Otopathogen interactions in the nasopharynx of children, and the predictive value of nasopharyngeal aspirate culture for the aetiology of upper respiratory infections.

AIM: To evaluate nasopharyngeal aspirate cultures for screening otopathogen carriage in the adenoid in children 2-7 years of age. METHODS: Thirty-seven children, 2-7 years of age, scheduled for adenoidectomy were enrolled into this prospective study at Rockhampton, Australia. Adenoid biopsy and nasopharyngeal aspirate bacteriology were assessed by conventional culture. Demographic and environmental data were collected by questionnaire. Statistical analyses for descriptive, comparison and logistic regression tests between microbial, demographic, environmental and clinical groups were applied. RESULTS: Streptococcus pneumoniae, Staphylococcus aureus, non-typeable Haemophilus influenzae and Moraxella catarrhalis were detected in 38, 38, 35 and 24% of cases, respectively. Streptococcus pneumoniae was an independent determinant for non-typeable H. influenzae and S. aureus colonisation, and S. aureus was an independent determinant for S. pneumoniae colonisation. The nasopharyngeal aspirate otopathogen cultures were strong predictors for otopathogens in the adenoid, with moderate-high test accuracy for all otopathogens (receiver operator characteristics area under the curve ranging from 71 to 97% for the otopathogens tested). Children with positive non-typeable H. influenzae, M. catarrhalis, S. pneumoniae and S. aureus nasopharyngeal aspirate cultures were more likely to have the equivalent species in adenoid cultures (positive likelihood ratios = undefined, 15.0, 9.09 and 5.85, respectively). CONCLUSIONS: This study provides evidence that nasopharyngeal aspirate cultures are an indicator of otopathogens in the adenoid. Nasopharyngeal aspirate cultures may provide clinicians with information that informs clinical management. Strategies for improved management to reduce otopathogen carriage could reduce the prevalence of chronic upper respiratory infections that contribute to adenoidectomy.

Effectiveness of engineering the nontypeable Haemophilus influenzae antigen Omp26 as an S-layer fusion in bacterial ghosts as a mucosal vaccine delivery.

The potential of empty bacterial cell envelopes (ghosts) as a delivery system for mucosal immunization was assessed in a rat model and different routes of immunization were evaluated. Animals were mucosally immunized targeting either gut only or gut and lung mucosal sites with Escherichia coli ghosts harbouring the nontypeable Haemophilus influenzae (NTHi) antigen Omp26. Omp26 was expressed as either a part of an S-layer fusion or as a soluble protein in the periplasm. In the gut/lung regime two initial gut targeted inoculations with the ghosts were followed by an intratracheal (IT) boost with purified Omp26. The gut only immunization regime showed a moderate enhancement of bacterial clearance following pulmonary challenge whereas the gut/lung immunization regime resulted in significantly enhanced pulmonary clearance of NTHi. Both immunization regimes induced high levels of Omp26 specific antibodies in the serum of immunized rats, with higher levels in the groups that received the IT boost with purified Omp26. Analysis of IgG isotypes present in serum suggest that the immune response was predominantly of a T-helper1 type. Additionally, immunization induced a significant cellular immune response with lymphocytes from animals vaccinated using the gut/lung regime responding significantly to Omp26 when compared to control groups. The results of this study show that mucosal immunization with recombinant Omp26 in E. coli ghosts followed by a boost with purified Omp26 can induce a specific and protective immune response in a rodent model of acute lung infection.

Developmental profiles of mucosal immunity in pre-school children.

This study investigated the effect of attending pre-school on mucosal immunity. Children 3.5 to 5 years of age who attended pre-school were observed for a 10 month period. Demographic information was collected on previous childcare experiences, the home environment and clinical information relating to the child and the family. A daily illness log was kept for each child. A multivariate longitudinal analysis of the relation between immunoglobulins in saliva and age, gender, childcare experience, pre-school exposure, number of siblings, environmental tobacco smoke (ETS), atopy and hospitalisation was conducted. There was a positive association of higher IgA levels with the winter season and with children being older than 4 years (P < .001), having attended childcare prior to commencing pre-school (P < .05), and having been exposed to ETS at home (P < .05). Lower IgA levels were associated with being atopic (P < .05). Higher IgG levels were associated with exposure to ETS (P < .001), while lower levels were associated to having atopy. Higher IgM levels were associated with previous childcare experience (P < .01) whilst having been hospitalised was associated with having low salivary IgM levels (P < .01). Lagged analyses demonstrated that immunological parameters were affected by the number of respiratory infections in the preceding 2 months.

