Susceptibility of nontypeable Haemophilus influenzae to human beta-defensins is influenced by lipooligosaccharide acylation

Nontypeable Haemophilus influenzae (NTHI) lipooligosaccharide htrB mutants exhibited greater than 45-fold-increased sensitivity to human beta-defensin 2 (HBD-2) compared to the wild type. Complementation by htrB in trans to acylation competence reversed this increased sensitivity. In contrast, NTHI was more susceptible to HBD-3 and showed no changes in sensitivity as a result of lipooligosaccharide mutations in oligosaccharide and lipid A biosynthesis genes.     

Differential involvement of toll-like receptors 2 and 4 in the host response to acute respiratory infections with wild-type and mutant Haemophilus influenzae strains

We used a mouse model of acute respiratory infections to investigate the role of Toll-like receptor 2 (TLR2) and TLR4 in the host response to Haemophilus influenzae. Acute aerosol exposures to wild-type strains of H. influenzae showed that TLR4 function was essential for TNF-alpha induction, neutrophil influx, and bacterial clearance. To determine how lipooligosaccharide (LOS) modifications would affect the role of TLR4 in inducing the host response, we used acute infections with an H. influenzae strain expressing a mutation in the htrB gene. This mutant strain expresses an LOS subunit with decreased acylation. In response to H. influenzae htrB infection, tumor necrosis factor alpha (TNF-alpha) secretion remained TLR4 dependent. But the decrease in LOS acylation made the neutrophil influx and the bacterial clearance also dependent on TLR2, as shown by the decreased host response elicited in TLR2 knockout mice compared to C57BL/6 mice. A subsequent analysis of TLR2 and TLR4 gene expression by quantitative PCR indicated that TLR4 function induces TLR2 expression and vice versa. These results indicate that some changes in the LOS subunit of H. influenzae can favor signaling through non-TLR4 receptors, such as TLR2. The results also indicate a close interaction between TLR4 and TLR2 that tightly regulates the expression of both receptors.     

Evaluation of the virulence of nontypeable Haemophilus influenzae lipooligosaccharide htrB and rfaD mutants in the chinchilla model of otitis media.

Considerable evidence has implicated nontypeable Haemophilus influenzae (NTHi) lipooligosaccharide (LOS) in the pathogenesis of otitis media (OM); however, its exact role has not been conclusively established. Recently, two NTHi LOS-deficient mutants have been created and described. Strain 2019-DK1, an rfaD gene mutant, expresses a truncated LOS consisting of only three deoxy-D-manno-octulosonic acid residues, a single heptose, and lipid A. Strain 2019-B29, an isogenic htrB mutant, possesses an altered oligosaccharide core and an altered lipid A. Each strain's ability to colonize the nasopharynx and to induce OM subsequent to transbullar inoculation was evaluated in the chinchilla model. Nasopharyngeal colonization data indicate that the parent strain and both mutants are able to colonize the nasopharynx and exhibit comparable clearance kinetics. Compared with the parent and each other, however, the mutants demonstrated marked differences in virulence regarding their relative abilities to induce OM and persist in the middle ear post-transbullar inoculation. Strain B29 required a 3-log-greater dose to induce OM than the parent strain and did not exhibit evidence of sustained multiplication but persisted for the same duration as the parent. Conversely, strain-DK1, even when inoculated at a dose 4 logs greater than the parent dose, was eliminated from the middle ear 72 h after challenge. A comparison of the relative pathogenicities of these isolates provides the opportunity to address fundamental questions regarding the contribution of LOS to pathogenesis issues at the molecular level. Specifically, the impact of these LOS gene disruptions on OM pathogenesis can be defined and may thus provide potential new targets for future protection and intervention strategies.     

