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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.

Previous studies using rodent respiratory infection models of nontypeable Haemophilus influenzae (NTHi) infection have established the 26-kDa outer membrane protein of the bacterium, OMP26, as a potential vaccine antigen for NTHi. This study undertook a comprehensive immunological identification of OMP26 T- and B-cell epitopes. A series of OMP26 peptides were constructed and regions of the OMP26 antigen involved in recognition by lymphocyte receptors and induction of acquired immune responses were identified. The dominant T-cell epitopes for OMP26 were located toward the C-terminus between amino acid residues 95 and 197 (T3+T4 region) as mapped using antigen-specific lymphocyte proliferation assays. The newly identified T-cell epitopes exhibited strong capacity for efficient T-cell activation, suggesting that, compared with other OMP26 regions; epitopes within the T3+T4 region have the highest affinity for binding to major histocompatibility complex molecules. In contrast, the predominant B-cell epitopes of OMP26 were located more centrally within the molecule between amino acid residues 45 and 145 (T2+T3 region) as determined using enzyme-linked immunosorbent assay and surface plasmon resonance assays. The T2+T3 region was immunodominant in several species including chinchilla, mice and rats when assessed using both mucosal and parenteral immunization regimes. In addition, the antibodies directed against the T2+T3 region bound to intact NTHi cell surface, according to flow cytometry. Collectively, these results specifically locate the amino acid sequences containing the OMP26 T- and B-cell epitopes, which, as newly mapped antigenic epitopes for lymphocyte recognition, will be useful to improve existing NTHi vaccine strategies. Comprehensive definition of the minimum epitope length required for optimal B- and T-cell responses requires further study.

Pseudomonas aeruginosa is an important prognostic determinant in cystic fibrosis (CF). Little is known however, about P. aeruginosa induced local mucosal and systemic immune responses. Twenty CF children were categorized according to their P. aeruginosa status: (1) chronic lower respiratory tract infection (LRTI), (2) prior successfully treated initial LRTI, (3) isolated upper respiratory tract (URT) colonization, and (4) no known URT colonization or previous LRTI. Their antibody responses, and those of six non-CF disease controls, in serum and bronchoalveolar lavage (BAL) fluid to potential P. aeruginosa vaccine antigens outer membrane protein F (OprF), outer membrane protein H (OprH), catalase A (KatA) and a whole killed cell (WKC) extract were evaluated. Outer membrane protein G (OprG) responses were also measured in blood. Natural exposure, colonization and infection resulted in detectable antibody levels in BAL and serum in all CF groups. Both chronically infected and URT colonized CF children had substantially elevated immunoglobulin A antibody levels in the BAL fluid and sera toward the WKC extract and OprF antigen compared with the other groups of CF children and non-CF controls. The serum levels of specific P. aeruginosa antibodies involving immunoglobulin G and M isotypes increased with chronic LRTI, especially antibody levels to KatA, OprH and WKC extract, which were substantially greater in chronically infected children compared with all other groups. In conclusion, natural exposure, URT colonization and LRTI with P. aeruginosa all induce substantial mucosal and systemic antibody responses to potential vaccine antigens with chronically infected CF children having the highest levels.

Moraxella catarrhalis is a significant cause of respiratory tract infection against which a vaccine is sought. Several outer membrane proteins are currently under investigation as potential vaccine antigens, including the porin M35. We have previously shown that the third external loop of M35 was immunodominant over the remainder of the protein for antibody produced in mice against the refolded recombinant protein. However, as this loop is predicted to fold inside the porin channel we also predicted that it would not be accessible to these antibodies when M35 is expressed on the surface of the bacteria in its native conformation. This study investigated the functional activity of antibodies against M35 and those specific for the loop 3 region of M35 in vitro and in vivo. Antisera from mice immunized with M35 or the loop 3-deletion, M35loop3(-), recombinant proteins were not bactericidal but did have enhanced opsonic activity, whereas antibodies raised against the loop 3 peptide were not opsoniszing indicating that the immunodominant loop 3 of M35 was not accessible to antibody as we had previously predicted. Mucosal immunization with M35, M35 that had an antigenically altered loop 3 [M35(ID78)] and M35loop3(-) enhanced the clearance of M. catarrhalis from the lungs of mice challenged with live M. catarrhalis. The in vivo clearance of bacteria in the mice with the M35-derived protein constructs correlated significantly (p < 0.001) with the opsonic activity assessed an in vitro opsonophagocytosis assay. This study has demonstrated that the immunodominant B-cell epitope to loop 3 of the M. catarrhalis outer membrane protein M35 is not associated with immune protection and that M35-specific antibodies are not bactericidal but are opsoniszing. The opsoniszing activity correlated with in vivo clearance of the bacteria suggesting that opsoniszing antibody may be a good correlate of immune protection.