Nontypeable Haemophilus influenzae as a pathogen in children.

Nontypeable Haemophilus influenzae is a significant pathogen in children, causing otitis media, sinusitis, conjunctivitis, pneumonia, and occasionally invasive infections. H. influenzae type b conjugate vaccines have no effect on infections caused by nontypeable strains because nontypeable strains are nonencapsulated. Approximately, one-third of episodes of otitis media are caused by nontypeable H. influenzae and the bacterium is the most common cause of recurrent otitis media. Recent progress in elucidating molecular mechanisms of pathogenesis, understanding the role of biofilms in otitis media and an increasing understanding of immune responses have potential for development of novel strategies to improve prevention and treatment of otitis media caused by nontypeable H. influenzae. Feasibility of vaccination for prevention of otitis media due to nontypeable H. influenzae was recently demonstrated in a clinical trial with a vaccine that included the surface virulence factor, protein D.

Regulatory T lymphocytes are associated with increased nasopharyngeal colonization in children.

OBJECTIVES: Regulatory T lymphocytes (Treg) have been linked to survival of commensal bacteria at mucosal sites, but their presence and role in chronic otitis media (COM) and their response to otopathogens has not been evaluated previously. We investigated the association between Treg lymphocytes and otopathogens in COM prone and non-COM prone children. METHODS: Forty children, 2-7 years of age, scheduled for adenoidectomy were enrolled into COM (n = 20) or non-COM (n = 20) groups. Adenoid biopsy and nasopharyngeal aspirate bacteriology were assessed by conventional culture techniques. Peripheral blood and adenoid lymphocytes were stained with viability stain, monoclonal anti-CD19, anti-CD3, anti-CD4, anti-CD8, anti-CD25 and anti-CD127. Cells were stained intracellularly with monoclonal anti-FoxP3 and then quantified by flow cytometry. RESULTS: Children with nasopharyngeal otopathogen-positive culture had significantly more circulating CD3+CD4+FoxP3+CD25hi+CD127lo+ lymphocytes (M = 4.4%) compared to culture-negative children (M = 3.1%, p = 0.005). Circulating CD19+ lymphocytes were significantly increased in children with positive Moraxella catarrhalis nasopharyngeal culture (M = 12.4%) compared to culture-negative children (M = 8.6%, p = 0.006). Adenoid-derived lymphocytes were not significantly different in children with any positive nasopharyngeal culture compared to negative culture. Lymphocyte subsets were not significantly different between COM and non-COM prone children. CONCLUSION: Clinically-detectable otopathogen nasopharyngeal culture is positively associated with Treg lymphocytes, potentially inducing suppressive effector responses to promote colonization and infection chronicity. This finding supports further investigation of Treg lymphocyte activity and influence on upper airway colonization of young children.

Moraxella catarrhalis M35 is a general porin that is important for growth under nutrient-limiting conditions and in the nasopharynges of mice.

Moraxella catarrhalis is a gram-negative respiratory pathogen that is an important causative agent for otitis media and exacerbations of chronic obstructive pulmonary disease. We have previously predicted the outer membrane protein M35 to be a general porin, and in the current study, we have investigated the function of M35 and its importance for survival of M. catarrhalis in vivo. Lipid bilayer experiments reveal that refolded M35 functions as a channel that is typical of gram-negative bacterial porins. M35 forms wide and water-filled channels with a single-channel conductance of about 1.25 nS in 1 M KCl solution and has only a small selectivity for cations over anions. When the in vitro growth characteristics of two M35 deletion mutant strains of M. catarrhalis were compared to the wild-type parent isolates, the growth of the mutant strains was inhibited only under nutrient-poor conditions. This growth defect could be eliminated by additional glutamic acid, but not additional aspartic acid, glycine, sucrose, or glucose. The mutant strains compensated for the lack of M35 by enhancing their uptake of glutamic acid, and this enhanced rate of glutamic acid uptake was attributed to the compensatory upregulation of a protein of approximately 40 kDa. M35 was also found to be essential for nasal colonization of mice, demonstrating that its presence is essential for survival of M. catarrhalis in vivo. These results suggest that M35 is a general porin that is necessary for the uptake of important energy sources by M. catarrhalis and that it is likely that M35 is an essential functional protein for in vivo colonization.

Challenges for the development of vaccines against Haemophilus influenzae and Neisseria meningitidis.