Involvement of lipooligosaccharides of Haemophilus influenzae and Neisseria meningitidis in defensin-enhanced bacterial adherence to epithelial cells

Stimulated neutrophils release a variety of antimicrobial peptides, including neutrophil defensins (HNP1-4). We have previously reported that neutrophil defensins enhanced the adherence of Haemophilus influenzae and Neisseria meningitidis to cultured respiratory epithelial cells. In this study, the effect of defensins on the adherence of H. influenzae and N. meningitidis lipooligosaccharide (LOS) mutants to epithelial cells was tested. Neutrophil defensins enhanced the adherence of the oligosaccharide mutants of H. influenzae and N. meningitidis, whilst the adherence of the lipid A mutants B29 of H. influenzae and lpxL1 and lpxL2 of N. meningitidis was not or only moderately stimulated by neutrophil defensins. The adherence of the N. meningitidis LOS negative mutant lpxA was not enhanced by defensins. These findings suggested that the secondary fatty acids of lipid A were involved in the defensin-enhanced adherence. LOS from strain H44/76 or HNP-LOS complexes did not affect or stimulate the adherence of N. meningitidis, although the defensin-enhanced adherence is specific for certain bacterial species having LOS in their outer membrane. These results indicated that LOS is involved in the defensin-enhanced adherence. However, the mechanism by which defensins and LOS interact with epithelial cells to promote bacterial adherence remains to be resolved.     

Pharyngeal colonization dynamics of Haemophilus influenzae and Haemophilus haemolyticus in healthy adult carriers

Haemophilus influenzae is an important cause of respiratory infections, including acute otitis media, sinusitis, and chronic bronchitis, which are preceded by asymptomatic H. influenzae colonization of the human pharynx. The aim of this study was to describe the dynamics of pharyngeal colonization by H. influenzae and an intimately related species, Haemophilus haemolyticus, in healthy adults. Throat specimens from four healthy adult carriers were screened for Haemophilus species; 860 isolates were identified as H. influenzae or H. haemolyticus based on the porphyrin test and on dependence on hemin and NAD for growth. Based on tests for hemolysis, for the presence of the 7F3 epitope of the P6 protein, and for the presence of iga in 412 of the isolates, 346 (84%) were H. influenzae, 47 (11%) were H. haemolyticus, 18 (4%) were nonhemolytic H. haemolyticus, and 1 was a variant strain. Carriers A and B were predominantly colonized with nontypeable H. influenzae, carrier C predominantly with b(-) H. influenzae mutants, and carrier D with H. haemolyticus. A total of 358 H. influenzae and H. haemolyticus isolates were genotyped by pulsed-field gel electrophoresis (PFGE) following SmaI or EagI digestion of their DNA, and the carriers displayed the following: carrier A had 11 unique PFGE genotypes, carrier B had 15, carrier C had 7, and carrier D had 10. Thus, adult H. influenzae and H. haemolyticus carriers are colonized with multiple unique genotypes, the colonizing strains exhibit genetic diversity, and we observed day-to-day and week-to-week variability of the genotypes. These results appear to reflect both evolutionary processes that occur among H. influenzae isolates during asymptomatic pharyngeal carriage and sample-to-sample collection bias from a large, variable population of colonizing bacteria.     

Two-component systems in Haemophilus influenzae: a regulatory role for ArcA in serum resistance

Knockout mutations were constructed in the arcA gene of a virulent type b strain of Haemophilus influenzae, and the behavior of the resulting mutants was investigated in a number of conditions that mimicked distinct steps in the natural infection pathway. In arcA mutants, synthesis of capsule and lipooligosaccharide (LOS) and growth in synthetic media were unaltered compared to synthesis of capsule and LOS and growth in synthetic media in the wild-type H. influenzae type b parent strain. However, the virulence of the arcA mutants for BALB/c mice was significantly reduced. Upon exposure to human blood or serum, the arcA mutants showed markedly reduced survival compared with the survival of its wild-type parent. Serum resistance could be fully restored by complementation in cis with the H. influenzae arcA gene but not by complementation in cis with the homologous gene from Escherichia coli. The proteomes of wild-type and mutant bacteria were markedly different, especially under anaerobic conditions, underscoring the global regulatory role of ArcAB in H. influenzae. Evaluation of antibody titers and classical complement activities in various serum samples pointed to complement-mediated bactericidal activity as the factor that distinguishes between the arcA mutant and wild-type phenotypes. Comparative analysis of the membrane fractions of the arcA mutants and the wild-type strain revealed several ArcA-regulated proteins, some of which may be implicated in the serum hypersensitivity phenotype.     