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.

Otitis media (OM) is a highly prevalent paediatric disease with both bacterial and viral triggers of infection. This study has investigated how combinations of bacteria associated with nasal colonisation and the occurrence and absence of viral infection (Sendai virus) induce OM in a mouse nasal colonisation model. The respiratory virus significantly contributed to bacterial OM for all bacterial combinations (p<0.001). Streptococcus pneumoniae consistently dominated as the causative bacterium of OM and when co-infected with S. pneumoniae, Moraxella catarrhalis more significantly affected pneumococcal OM than did non-typeable Haemophilus influenzae (p<0.001) by increasing the incidence rate, infection bacterial load and duration of infection. Nitric oxide levels in the middle ear, an indicator of inflammation, peaked at day 3 in single bacterium groups, but at day 1 in mixed bacterial groups and was produced in all bacteria inoculated groups even in the absence of viable bacterial recovery. Phagocytic cells were recruited rapidly to the ear following nasal inoculation but over time their numbers did not correlate with persistence of bacterial infection. The study has shown that the composition of bacteria in the nasal cavity and respiratory viral infection significantly affected the OM incidence rate, duration of infection and bacterial load (severity).

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.

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.

The receptors involved in the sampling of particulate microbial antigens by the gut are largely unknown. Here we demonstrate for the first time in an in vitro M-cell model and in situ in isolated murine intestinal segments that the receptors TLR-4, PAF-R, and alpha5beta1 integrin are all involved in mediating bacterial uptake associated with transcytosis. The pattern of expression of TLR-4 and alpha5beta1 integrin differed between M cells and enterocytes. There was increased apical expression of TLR-4 in M-cell cultures, and it was present on the apical surface of murine M cells but not enterocytes in situ. In contrast, PAF-R was expressed equally by both cell types in vitro and was abundantly expressed throughout the intestinal epithelium. Inhibition of TLR-4 and PAF-R, but not TLR-2, reduced gram-negative bacterial uptake by both cell types, whereas inhibition of the apically expressed alpha5beta1 integrin significantly reduced the ability of M cells to translocate bacteria. Hence, the involvement of each receptor was dependent not only on differences in the level of receptor expression but the cellular localization. Using bacteria that had mutations that affected the bacterial lipooligosaccharide structure indicated that the oligosaccharide moiety was important in bacterial uptake. Taken together, the data suggest that pathogen-associated molecular pattern interactions with pattern recognition receptors are key factors in M-cell recognition of intestinal antigens for mucosal immune priming.

Moraxella catarrhalis is a gram-negative bacterium that is mainly responsible for respiratory tract infections. In this study we report a novel outer membrane protein (OMP), designated M35, with a molecular mass of 36.1 kDa. This protein was structurally homologous to classic gram-negative porins, such as OMP C from Escherichia coli and OMP K36 from Klebsiella pneumoniae, with a predicted structure of 8 surface loops and 16 antiparallel beta-sheets. The DNA sequences of the genes from 18 diverse clinical isolates showed that the gene was highly conserved (99.6 to 100% of nucleotides), with only one isolate (ID78LN266) having base variations that resulted in amino acid substitutions. Electrophoresis and analysis of recognition of the protein using mouse anti-M35 sera showed that M35 was expressed on the bacterial surface and constitutively expressed across M. catarrhalis isolates, with only ID78LN266 showing poor antibody recognition. Our results showed that the single amino acid mutation in loop 3 significantly affected antibody recognition, indicating that loop 3 appeared to contain an immunodominant B-cell epitope. The antibody specificity to loop 3 may be a potential mechanism for evasion of host immune responses targeted to M35, since loop 3 should theoretically orientate into the porin channel. Thus, M35 is a highly conserved, surface-expressed protein that is of significance for its potential functional role as an M. catarrhalis porin and is of interest as a vaccine candidate.