The development of protein-polysaccharide conjugate vaccines has had a major impact on Haemophilus influenzae type b disease. The application of this technology to Neisseria meningitidis is also striking, particularly for serogroup C. However, significant challenges exist for the development of vaccines against non-typeable H. influenzae and against N. meningitidis serogroup B. Issues such as non-vaccine-strain replacement and correlates of protection need to be addressed as well as the longer-term implications of vaccination against what are essentially 'normal' microflora.

Matrix effect and cross-reactivity of select amphetamine-type substances, designer analogues, and putrefactive amines using the Bio-Quant direct ELISA presumptive assays for amphetamine and methamphetamine.

The aim of this study was to evaluate the Bio-Quant Direct ELISA assays for amphetamine and methamphetamine in the routine presumptive screening of biological fluids. Standard concentration curves of the target analytes were assayed to assess sensitivity, and known concentrations of common amphetamine-type substances (ephedrine, pseudoephedrine, phentermine), designer analogues (MDA, MDMA, MDEA, MBDB, PMA, 4-MTA, 2CB), and putrefactive amines (phenylethylamine, putrescine, tryptamine, tyramine) were analyzed to determine cross-reactivity. Results of the standard curve studies show the capacity of both Direct ELISA kits to confidently detect down to 3 ng/mL interday (PBS matrix; CVs 6.3-15.5%). Cross-reactivity relative to that of 50 ng/mL preparations of the target compounds demonstrated that the Direct ELISA kit for amphetamine also detected MDA (282%), PMA (265%), 4-MTA (280%), and phentermine (61%), and the Direct ELISA for methamphetamine also assayed positive for MDMA (73%), MDEA (18%), pseudoephedrine (19%), MBDB (8%), and ephedrine (9%). Matrix studies demonstrated that both ELISA kits could be applied to screening of blood, urine, and saliva to a concentration of 6 ng/mL or lower. In conclusion, the Bio-Quant Direct ELISA kits for amphetamine and methamphetamine are fast and accurate and have demonstrated themselves to be useful tools in routine toxicological testing.

Panel 5: Immunology.

Objective To perform a state-of-the-art review of the literature from January 2012 through May 2015 on studies that advanced our knowledge of the innate and adaptive immunology related to otitis media. This review also proposes future directions for research in this area. Data Sources PubMed database of the National Library of Medicine. Review Methods Three subpanels comprising experts in the field focused on sections relevant to cytokines, innate immunity, and adaptive immunity. The review focused on animal, cell line, and human studies and was critical in relation to the recommendations from the previous publication and for determination of the proposed goals and priorities. The panel met at the 18th International Symposium on Recent Advances in Otitis Media in June 2015 to consolidate its prior search results and discuss, plan, and refine the review. The panel approved the final draft. Conclusion From 2012 to 2014, tremendous progresses in immunology of otitis media were established-especially in the areas of innate immunity associated with the pathogenesis of otitis media. Implications for Practice The advances of the past 4 years formed the basis for a series of short- and long-term research goals in an effort to guide the field. Accomplishing these goals will provide opportunities for the development of novel interventions, including new ways to better treat and prevent otitis media, especially for recurrent otitis media.

A demonstration of the use of ultra-performance liquid chromatography-mass spectrometry [UPLC/MS] in the determination of amphetamine-type substances and ketamine for forensic and toxicological analysis.

We have recently seen the emergence of ultra-performance liquid chromatography (UPLC) coupled to mass spectrometry as an alternative to traditional high-performance liquid chromatography techniques. The strengths of UPLC technology promote the ability to separate and identify drug compounds with significant gains in resolution and sensitivity and marked reductions in the overall time of analysis. As increased throughput is the desire of the practical toxicology laboratory, the aim of this study was to trial commercially available technology by assessment of the separation of several commonly encountered amphetamine-type substances. From injection of a poly-drug reference standard and whole blood extract, we successfully separated and identified amphetamine, methamphetamine, ephedrine, pseudoephedrine, phentermine, MDA, MDMA, MDEA and ketamine in less than 3 min using the Acquity UPLC-Micromass Quattro Micro API MS instrumentation (Waters Corporation, USA). In addition to this significant reduction in overall run time, all peaks exhibited acceptable resolution using selected ion recording (SIR), with analysis indicating the capability to separate 5-11 peaks in 1.75 min using the current system parameters. From this introductory data, it is therefore indicated that the technological advancements defining ultra-performance liquid chromatography will allow it to serve as a powerful analytical tool for rapid throughput analysis.