Lipooligosaccharides (LOS) of some Haemophilus species mimic human glycosphingolipids, and some LOS are sialylated.

The lipooligosaccharides (LOS) of strains of Haemophilus ducreyi, Neisseria gonorrhoeae, Neisseria meningitidis, and Neisseria lactamica contain epitopes that are antigenically and structurally similar to carbohydrates present in human glycosphingolipids. LOS from strains of Haemophilus influenzae and H. influenzae biogroup aegyptius were tested for the binding of monoclonal antibodies (MAbs) that bind to human glycosphingolipids possessing Gal beta 1-4GlcNAc (MAb 3F11) and Gal alpha 1-4Gal beta 1-4Glc (MAb anti-Pk). In solid-phase radioimmunoassays, the LOS of 18 of 19 H. influenzae type b (Hib), 8 of 19 nontypeable H. influenzae, and 10 of 20 H. influenzae biogroup aegyptius strains bound MAb anti-Pk. The LOS of 13 of 19 Hib, 10 of 16 nontypeable H. influenzae, and 2 of 18 H. influenzae biogroup aegyptius strains bound MAb 3F11. Neuraminidase treatment of the strains increased the binding of MAb 3F11 by more than twofold in 47% of the H. influenzae strains, suggesting that sialic acid occluded the LOS structure recognized by MAb 3F11. The material released from neuraminidase-treated Hib LOS was confirmed to be sialic acid by high-performance anion-exchange chromatography. A recombinant plasmid containing genes involved in Hib LOS biosynthesis directed the expression (assembly) of the 3F11 epitope in Escherichia coli. These studies demonstrate that H. influenzae and H. influenzae biogroup aegyptius express at least two LOS epitopes that are similar to those present in human glycosphingolipids. Sialic acid was present on the LOS of some H. influenzae strains and prevented the binding of MAb 3F11 to its epitope. The oligosaccharide portion of sialylated LOS may also resemble sialylated oligosaccharides present in human glycosphingolipids (gangliosides).     

Lipooligosaccharide epitopes shared among gram-negative non-enteric mucosal pathogens.

The non-enteric Gram-negative human pathogens, B. catarrhalis, H. ducreyi, H. influenzae, N. gonorrhoeae and N. meningitidis, do not have repeating O-antigens as part of their principle surface glycolipid, the lipooligosaccharide (LOS). Because they have similar LOS structures, we studied the conservation of LOS oligosaccharide epitopes among these organisms. Twenty-one monoclonal antibodies (mAbs) generated by immunizing mice with H. influenzae, N. gonorrhoeae and N. meningitidis were studied for cross reactivity. Five mAbs generated against non-typable H. influenzae were the only strain-specific antibodies. Ten mAbs reacted to LOS epitope(s) common to a genera or species, and six mAbs bound to epitope(s) on the LOS of strains from different genera. Some cross reactive mAbs bound to LOS bands of similar molecular weights, while others bound to bands of varying molecular weights. mAb 3F11, whose epitope mimics a human blood-group antigen, bound to a 4.8 kDa LOS band in N. gonorrhoeae and H. ducreyi, two pathogens that infect genital epithelium. mAb 3D9, whose epitope consists of 2-keto-3-deoxyoctulosonic acid (KDO), reacted with different LOS bands in N. gonorrhoeae, H. influenzae and some R mutants of S. minnesota. A 14 kb restriction fragment containing lipooligosaccharide synthesis genes responsible for the assembly of the 3D9 epitope in H. influenzae hybridized to all H. influenzae strains tested but did not hybridize to gonococcal and S. minnesota strains that expressed this epitope. These studies demonstrate that conserved LOS epitope(s) exist among different species and genera of non-enteric human pathogens and that different genetic mechanisms may have evolved in these pathogens to assemble some of these conserved epitopes.     

