriae) displayed an intermediate ciliary binding capacity throughout the coincubation. A B. bronchiseptica strain deficient in adenylate cyclase-hemolysin toxin also displayed an intermediate level of adherence between Bvg+ and Bvg- strains and had the lowest ciliary affinity of any of the Bvg+ phase strains tested. A B. bronchiseptica strain that was missing dermonecrotic toxin also displayed intermediate binding; however, this strain displayed ciliary binding significantly higher than most of the adhesin knockouts tested. Taken together, these findings suggest that virulent-state B. bronchiseptica expresses multiple adhesins with overlapping contributions to ciliary adhesion and that host production of SP-A can provide innate immunity by blocking bacterial adherence to the ciliated epithelium.     

Controlled Inflammatory Responses in the Lungs Are Associated with Protection Elicited by a Pneumococcal Surface Protein A-Based Vaccine against a Lethal Respiratory Challenge with Streptococcus pneumoniae in Mice

Streptococcus pneumoniae is a pathogen of great importance worldwide. We have previously described the efficacy of a nasal vaccine composed of the pneumococcal surface protein A and the whole-cell pertussis vaccine as an adjuvant against a pneumococcal invasive challenge in mice. Spread of bacteria to the bloodstream was probably prevented by the high levels of systemic antibodies induced by the vaccine, but bacteria were only cleared from the lungs 3 weeks later, indicating that local immune responses may contribute to survival. Here we show that a strict control of inflammatory responses in lungs of vaccinated mice occurs even in the presence of high numbers of pneumococci. This response was characterized by a sharp peak of neutrophils and lymphocytes with a simultaneous decrease in macrophages in the respiratory mucosa at 12 h postchallenge. Secretion of interleukin-6 (IL-6) and gamma interferon (IFN-γ) was reduced at 24 h postchallenge, and the induction of tumor necrosis factor alpha (TNF-α) secretion, observed in the first hours postchallenge, was completely abolished at 24 h. Before challenge and at 12 h postchallenge, vaccinated mice displayed higher numbers of CD4(+) T, CD8(+) T, and B lymphocytes in the lungs. However, protection still occurs in the absence of each of these cells during the challenge, indicating that other effectors may be related to the prevention of lung injuries in this model. High levels of mucosal anti-PspA antibodies were maintained in vaccinated mice during the challenge, suggesting an important role in protection.     

Characterization of Protective Mucosal and Systemic Immune Responses Elicited by Pneumococcal Surface Protein PspA and PspC Nasal Vaccines against a Respiratory Pneumococcal Challenge in Mice

Pneumococcal surface protein A (PspA) and PspC are virulence factors that are involved in the adhesion of Streptococcus pneumoniae to epithelial cells and/or evasion from the immune system. Here, the immune responses induced by mucosal vaccines composed of both antigens as recombinant proteins or delivered by Lactobacillus casei were evaluated. None of the PspC vaccines protected mice against an invasive challenge with pneumococcal strain ATCC 6303. On the other hand, protection was observed for immunization with vaccines composed of PspA from clade 5 (PspA5 or L. casei expressing PspA5) through the intranasal route. The protective response was distinguished by a Th1 profile with high levels of immunoglobulin G2a production, efficient complement deposition, release of proinflammatory cytokines, and infiltration of neutrophils. Intranasal immunization with PspA5 elicited the highest level of protection, characterized by increased levels of secretion of interleukin-17 and gamma interferon by lung and spleen cells, respectively, and low levels of tumor necrosis factor alpha in the respiratory tract.Pneumococcal diseases kill more than 1 million children worldwide every year. The situation is worse in developing countries, where 90% of deaths occur. In Latin America, there are at least 1.6 million cases of pneumococcal disease every year, killing 18,000 children (53). While appropriate treatment, including the use of antibiotics and good nutrition, lowers the incidence of pneumococcal diseases, vaccines are the most efficacious way of preventing them. The existing pneumococcal conjugate vaccine dramatically reduces diseases, disabilities, and deaths, but elevated cost and protection restricted to included serotypes have prevented its implementation in large-scale immunization programs in developing countries. For these reasons, there is considerable interest in using conserved pneumococcal protein antigens as vaccines to provide cost-effective broad protection in all age groups. A number of leading candidates have been shown to elicit protection in mice (10, 51); among these antigens, two of the most promising candidates are pneumococcal surface protein A (PspA) and PspC.An additional concern in the development of cost-effective vaccines against pneumococcal disease is the route of immunization. Human vaccines are traditionally administered intramuscularly by needle inoculation, which brings the risk of transmitting blood-borne pathogens such as human immunodeficiency virus and hepatitis viruses (20). Furthermore, the cost of equipment and well-trained personnel for delivering vaccines by parenteral routes is several times higher than the cost of the vaccines themselves. This aspect is extremely important for vaccine implementation in large-scale immunization programs for developing countries. Mucosal delivery of pediatric vaccines has become an explicit goal of the WHO (20). Immunization via mucosal surfaces would greatly increase the ease of vaccination and would be more readily acceptable than parenteral immunization in many populations. Therefore, the move from injection to mucosal application would be very positive from economical, logistical, and safety standpoints. Mucosal immune responses are also important for the prevention of many infectious diseases because they represent the first barrier from the hosts that pathogens must evade.Research into the host immune response to pneumococcal diseases has focused primarily on the role of innate and adaptative humoral immune responses. However, in the last few years, attention has been drawn to cellular immune responses against Streptococcus pneumoniae, with interesting results. The majority of these studies analyzed cellular aspects of innate immunity and proposed that lymphocytes, neutrophils, and macrophages orchestrate effective immune responses without the presence of specific antibodies. In this context, proinflammatory cytokines promote an adequate milieu for pneumococcal clearance (22, 24, 25, 31, 34, 50, 54). A Th1-biased immune response has also been shown to be engaged in the resolution of pneumococcal infection in humans (21). Nevertheless, inflammatory cell influx into the lung and mucosal responses must be regulated to avoid exacerbated tissue injury. This is evidenced in recent studies of the role of γδ T cells and/or anti-inflammatory cytokines, such as interleukin-10 (IL-10), in pneumococcal infection (26, 42, 55).Protective immune responses against invasive pneumococcal disease and colonization were shown using pneumococcal whole-cell vaccines (28, 46) or recombinant proteins as mucosal vaccines (2, 6, 7, 9, 40). In recent approaches, lactic acid bacteria (3, 11, 18, 37), which are able to activate and modulate the innate immune system (35, 42), were used for pneumococcal antigen presentation, with promising results.Very few works compared pulmonary and systemic immune responses induced by pneumococcal antigens using parenteral and mucosal immunizations (13). The present study aims at investigating local and systemic cellular and humoral immune responses required for protection against invasive intranasal (i.n.) challenge with S. pneumoniae strain ATCC 6303 using PspA and PspC antigens administered by both routes, without the use of adjuvants, or presented by Lactobacillus casei through the nasal route.