Enterocyte and M-cell transport of native and heat-denatured bovine beta-lactoglobulin: significance of heat denaturation.

The three-dimensional structure, digestibility, and immunological properties of bovine beta-lactoglobulin (beta-lg) are modified by heat treatments used in processing of liquid milk products. Because it is not known if such treatments also modify the intestinal transport properties of beta-lg, the transport of native and heat-denatured bovine beta-lg was investigated in experimental cell models using Caco-2 cells and M cells. Transport of beta-lg labeled with a fluorescent marker was followed with fluorometric measurements, electrophoretic analyses, and fluorescence microscopy. The data show that both cell types transported native beta-lg more efficiently than they did heat-denatured beta-lg. In addition, M cells transported native beta-lg more than Caco-2 cells. Transport of native and heat-denatured beta-lg was transcellular. The electrophoretic data also suggest that heat-denatured beta-lg may have degraded more than native beta-lg during the transport.

Impact of saliva collection methods on sIgA and cortisol assays and acceptability to participants.

In community-based studies of stress and immunity, saliva samples offer a non-intrusive way of gathering biological data. Cotton-based devices are widely used in cortisol research, but some may affect assay results. We compared assay reliability and perceived acceptability of three saliva collection methods: passive, cotton 'salivettes' and cellulose-cotton tip 'eyespears'. Compared to passive collection, salivettes reduced the concentration of cortisol (p = .001) and sIgA (p = .002). Eyespears did not reduce cortisol or sIgA concentration, and showed less interference in the rank ordering of cortisol (r(eyespear with passive) = .90) and sIgA scores (r(eyespear with passive) = .96) compared to salivettes (r cortisol(salivette with passive) = .79; r sIgA(salivette with passive) = .66). The comfort and acceptability of both cotton-based devices were rated positively. Cotton-cellulose eyespears could offer methodological advantages for collecting saliva to measure cortisol and sIgA levels, and, because they can be held during sampling, may be useful for research with children and the frail elderly.

Mucosal immunisation with novel Streptococcus pneumoniae protein antigens enhances bacterial clearance in an acute mouse lung infection model.

Streptococcus pneumoniae contains many proteins that have not been evaluated as potential protective vaccine antigens. In this study we isolated proteins from a serotype 3 strain of S. pneumoniae for use in mouse immunisation studies. Separation of the protein mix was achieved by SDS-PAGE electrophoresis followed by electro-elution to isolate individual proteins. This procedure successfully separated 21 fractions from which six proteins were selected based on purity and quantity and were initially denoted by their molecular masses: 14-, 34-, 38-, 48-, 57- and 75-kDa. The immunogenicity of these proteins was investigated in a mucosal immunisation model in mice involving a primary inoculation to the intestinal Peyer's patches followed by an intra-tracheal boost two weeks later. The immune response was assessed by enhancement of pulmonary clearance of infection, recruitment of phagocytes to the lungs and induction of an antibody response. Two of the proteins, the 14-kDa identified as a L7/L12 ribosomal protein, and the 34-kDa identified as glyceraldehyde-3-phosphate dehydrogenase resulted in up to 99% and 94%, respectively, enhanced clearance of infection within 5 h following pulmonary challenge with S. pneumoniae. This study has shown that novel pneumococcal proteins have the potential to be vaccine candidates to enhance clearance of an acute mucosal S. pneumoniae infection.

Short tandem repeat (STR) genotyping of keratinised hair. Part 1. Review of current status and knowledge gaps.

We present a review of the literature on procedures for obtaining short tandem repeat (STR) genotypes from keratinised hair, being either hair shaft or telogen phase (naturally shed) hairs without associated scalp, follicle or sheath cells. Both the hair shaft and the telogen hair club have been subjected to the DNA-degrading keratinisation process and are more likely to be found at a crime scene than anagen (plucked) or catagen phase hairs. We discuss human hair structure, the human hair growth cycle, the keratinisation process and their implications for DNA extraction procedures, PCR amplification strategies and the interpretation of STR genotypes. Knowledge gaps and areas requiring research are identified and are the subject of a second article in this series.

Short tandem repeat (STR) genotyping of keratinised hair. Part 2. An optimised genomic DNA extraction procedure reveals donor dependence of STR profiles.