Enhanced nasopharyngeal colonization of rats by piliated Haemophilus influenzae type b.

Piliated Haemophilus influenzae type b strains display an enhanced adherence to human epithelial cells in vitro. However, clinical isolates, even from mucosal sites, are seldom piliated, although piliated populations can be selected from them. Experiments with rats have led some authors to suggest that piliation does not implement colonization by H. influenzae type b. Piliated populations were obtained from 35 strains by selection for adherence to human erythrocytes. One strain, H. influenzae H305, simultaneously acquired an increased adherence to rat erythrocytes and buccal epithelial cells. In contrast to other strains, H. influenzae H305 in piliated form was more effective than in nonpiliated form in the colonization of rats by intranasal inoculation. After the piliated inoculum, however, the colonies cultured from the nasal washes were negative for erythrocyte adherence. Thus, piliated H. influenzae type b strains have an apparent advantage to initiating colonization in the rat model but may give rise to nonpiliated progeny that are more readily cultivable from the mucosal surface.     

Evaluation of phase variation of nontypeable Haemophilus influenzae lipooligosaccharide during nasopharyngeal colonization and development of otitis media in the chinchilla model

Nontypeable Haemophilus influenzae (NTHI) has four loci, lic-1 to lic-3 and lgtC, that generate phase-variable lipooligosaccharide (LOS) structures. lic-1, which is required for the expression of phosphorylcholine (ChoP), is the best characterized and is associated with an enhanced ability of H. influenzae to persist within the nasopharynges of infant rats. Recent data indicate that LOS impacts various aspects of NTHI virulence in the chinchilla model of nasopharyngeal colonization and otitis media (OM). In this study the effects of ChoP expression and the sequences of lic-1 to lic-3 and lgtC of NTHI strain 2019 were evaluated in the chinchilla OM model. Nasopharyngeal colonization data showed that a switch from the ChoP(-) to the ChoP(+) phenotype was observed as early as day 3 after intranasal inoculation. Chinchillas colonized by strains with the ChoP(+) phenotype demonstrated a significantly higher level of NTHI 2019 per milliliter of nasal lavage fluid than chinchillas colonized with predominantly the ChoP(-) variant (P < 0.05). The concentration of cells with the ChoP(+) phenotype in the middle ear was 3 log units higher than that of cells with the ChoP(-) variant (P < 0.01). There was a statistically significant association between ChoP(+) expression in the nasal lavage and the development of OM with culture-positive middle ear fluids in this model. These data suggest that expression of the ChoP(+) phenotype promotes enhanced nasopharyngeal colonization and development of OM.     

Serum amyloid P component bound to gram-negative bacteria prevents lipopolysaccharide-mediated classical pathway complement activation

Although serum amyloid P component (SAP) is known to bind many ligands, its biological function is not yet clear. Recently, it was demonstrated that SAP binds to lipopolysaccharide (LPS). In the present study, SAP was shown to bind to gram-negative bacteria expressing short types of LPS or lipo-oligosaccharide (LOS), such as Salmonella enterica serovar Copenhagen Re and Escherichia coli J5, and also to clinical isolates of Haemophilus influenzae. It was hypothesized that SAP binds to the bacteria via the lipid A part of LPS or LOS, since the htrB mutant of the nontypeable H. influenzae strain NTHi 2019-B29-3, which expresses a nonacetylated lipid A, did not bind SAP. This was in contrast to the parental strain NTHi 2019. The binding of SAP resulted in a clear inhibition of the deposition of complement component C3 on the bacteria. SAP inhibited only the activation of the classical complement pathway; the alternative route remained unaffected. In the classical route, SAP prevented the deposition of the first complement component, Clq, probably by interfering with the binding of Clq to LPS. Since antibody-mediated Clq activation was not inhibited by SAP, SAP seems to inhibit only the LPS-induced classical complement pathway activation. The SAP-induced inhibition of C3 deposition strongly diminished the complement-mediated lysis as well as the phagocytosis of the bacteria. The binding of SAP to gram-negative bacteria, therefore, might influence the pathophysiology of an infection with such bacteria.     