The rat middle ear and lung clearance model has been used to show that the nontypeable Haemophilus influenzae 26-kDa outer membrane protein OMP26 is highly efficacious as a mucosal immunogen, inducing significantly enhanced clearance in immunized rats upon direct challenge of these two anatomic sites. Similarly, the chinchilla model of middle ear and nasopharyngeal clearance has been used to show that two P5 fimbrin adhesin-derived immunogens, LB1 and lipoprotein D (LPD)-LB1(f)(2,1,3), are highly efficacious as parenteral immunogens. Both induced significantly augmented clearance of nontypeable H. influenzae upon challenge of these sites. Here, these three nontypeable H. influenzae immunogens in addition to six bovine serum albumin and keyhole limpet hemocyanin conjugates of the synthetic peptide LB1(f) were assayed for relative efficacy in the reciprocal rodent model system. OMP26 was assayed in the chinchilla host by a parenteral immunization route, with clearance of the middle ear and nasopharynx used as outcome measures. Both LB1 and LPD-LB1(f)(2,1,3) were assayed in the rat host with a mucosal immunization route and clearance of nontypeable H. influenzae from the lungs and middle ears as outcome measures. Both of the immunogens were found to induce a high-titered and specific immune responses in the heterologous host system. Moreover, each was found to be highly efficacious in the reciprocal host system, providing strong support for the continued development and inclusion of both OMP26 and P5 fimbrin-derived peptides as candidate vaccine antigens directed at otitis media caused by nontypeable H. influenzae.

A rodent respiratory experimental model has proved useful for investigating the immune mechanisms responsible for clearance of bacteria from the lungs. Immunohistochemical studies in immune and nonimmune rats have identified the cellular kinetics of response to bacterial pulmonary infection for CD8+, CD4+, and gammadelta+ T cells; B cells; and the expression of major histocompatibility complex class II (MHC-II). During the course of bacterial clearance, there was no apparent proliferation or extravasation of lymphocytes, nor was there increased expression of MHC-II in nonimmune animals despite an influx of polymorphonuclear leukocytes, whereas in immunized animals there was an early influx of CD8+ and gammadelta+ T cells, followed by enhanced expression of the MHC-II marker, cellular infiltration by polymorphonuclear leukocytes, and finally an increased number of CD4+ T cells. Depletion of CD8+ T cells confirmed their vital contribution in the preprimed immune response to pulmonary infection by significantly decreasing the animals' ability to clear bacteria following challenge.

A 26-kDa protein (OMP26) isolated and purified from nontypeable Haemophilus influenzae (NTHI) strain 289 has been shown to enhance clearance of infection following pulmonary challenge with NTHI in rats. DNA sequence analysis revealed that it was 99% identical to a gene encoding a cell envelope protein of the H. influenzae Rd strain (TIGR accession no. HI0916). The deduced amino acid sequence revealed a hydrophilic polypeptide rich in basic amino acids. Restriction fragment length polymorphism analysis suggested that the OMP26 gene was relatively conserved among isolates of NTHI. Analysis of the deduced amino acid sequence of the OMP26 gene from 20 different isolates showed that similarity with NTHI-289 ranged from 96.5% (1 isolate) to 99.5% (14 isolates). Two recombinant forms of OMP26, a full length 28-kDa protein (equivalent to preprotein) and a 26-kDa protein lacking a 23-amino-acid leader peptide (equivalent to processed protein), were assessed in immunization studies for the ability to induce an immune response that would be as effective as the native protein in enhancing the clearance of NTHI following pulmonary challenge in rats. Immunization with the recombinant protein that included the leader peptide was more effective in enhancing pulmonary clearance, and it induced a better cell-mediated response and higher titers of systemic and mucosal antibody. This study has characterized a 26-kDa protein from NTHI that shows significant potential as a vaccine candidate.

