Detoxified lipooligosaccharide from nontypeable Haemophilus influenzae conjugated to proteins confers protection against otitis media in chinchillas.

Detoxified-lipooligosaccharide (dLOS)-protein conjugates from nontypeable Haemophilus influenzae (NTHi) elicited a significant rise of anti-LOS antibodies with bactericidal activity in rabbits (X.-X. Gu, C.-M. Tsai, T. Ueyama, S. J. Barenkamp, J. B. Robbins, and D. J. Lim, Infect. Immun. 64:4047-4053, 1996). In this study, we evaluated whether vaccination with the conjugates would protect against NTHi otitis media in chinchillas. Fifty-eight chinchillas received three subcutaneous or intramuscular injections of dLOS-conjugated tetanus toxoid, dLOS-conjugated high-molecular-weight proteins from NTHi, or saline (control) in Freund's adjuvant and then were challenged by intrabullar inoculation with 140 CFU of NTHi. All vaccinated animals responded with elevated serum titers of anti-LOS antibody, and 49% (19 of 39) demonstrated bactericidal activity against the homologous strain. Otitis media with culture-positive NTHi effusions developed in all 19 controls and 56% (22 of 39) of the vaccinated animals during a period of 21 days (P < 0.001). Bacterial counts of the middle ear effusions were lower in the vaccine groups than in the controls (P < 0.01). The incidences of infection in the unchallenged ear or inner ear were 26 or 28% in the vaccine groups and 53 or 58% in the controls (P < 0.05). The signs of infection observed by otoscopy were less severe in the vaccine groups than in the controls. There was no significant difference between the two vaccine groups. These data indicate that active immunization with LOS-based conjugates reduces the incidence of NTHi-induced otitis media.     

Immunization with high-molecular-weight adhesion proteins of nontypeable Haemophilus influenzae modifies experimental otitis media in chinchillas.

Prevention of nontypeable Haemophilus influenzae otitis media by vaccination is an important health care goal. Proteins important in bacterial adherence deserve consideration as potential vaccine candidates. Two colleagues and I previously identified a family of immunogenic high-molecular-weight proteins important in adherence of nontypeable H. influenzae to human epithelial cells (J.W. St. Geme III, S. Falkow, and S.J. Barenkamp, Proc. Natl. Acad. Sci. USA, 90:2875-2879, 1993). In the work described here, I determined whether immunization with two such adherence proteins, HMW1 and HMW2, purified from prototype nontypeable Haemophilus strain 12, would modify the course of experimental otitis media caused by the homologous strain. Chinchillas received three monthly subcutaneous injections with 40 microgram of an HMW1/HMW2 protein mixture in Freud's adjuvant. One month after the last injection, animals were challenged by intrabullar inoculation with 300 CFU of nontypeable H. influenzae 12. Infection developed in five of five control animals versus 5 of 10 immunized animals (P = 0.08, Fisher exact, one-tailed). Among infected animals, bacterial counts in middle ear fluid specimens 7 days postchallenge were significantly greater in control animals than in immunized animals (P = 0.014, Mann-Whitney U test). Serum antibody titers following immunization were comparable in uninfected and infected animals. However, infection in immunized animals was uniformly associated with the appearance of bacteria downregulated in expression of the high-molecular-weight proteins, suggesting bacterial selection in response to immunologic pressure. Although protection following immunization was incomplete, these data suggest that the high-molecular-weight adhesion proteins are potentially important protective antigens which might represent one component of a multicomponent nontypeable Haemophilus vaccine.     

Variability of outer membrane protein P1 and its evaluation as a vaccine candidate against experimental otitis media due to nontypeable Haemophilus influenzae: an unambiguous, multifaceted approach

Candidate vaccine antigens for preventing otitis media caused by nontypeable Haemophilus influenzae (NTHI) should possess one or more conserved epitopes. We sought to evaluate the candidacy of P1, a surface-expressed outer membrane protein knowing that this antigen is subject to diversifying selection. Therefore, we selected NTHI strains from among >500 phylogenically variant isolates representative of the diversity found in natural populations of H. influenzae. Twenty-three variants of P1 (相似文献   

Partial analysis of the genomes of two nontypeable Haemophilus influenzae otitis media isolates