Cloning and expression in Escherichia coli of a Haemophilus influenzae type b lipooligosaccharide synthesis gene(s) that encodes a 2-keto-3-deoxyoctulosonic acid epitope.

The composition of lipooligosaccharide (LOS) can modify the virulence of Haemophilus influenzae type b (Hib). A genomic library of Hib strain A2 was constructed in the lambda bacteriophage EMBL3. Twenty-six phage clones expressed a Hib LOS oligosaccharide epitope in Escherichia coli that was detected by the monoclonal antibody (MAb) 6E4. None of the clones bound a polyclonal sera specific for Hib A2 LOS or an anti-H. influenzae lipid A MAb. One clone, designated EMBLOS-1, assembled an oligosaccharide with an apparent molecular weight of 1,400 (the 1.4K oligosaccharide) on a 4.1K lipopolysaccharide (LPS) species in E. coli LE392 and produced a novel 5.5K LPS that bound 6E4. Binding of 6E4 to the 5.5K EMBLOS-1 LPS band was abolished by treatment with sodium metaperiodate but was not affected by digestion with proteinase K, confirming the carbohydrate nature of the epitope. The EMBLOS-1 Haemophilus insert hybridized to similar restriction fragments in type b and nontypeable strains regardless of whether they expressed the 6E4 epitope. The 6E4 epitope did not undergo phase variation in Hib strain A2 at a frequency of greater than 10(-3). The oligosaccharide of the Salmonella minnesota Re mutant and 2-keto-3-deoxyoctulosonic acid (KDO) inhibited binding of 6E4 to Hib A2 LOS. We conclude that a gene(s) encoding an enzyme(s) that assembles a stable Hib LOS epitope containing KDO is conserved in H. influenzae and that the cloned Hib LOS synthesis gene products assemble a Hib LOS epitope on an E. coli K-12 LPS core.     

Contribution of the major and minor subunits to fimbria-mediated adherence of Haemophilus influenzae to human epithelial cells and erythrocytes.

Fimbriae are colonization factors of the human pathogen Haemophilus influenzae in that they mediate bacterial adherence to human eukaryotic cells. The contribution of the major (HifA) and putative minor (HifD and HifE) subunits of H. influenzae fimbriae to fimbria-specific adherence was studied by using mutants that were inactivated in distinct fimbrial genes. Both the major and minor subunits were required for adherence of H. influenzae to oropharyngeal epithelial cells and human erythrocytes carrying the AnWj antigen. Cloning of defined H. influenzae fimbrial genes in an Escherichia coli strain with type 1 fimbriae yielded recombinants expressing high amounts of HifA-containing H. influenzae fimbriae either with or without coexpression of both H. influenzae minor subunits. Both clones exhibited the specific adherence properties of H. influenzae fimbriae, implying that the minor H. influenzae subunits are dispensable for adherence and that the adhesive domain resides in the major subunit, HifA. In H. influenzae itself, the minor subunits probably affect adherence by raising the number of fimbriae above the minimal level required to establish adherence.     