A feasibility study of short tandem repeat (STR) genotyping of telogen phase hairs in particular, and hair shaft in general, is presented. A number of extraction procedures in common use were investigated and the quantities of nuclear DNA (nuDNA) delivered were quantified via a real-time PCR assay. The extracts were subjected to two variations on AmpFlSTR Profiler Plus PCR amplification strategies (extended cycles, two rounds of PCR) and the genotypes compared. Nuclear DNA was found to persist in human hair shafts, albeit at very low levels. Full Profiler Plus profiles consistent with the hair donor were obtained from 100 mg hair shaft samples (bleached and unbleached). These were, however, mixed profiles, indicating low copy number (LCN) contamination in the extracts. Single telogen hair clubs and single hair shafts delivered partial profiles with usually only one allele of heterozygous loci. Telogen phase hairs yielded the same amount of nuDNA (and no more) as hair shafts (either anagen or telogen). Whether hair shafts dissolved or not in lysis buffer had no effect on either the quantitated yield of DNA or on the chance of obtaining a correct genotype. These results provide evidence that genomic DNA resides on the exterior of the hair shaft and we use this information to suggest an optimal procedure for nuDNA extraction from keratinised hair samples: soaking hairs in simple digestion buffers containing Tris-HCl, a salt and a chelating agent without prior cleaning of the hair shafts. It is proposed that cleaning removes most of the recoverable DNA. This procedure was applied to obtain genotypes from 3 cm hair shafts which matched reference profiles from the donors at up to 9 out of 10 AmpFlSTR Profiler Plus STR loci. When the genotyping success was measured by counting the number of matches between the two dominant alleles at each locus for each extract with the reference DNA profile of the hair donor, the success was found to be highly dependent on the donor. The number of matching alleles varied between not less than 10 for one donor to no more than two for another donor. These results may well be linked to the environmental experience of the hairs from each donor prior to removal.

Vaccination against respiratory Pseudomonas aeruginosa infection.

Respiratory infections caused by Pseudomonas aeruginosa are a major clinical problem globally, particularly for patients with chronic pulmonary disorders, such as those with cystic fibrosis (CF), non-CF bronchiectasis (nCFB) and severe chronic obstructive pulmonary disease (COPD). In addition, critically ill and immunocompromised patients are also at significant risk of P. aeruginosa infection. For almost half a century, research efforts have focused toward development of a vaccine against infections caused by P. aeruginosa, but a licensed vaccine is not yet available. Significant advances in identifying potential vaccine antigens have been made. Immunisations via both the mucosal and systemic routes have been trialled in animal models and their effectiveness in clearing acute infections demonstrated. The challenge for translation of this research to human applications remains, since P. aeruginosa infections in the human respiratory tract can present both as an acute or chronic infection. In addition, immunisation prior to infection may not be possible for many patients with CF, nCFB or COPD. Therefore, development of a therapeutic vaccine provides an alternative approach for treatment of chronic infection. Preliminary animal and human studies suggest that mucosal immunisation may be effective as a therapeutic vaccine against P. aeruginosa respiratory infections. Nevertheless, more research is needed to improve our understanding of the basic biology of P. aeruginosa and the mechanisms needed to upregulate the induction of host immune pathways to prevent infection. Recognition of variability in the host immune responses for a range of patient health conditions at risk from P. aeruginosa infection is also required to support development of a successful vaccine delivery strategy and vaccine. Activation of mucosal immune responses may provide improved efficacy of vaccination for P. aeruginosa during both acute exacerbations and chronic infection.

Mucosal immunization against respiratory bacterial pathogens.

Bacterial respiratory diseases remain a major cause of morbidity and mortality throughout the world. The young and the elderly are particularly susceptible to the pathogens that cause these diseases. Therapeutic approaches remain dependent upon antibiotics contributing to the persistent increases in antibiotic resistance. The main causes of respiratory disease discussed in this review are Mycobacterium tuberculosis, Corynebacterium diphtheriae, Bordatella pertussis, Streptococcus pneumoniae, non-typeable Haemophilus influenzae, Moraxella catarrhalis and Pseudomonas aeruginosa. All these organisms initiate disease at the mucosal surface of the respiratory tract and thus the efficacy of the host's response to infection needs to be optimal at this site. Vaccines available for diseases caused by many of these pathogens have limitations in accessibility or efficacy, highlighting the need for improvements in approaches and products. The most significant challenges in both therapy and prevention of disease induced by bacteria in the respiratory tract remain the development of non-injectable vaccines and delivery systems/immunization regimens that improve mucosal immunity.