In 1995, The Institute for Genomic Research completed the genomic sequence of a rough derivative of Haemophilus influenzae serotype d, strain KW20. This sequence, though extremely useful in understanding the basic biology of H. influenzae, has yet to provide significant insight into our understanding of disease caused by nontypeable H. influenzae (NTHI), because serotype d strains are not generally pathogens. In contrast, NTHI strains are frequently mucosal pathogens and are the primary pathogens of chronic otitis media as well as a significant cause of acute otitis media in children. Thus, it is of great importance to further understand their biology. We used a DNA-based microarray approach to identify genes present in a clinical isolate of NTHI that were absent from strain Rd. We also sequenced the genome of a second NTHI isolate from a child with chronic otitis media to threefold coverage and then used an array of bioinformatics tools to identify genes present in this NTHI strain but absent from strain Rd. These methods were complementary in approach and results. We identified, in both strains, homologues of H. influenzae lav, an autotransported protein of unknown function; tnaA, which encodes tryptophanase; as well as a homologue of Pasteurella multocida tsaA, which encodes an alkyl peroxidase that may play a role in protection against reactive oxygen species. We also identified a number of putative restriction-modification systems, bacteriophage genes and transposon-related genes. These data provide new insight that complements and extends our ongoing analysis of NTHI virulence determinants.     

Synergistic effect of adenovirus type 1 and nontypeable Haemophilus influenzae in a chinchilla model of experimental otitis media.

We recently reported the development of a chinchilla model of experimental otitis media (OM) that uses a pediatric clinical isolate of adenovirus type 1 (4) and in which an active infection with the wild-type strain was demonstrated. To expand upon these findings, this study was designed to determine whether we could demonstrate adenovirus infection-induced predisposition to bacterial OM in the chinchilla, as has been shown in human epidemiological studies (D. A. Clements, F. W. Henderson, and E. C. Neebe, p. 27-29, in D. J. Lim, C. D. Bluestone, J. O. Klein, D. J. Nelson, and P. L. Ogra, ed., Proceedings of the Fifth International Symposium on Recent Advances in Otitis Media, 1993; F. W. Henderson, A. M. Collier, M. A. Sanyai, et al., N. Engl. J. Med. 306:1377-1383, 1982). In addition, we were interested in determining whether altering the order of pathogen acquisition would further affect the outcome of disease incidence and severity. Toward this end, cohorts of chinchillas were inoculated intranasally with a strain of nontypeable Haemophilus influenzae (NTHi) (86-028NP) which colonizes the chinchilla nasopharynx but does not consistently induce culture-positive OM when inoculated intranasally (L. O. Bakaletz, T. M. Hoepf, D. J. Lim, and B. Tallan, Abstr. 90th Annu. Meet. Am. Soc. Microbiol. 1990, abstr. B-66, p. 37, 1990), adenovirus type 1 and then inoculated 7 days later with NTHi, NTHi and then inoculated 7 days later with adenovirus type 1, or both pathogens concurrently. All cohorts were observed over a 35-day period and assessed for incidence and severity of OM by several methodologies. The data collectively indicated that all animals receiving both pathogens developed OM of greater severity than those receiving only a single agent. Adenovirus inoculation followed 7 days later by NTHi inoculation was the order of pathogen acquisition which induced the most prolonged presence of NTHi in both the nasopharynx and the middle ear, the most severe tympanic membrane inflammation overall, and the most significant damage to and altered function of both middle ear and eustachian tube mucosae.     

Development of a chinchilla model to allow direct, continuous, biophotonic imaging of bioluminescent nontypeable Haemophilus influenzae during experimental otitis media

We transformed a nontypeable Haemophilus influenzae clinical isolate with a plasmid containing the luxCDABE operon driven by the H. influenzae outer membrane protein P2 promoter. Herein, we demonstrate the ability to detect bioluminescence and to monitor infection within the nasopharynges, eustachian tubes, and middle ears of chinchillas after intranasal and transbullar challenges.     