Cj1136 is required for lipooligosaccharide biosynthesis, hyperinvasion, and chick colonization by Campylobacter jejuni

Campylobacter jejuni is a major cause of bacterial food-borne enteritis worldwide, and invasion into intestinal epithelial cells is an important virulence mechanism. Recently we reported the identification of hyperinvasive C. jejuni strains and created a number of transposon mutants of one of these strains, some of which exhibited reduced invasion into INT-407 and Caco-2 cells. In one such mutant the transposon had inserted into a homologue of cj1136, which encodes a putative galactosyltransferase according to the annotation of the C. jejuni NCTC11168 genome. In the current study, we investigated the role of cj1136 in C. jejuni virulence, lipooligosaccharide (LOS) biosynthesis, and host colonization by targeted mutagenesis and complementation of the mutation. The cj1136 mutant showed a significant reduction in invasion into human intestinal epithelial cells compared to the wild-type strain 01/51. Invasion levels were partially restored on complementing the mutation. The inactivation of cj1136 resulted in the production of truncated LOS, while biosynthesis of a full-length LOS molecule was restored in the complemented strain. The cj1136 mutant showed an increase in sensitivity to the bile salts sodium taurocholate and sodium deoxycholate and significantly increased sensitivity to polymyxin B compared to the parental strain. Importantly, the ability of the mutant to colonize 1-day-old chicks was also significantly impaired. This study confirms that a putative galactosyltransferase encoded by cj1136 is involved in LOS biosynthesis and is important for C. jejuni virulence, as disruption of this gene and the resultant truncation of LOS affect both colonization in vivo and invasiveness in vitro.     

Cloning of a gonococcal DNA sequence that complements the lipooligosaccharide defects of Neisseria gonorrhoeae 1291d and 1291e.

An isogenic set of gonococcal lipooligosaccharide (LOS) mutants derived from pyocin treatment of Neisseria gonorrhoeae 1291 was used to identify cloned gonococcal DNA fragments. A gene bank from N. gonorrhoeae 1291c chromosomal DNA was made in pLEE10, a shuttle vector that replicates in the gonococcus and Escherichia coli. A plasmid (pSG30) that could transform the LOS mutants 1291d and 1291e to reactivity with monoclonal antibody 3F11 and to production of an LOS component with migration identical to that of the parent, 1291, was identified. pSG30 contains a 9-kb EcoRI fragment. Curing studies indicate that pSG30 encodes gene products that affect LOS biosynthesis in trans. Subcloning identified a 2.6-kb HincII fragment (pSG38) that retained the ability to modify the LOS of 1291d and 1291e. The DNA regions involved in modification of 1291d and 1291e were named lsi-4 and lsi-5, respectively. The region of pSG38 that was involved in LOS modification was further localized by the construction of exonuclease III deletion plasmids. Transformation of these constructs identified a 750-bp fragment that retains the ability to modify 1291e and a 540-bp fragment which retains the ability to modify 1291d.     

Increased chain length promotes pneumococcal adherence and colonization

Streptococcus pneumoniae is a mucosal pathogen that grows in chains of variable lengths. Short-chain forms are less likely to activate complement, and as a consequence they evade opsonophagocytic clearance more effectively during invasive disease. When grown in human nasal airway surface fluid, pneumococci exhibited both short- and long-chain forms. Here, we determined whether longer chains provide an advantage during colonization when the organism is attached to the epithelial surface. Chain-forming mutants and the parental strain grown under conditions to promote chain formation showed increased adherence to human epithelial cells (A549 cells) in vitro. Additionally, adherence to A549 cells selected for longer chains within the wild-type strain. In vivo in a murine model of colonization, chain-forming mutants outcompeted the parental strain. Together, our results demonstrate that morphological heterogeneity in the pneumococcus may promote colonization of the upper respiratory tract by enhancing the ability of the organism to bind to the epithelial surface.     

Factors affecting pharyngeal Haemophilus influenzae type b colonization rates in children.