Immunisation with non-integral OMPs promotes pulmonary clearance of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that can cause fatal acute lung infections in critically ill individuals. Lung damage due to chronic infections in cystic fibrosis sufferers is the major cause of morbidity and mortality in this group. The bacterium produces various immunomodulatory products that enable it to survive in the lung. Innate and increasing resistance to antibiotic therapy shown by this organism heightens the need for development of a vaccine. This study reports the identification of six non-integral protein antigens; Pa13, azurin, acyl carrier protein (ACP), amidase, aminopeptidase and KatE, purified from a mucoid strain of P. aeruginosa. N-terminal amino acid sequencing was used to identify these proteins and, based on their ascribed functions, determined that their normal cellular location was cytosolic. A rat model of acute pulmonary infection was used to investigate the ability of these protein antigens to enhance pulmonary clearance of a live P. aeruginosa challenge. Mucosal immunisation with four of the six antigens significantly enhanced bacterial clearance from both the lavage fluid and lung tissue. The greatest level of clearance was demonstrated for the antigens; KatE, aminopeptidase and amidase. Enhanced bacterial clearance was maintained when the antigens amidase and aminopeptidase were produced in recombinant form. When delivered parenterally, aminopeptidase demonstrated its continued efficacy as a vaccine candidate. This study has demonstrated that non-integral outer membrane proteins are antigenic and protective and warrant further investigation as potential components of a vaccine.

Pseudomonas aeruginosa-specific IgG1 and IgG2 subclasses in enhancement of pulmonary clearance following passive immunisation in the rat.

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen, which causes serious debilitating infections in patients with compromised lung function. The mechanism by which P. aeruginosa is cleared from the lung is not fully defined, although our previous studies have established a role for cellular immunity in protection against P. aeruginosa infections. This study aimed to evaluate the role of P. aeruginosa-specific IgG in protection against P. aeruginosa in a rat model of acute pulmonary infection. Immunoaffinity chromatography was used to purify total rat IgG from rat immune serum (rats immunised with P. aeruginosa) and non-immune serum. Untreated recipient rats were injected intravenously with different concentrations of pure IgG prepared from serum of unimmunised rats (non-immune IgG) or from rats immunised intestinally with killed P. aeruginosa (immune IgG) and infected intratracheally with P. aeruginosa 18 h later. The protective capability of the purified IgG against P. aeruginosa was assessed by measurement of reduction in P. aeruginosa infection in the lung 4 h after instillation of bacteria. Enhanced bacterial clearance induced by IgG was determined to be dose-dependent with a 1 mg dose failing to enhance clearance, whereas 5 mg of immune IgG enhanced clearance from the airways and the lung tissue. Measurement of the IgG1, IgG2a and IgG2b isotypes in serum and the lung lavage following transfer of P. aeruginosa-specific IgG found that all three were present. These results demonstrate that anti-P. aeruginosa IgG can enhance bacterial clearance from the airways in an acute infection and identify an important role for IgG in acute respiratory infections caused by P. aeruginosa.

Construction of recombinant S-layer proteins (rSbsA) and their expression in bacterial ghosts--a delivery system for the nontypeable Haemophilus influenzae antigen Omp26.

This study has investigated the feasibility of a combination of recombinant surface layer (S-layer) proteins and empty bacterial cell envelopes (ghosts) to deliver candidate antigens for a vaccine against nontypeable Haemophilus influenzae (NTHi) infections. The S-layer gene sbsA from Bacillus stearothermophilus PV72 was used for the construction of fusion proteins. Fusion of maltose binding protein (MBP) to the N-terminus of SbsA allowed expression of the S-layer in the periplasm of Escherichia coli. The outer membrane protein (Omp) 26 of NTHi was inserted into the N-terminal and C-terminal regions of SbsA. The presence of the fused antigen Omp26 was demonstrated by Western blot experiments using anti-Omp26 antisera. Electron microscopy showed that the recombinant SbsA maintained the ability to self-assemble into sheet-like and cylindrical structures. Recombinant E. coli cell envelopes (ghosts) were produced by the expression of SbsA/Omp26 fusion proteins prior to gene E-mediated lysis. Intraperitoneal immunization with these recombinant bacterial ghosts induced an Omp26-specific antibody response in BALB/c mice. These results demonstrate that the NTHi antigen, Omp26, was expressed in the S-layer self-assembly product and this construct was immunogenic for Omp26 when administered to mice in bacterial cell envelopes.