Multiple consecutive lavage samplings reveal greater burden of disease and provide direct access to the nontypeable Haemophilus influenzae biofilm in experimental otitis media

The typically recovered quantity of nontypeable Haemophilus influenzae (NTHi) bacteria in an ex vivo middle ear (ME) aspirate from the chinchilla model of experimental otitis media is insufficient for direct analysis of gene expression by microarray or of lipopolysaccharide glycoforms by mass spectrometry. This prompted us to investigate a strategy of multiple consecutive lavage samplings to increase ex vivo bacterial recovery. As multiple consecutive lavage samples significantly increased the total number of bacterial CFU collected during nasopharyngeal colonization or ME infection, this led us to evaluate whether bacteria sequentially acquired from consecutive lavages were similar. Comparative observation of complete ex vivo sample series by microscopy initially revealed ME inflammatory fluid consisting solely of planktonic-phase NTHi. In contrast, subsequent lavage samplings of the same infected ear revealed the existence of bacteria in two additional growth states, filamentous and biofilm encased. Gene expression analysis of such ex vivo samples was in accord with different bacterial growth phases in sequential lavage specimens. The existence of morphologically distinct NTHi subpopulations with varying levels of gene expression indicates that the pooling of specimens requires caution until methods for their separation are developed. This study based on multiple consecutive lavages is consistent with prior reports that NTHi forms a biofilm in vivo, describes the means to directly acquire ex vivo biofilm samples without sacrificing the animal, and has broad applicability for a study of mucosal infections. Moreover, this approach revealed that the actual burden of bacteria in experimental otitis media is significantly greater than was previously reported. Such findings may have direct implications for antibiotic treatment and vaccine development against NTHi.     

Invasive disease due to nontypeable Haemophilus influenzae among children in Arkansas

In this study, we reviewed cases of invasive disease due to nontypeable Haemophilus influenzae among children hospitalized at Arkansas Children's Hospital from 1993 to 2001. A total of 28 cases were examined, including 21 associated with bacteremia and 4 associated with meningitis. Of the patients examined, 86% were 相似文献   

Genetic diversity of paired middle-ear and pharyngeal nontypeable Haemophilus influenzae isolates from children with acute otitis media

Pulsed-field gel electrophoresis was used to determine genetic diversities of multiple nontypeable Haemophilus influenzae isolates from throat and ear specimens of eight children with otitis media. From five children, all ear and throat isolates were identical. The bacterial populations in these specimens showed less diversity than populations in throat isolates of healthy children.     

Protection by serum antibodies in experimental nontypable Haemophilus influenzae otitis media.

The chinchilla experimental model of otitis media was used to examine the importance of serum antibodies in protection against disease caused by nontypable Haemophilus influenzae. An immune serum pool was prepared by immunizing chinchillas with killed bacterial cells of nontypable H. influenzae 3245. Pooled preimmune or immune serum from these immunized animals was administered intravenously to a group of nonimmune chinchillas 1 day before intrabullar challenge with strain 3245. Of 5 animals receiving preimmune serum, 5 developed otitis media compared with 0 of 10 animals receiving immune serum (P = 0.008). The immune serum pool contained antibodies directed against both surface-exposed outer membrane proteins and lipopolysaccharide (LPS). The 39-kilodalton major outer membrane protein was the immunodominant surface protein. Anti-LPS antibodies were removed from the immune serum pool by affinity chromatography, and affinity-purified anti-LPS antibodies were recovered. Immune serum, immune serum absorbed of LPS antibodies, or affinity-purified LPS antibodies were then administered to another group of experimental animals 1 day before bacterial challenge. Of four animals that received the affinity-purified LPS antibodies, four developed otitis compared with zero of four animals that received the immune serum or zero of four animals that received the LPS-absorbed immune serum (P = 0.028). These studies indicate that passive immunization with immune serum is protective in experimental nontypable H. influenzae otitis media and that bacterial outer membrane proteins may be the principal targets of protective antibody.     

Intranasal immunization enhances clearance of nontypeable Haemophilus influenzae and reduces stimulation of tumor necrosis factor alpha production in the murine model of otitis media