Over 1,300 children were studied in an analysis of factors that might affect pharyngeal colonization with Haemophilus influenzae type b. Our semiquantitative methods for the culture of H. influenzae type b, consisting of inoculation of 0.001 ml of throat swab fluid on antiserum agar plates and division of the results into three grades of intensity, showed agreement as to intensity of colonization in over 80% of repeat throat cultures. Our data also suggest that throat swabs are more efficient than nasopharyngeal swabs for detecting colonization, particularly for older children. All 17 H. influenzae type b carriers found with either method were detected with throat swabs, but six had negative nasopharyngeal cultures; four of these six were lightly colonized older children. Furthermore, colony counts were apt to be higher on plates inoculated with throat swab fluids. The frequency of pharyngeal H. influenzae type b colonization in children visiting health department clinics and pediatricians' offices was low during the first 6 months of life (0.7%) but averaged 3 to 5% throughout the rest of childhood. Approximately two-thirds of the carriers were colonized at an intensity too low to be detected by standard laboratory techniques. No influence on colonization rates was found for sex, race, season, economic status, or common childhood infectious diseases such as coryza or otitis media.     

Chronic colonization of rat airways with Pseudomonas aeruginosa.

Colonization of the airways of rats by Pseudomonas aeruginosa was established by treating the animals with hexamethylphosphoramide (HMPA) and inoculating with P. aeruginosa. Male Sprague-Dawley rats were given tap water (controls) or HMPA in the drinking water at 2 or 4 mg/ml. The ciliated cells of the airway epithelium were denuded, and microulcerative lesions in the epithelium were induced in the HMPA-treated rats. After 2 weeks of treatment, the rats were inoculated by transoral intratracheal instillation with 5 X 10(7) CFU of P. aeruginosa obtained from a cystic fibrosis patient. Two weeks after inoculation, P. aeruginosa was cultured from the airways, and scanning and transmission electron microscopy showed bacilli adhering to or invading the injured airway epithelium. P. aeruginosa was present in tracheal and intrapulmonary tissue homogenates of 9% of the P. aeruginosa-inoculated control rats (n = 22) as compared with 61% of the 2-mg/ml (n = 18) and 65% of the 4-mg/ml (n = 20) HMPA-treated rats (P less than 0.05). No dose-response relationship was found between 2 and 4 mg of HMPA per ml and colonization. Contamination of 47% of all of the rats with Mycoplasma pulmonis, as indicated by a positive enzyme-linked immunosorbent assay for immunoglobulin G, had no discernible significant effect on colonization by P. aeruginosa. These results indicate that colonization of the rat airway by P. aeruginosa can be achieved experimentally by treating the animals with HMPA. This research supports the hypothesis that colonization by P. aeruginosa may occur in airways where the ciliated epithelium has been injured and epithelial lesions exist.     

Synthesis, characterization, and immunologic properties of detoxified lipooligosaccharide from nontypeable Haemophilus influenzae conjugated to proteins.

Nontypeable Haemophilus influenzae (NTHi) is an important cause of otitis media in children and of pneumonitis in adults with depressed resistance. Lipooligosaccharide (LOS) is a major surface antigen of NTHi and elicits bactericidal and opsonic antibodies. We prepared detoxified LOS (dLOS) protein conjugates from NTHi for use as experimental vaccines. LOS from NTHi 9274 was treated with anhydrous hydrazine and had its toxicity reduced to clinically acceptable levels. dLOS was bound to tetanus toxoid (TT) or high- molecular-weight proteins (HMPs) from NTHi through a linker of adipic acid dihydrazide to form dLOS-TT or dLOS-HMP. The molar ratio of the dLOS to protein carriers ranged from 26:1 to 50:1. The antigenicity of the conjugates was similar to that of the LOS alone as determined by double immunodiffusion. Subcutaneous or intramuscular injection of the conjugates elicited a 28- to 486-fold rise in the level of immunoglobulin G antibodies in mice to the homologous LOS after two or three injections and a 169- to 243-fold rise in the level of immunoglobulin G antibodies in rabbits after two injections. The immunogenicity of the conjugates in mice and rabbits was enhanced by formulation with monophosphoryl lipid A plus trehalose dimycolate. In rabbits, conjugate-induced LOS antibodies induced complement-mediated bactericidal activity against the homologous strain 9274 and prototype strain 3189. These results indicate that a detoxified LOS-protein conjugate is a candidate vaccine for otitis media and pneumonitis caused by NTHi.