Moraxella (Branhamella) catarrhalis is an important human respiratory tract pathogen. Outer membrane protein (OMP) CD is highly conserved among strains and has characteristics that indicate it may be an effective vaccine antigen. This study investigated the effect of immunization with OMP CD on pulmonary clearance following intratracheal challenge of mice with M. catarrhalis. Two routes of immunization were studied: mucosal immunization (intra-Peyer's patch followed by intratracheal boost) and intramuscular immunization with OMP CD. Both resulted in enhanced pulmonary clearance of M. catarrhalis compared with sham-immunized controls. Immunization with OMP CD induced specific antibodies in serum and bronchoalveolar lavage fluid and induced a specific lymphocyte proliferative response in T cells from mesenteric lymph nodes from mice mucosally immunized with OMP CD. On the basis of these results, OMP CD should undergo continued testing to determine whether it will induce a protective immune response in humans.

In this paper, we describe the ability of nontypeable Haemophilus influenzae (NTHi) to coexist with the human host and the devastating results associated with disruption of the delicate state of balanced pathogenesis, resulting in both acute and chronic respiratory tract infections. It has been seen that the strains of NTHi causing disease show a marked genetic and phenotypic diversity but that changes in the lipooligosaccharide (LOS) and protein size and antigenicity in chronically infected individuals indicate that individual strains of NTHi can remain and adapt themselves to avoid expulsion from their infective niche. The lack of reliance of NTHi on a single mechanism of attachment and its ability to interact with the host with rapid responses to its environment confirmed the success of this organism as both a colonizer and a pathogen. In vitro experiments on cell and organ cultures, combined with otitis media and pulmonary models in chinchillas, rats, and mice, have allowed investigations into individual interactions between NTHi and the mammalian host. The host-organism interaction appears to be a two-way process, with NTHi using cell surface structures to directly interact with the mammalian host and using secreted proteins and LOS to change the mammalian host in order to pave the way for colonization and invasion. Many experiments have also noted that immune system evasion through antigenic variation, secretion of enzymes and epithelial cell invasion allowed NTHi to survive for longer periods despite a specific immune response being mounted to infection. Several outer membrane proteins and LOS derivatives are discussed in relation to their efficacy in preventing pulmonary infections and otitis media in animals. General host responses with respect to age, genetic makeup, and vaccine delivery routes are considered, and a mucosal vaccine strategy is suggested.

A major outer membrane protein band of approximately 25 to 27 kDa is commonly observed in strains of Haemophilus influenzae. This study has investigated the potential of a 26-kDa protein (OMP26) from nontypeable H. influenzae (NTHI) as a vaccine candidate. OMP26 was used to immunize rats via intestinal Peyer's patches, followed by an intratracheal boost. Immunization was found to significantly enhance bacterial clearance following pulmonary challenge with both the homologous NTHI strain and a different NTHI strain. Significant levels of anti-OMP26 were found in the serum and bronchoalveolar lavage from immunized rats, and isotypes of immunoglobulin G (IgG) were also measured in serum. Analysis of IgG isotypes present in serum following OMP26-immunization suggest that predominantly a T-helper 1-type response was induced. The OMP26 protein was amino-terminally sequenced and found to have no homology with the P5 of H. influenzae type b P5 or the fimbrin protein of NTHI, both can migrate upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis at similar molecular masses but OMP26 has 100% homology with a segment of the H. influenzae Rd genome. The results of this study suggest that OMP26 may be a suitable vaccine candidate against NTHI infection and warrants continued investigation and characterization.

Moraxella (Branhamella) catarrhalis is a common respiratory tract pathogen in man. The bacterium shows a strong tendency to form aggregates in vitro. A variant strain of M. catarrhalis that showed a reduced tendency to form aggregates was selected by successive in-vitro passage in broth culture from which aggregates had settled. The non-clumping variant strain showed alteration in expression of outer-membrane antigens, including the HMW-OMP, an outer-membrane protein of c. 200 kDa, outer-membrane protein CD and lipo-oligosaccharide. A mouse model for pulmonary challenge with M. catarrhalis revealed significant differences in the rate of clearance of the isogenic variant strains from the lung. The parent strain caused enhanced recruitment of neutrophils to the lung and more rapid clearance of bacteria from the lungs in comparison to the non-clumping variant. It is concluded that alteration of expression of surface molecules by M. catarrhalis has a significant impact in an in-vivo model of pulmonary clearance.

Nontypeable Haemophilus influenzae (NTHi) is a common cause of infection of the respiratory tract in children and adults. The search for an effective vaccine against this pathogen has focused on components of the outer membrane, and peptidoglycan-associated lipoprotein P6 is among the proposed candidates. This study investigated the immunogenicity of P6 in a rat respiratory model. P6 was purified from two strains of NTHi, one capsule-deficient strain and an H. influenzae type b strain, and assessed for clearance of both homologous and heterologous bacterial strains following mucosal immunization. A protective immune response was determined by enhancement of pulmonary clearance of live bacteria and an increased rate of recruitment of phagocytic cells to the lungs. This was most effective when Peyer's patch immunization was accompanied by an intratracheal (IT) boost. However, the rate of bacterial clearance varied between strains, which suggests some differences in anti-P6 immunological defenses recognizing the expression of the highly conserved P6 lipoprotein on the bacterial surface in some strains. P6-specific antibodies in both serum and bronchoalveolar lavage fluid were cross-reactive and did not differ significantly in strain specificity, demonstrating that difference in clearance was unlikely due to differences in P6-specific antibody levels. Serum homologous and heterologous P6-antibody was bactericidal against NTHi even when enhanced clearance had not been observed. Peyer's patch immunization induced P6-specific CD4+ T-helper cell proliferation in lymphocytes isolated from the mesenteric lymph nodes. An IT boost increased the level of P6-specific antibodies in serum and bronchoalveolar lavage fluid, and P6-specific mesenteric node lymphocyte proliferation. Cells from rats immunized with P6 demonstrated proliferation following stimulation with P6 from nonhomologous strains; however, there was some variation in proliferative responses to P6 from different strains in lymphocytes isolated from animals immunized with killed bacteria. The increase in P6-specific antibodies and T-helper cell responses following an IT boost correlated with an increased rate of recruitment of phagocytic cells and enhanced bacterial clearance of both homologous and heterologous bacteria in the lungs. The data suggests that P6 has the potential to afford protection against pulmonary infection by NTHi following the induction of effective antigen-specific B- and T-cell responses in mucosal tissues.

Outer membrane proteins P2, P4, and P6 and two with molecular masses of 26 and 28 kDa have been purified from a strain of nontypeable Haemophilus influenzae by a preparative form of polyacrylamide gel electrophoresis (PAGE). Outer membrane protein P6, with a molecular mass of 16 kDa (determined by sodium dodecyl sulfate [SDS]-PAGE) was purified by both native PAGE and SDS-PAGE from three strains of nontypeable H. influenzae and one strain of type b H. influenzae. The same conditions were required for purification from each strain. The suitability of proteins isolated by these methods was assessed by studying the immune response of rats immunized with P6 in incomplete Freund's adjuvant into the Peyer's patches. P6 purified by either native PAGE or SDS-PAGE did not differ significantly from P6 purified by gel filtration and anion-exchange chromatography in the ability to enhance pulmonary clearance of live bacteria. This study also investigated the effects of SDS on P2 immunological responses in vivo and the effects of the reagents Zwittergent and sodium lauryl sarcosinate on outer membrane protein lymphocyte-proliferative responses in vitro. It was found that the presence of SDS in the immunization emulsion enhanced the antigen-specific cell-mediated response but suppressed the antigen-specific antibody responses. The presence of residual traces of Zwittergent in an outer membrane protein preparation inhibited antigen-specific cell-mediated proliferation, whereas extraction of outer membrane proteins with sodium lauryl sarcosinate did not inhibit antigen-specific proliferation. These results demonstrate that preparative PAGE is a suitable method for the purification of proteins from the outer membrane of H. influenzae required for investigation of their immunological significance as vaccine candidates and that traces of reagents used during protein purification may play an important role in determining the success of in vivo and in vitro studies.