Nontypeable Haemophilus influenzae (NTHi) is a major pathogen causing otitis media (OM). One of the outer membrane proteins of NTHi, P6, is a common antigen to all strains and is considered a candidate for mucosal vaccine. We have previously reported that intranasal immunization with P6 and cholera toxin (CT) could induce P6-specific immunoglobulin A (IgA) antibodies in the middle ear. In the present study, we assessed the effect of intranasal immunization for the protection against NTHi-induced OM. Mice were immunized intranasally with P6 and CT as an adjuvant on days 0, 7, and 14. Control mice were given phosphate-buffered saline (PBS) without antigen. One week after the final immunization, a suspension of live NTHi (10(7) CFU) was injected into the tympanic cavity to induce experimental OM. On days 3 and 7 after bacterial challenge, mice were killed and middle ear effusions (MEEs) were collected. All immunized mice showed elevated titers of P6-specific antibodies in MEEs. The rank order of specific antibody included, from highest to lowest levels, IgG, IgA, and IgM. In addition, immunized mice showed enhanced clearance of NTHi from the middle ear and the number of NTHi in MEEs of immunized mice was reduced by 97% on day 3 and by 92% on day 7 after bacterial challenge relative the number in the MEEs of control mice. The protective effect of intranasal immunization on the incidence of NTHi-induced experimental OM was evident on day 7 after challenge. By day 7, the number of MEEs in immunized mice was 64% less than that in control mice and the incidence of NTHi culture-positive MEEs in immunized mice was 56% less than that in control mice. Less stimulation of tumor necrosis factor alpha (TNF-alpha) production in the middle ear was evident on day 3 after challenge. Immunized mice showed lower concentrations of TNF-alpha in MEEs. These results indicate that intranasal immunization affords protection against experimental OM as evidenced by enhanced clearance of NTHi and less stimulation of TNF-alpha production in the middle ear. These findings suggest that a nasal vaccine might be useful for preventing OM.     

Identification of new genetic regions more prevalent in nontypeable Haemophilus influenzae otitis media strains than in throat strains

Nontypeable (NT) Haemophilus influenzae strains cause significant respiratory illness and are isolated from up to half of middle ear aspirates from children with acute otitis media. Previous studies have identified two genes, lic2B and hmwA, that are associated with NT H. influenzae strains isolated from the middle ears of children with otitis media but that are not associated with NT H. influenzae strains isolated from the throats of healthy children, suggesting that they may play a role in virulence in otitis media. In this study, genomic subtraction was used to identify additional genetic regions unique to middle ear strains. The genome of NT H. influenzae middle ear strain G622 was subtracted from that of NT H. influenzae throat strain 23221, and the resultant gene regions unique to the middle ear strain were identified. Subsequently, the relative prevalence of the middle ear-specific gene regions among a large panel of otitis media and throat strains was determined by dot blot hybridization. By this approach, nine genetic regions were found to be significantly more prevalent in otitis media strains. Classification tree analysis of lic2B, hmwA, and the nine new potential otitis media virulence genes revealed two H. influenzae pathotypes associated with otitis media.     

Evaluation of mixtures of purified Haemophilus influenzae outer membrane proteins in protection against challenge with nontypeable H. influenzae in the chinchilla otitis media model.

Nontypeable Haemophilus influenzae (NTHi) is one of the leading causative agents of bacterial otitis media, and no vaccine has been shown to be effective against it. Three outer membrane lipoproteins of NTHi have been investigated extensively and are leading candidates for inclusion in a vaccine against this organism. Hi-PAL (P6), recombinant PCP (rPCP), and e (P4) proteins are antigenically conserved among NTHi strains and elicit bactericidal and protective antibodies. A genetic fusion of the rPCP and Hi-PAL proteins has also been reported. Mixtures of these proteins were used for active immunization experiments in the chinchilla model of otitis media. Chinchillas were immunized either with a mixture of all three lipoproteins or with the mixture of rPCP-PAL hybrid plus e protein. When these animals were challenged with a NTHi strain injected directly into the middle ears, no protection from infection or disease, as measured by otoscopy, was observed in either group. However, effusion and inflammation measured by tympanometry were significantly reduced in animals immunized with the three lipoproteins. Animals that had been immunized with either whole NTHi cells or total outer membranes and then challenged with the homologous strain were significantly protected from both infection and disease, as determined by tympanometry and otoscopy. Unlike other animals antisera, chinchilla antisera against the purified proteins had no bactericidal activity against NTHi but did fix complement on the cell surface. Thus, the chinchilla immune responses to mixtures of these lipoproteins differ from the immune responses observed in other animal species. Further evaluation of these proteins for their vaccine potential remains to be done.     

Otitis media in children: local immune response to nontypeable Haemophilus influenzae.

Twenty children experienced 30 episodes of otitis media with effusion due to nontypeable (NT) Haemophilus influenzae in the first 2 years of life. The local and systemic immune responses to homologous strains of NT H. influenzae were determined by an immunodot assay. Strain-specific immunoglobulin G (IgG) antibody predominated in the middle ear fluid (MEF). It was detected in 91% of the children, compared with IgM in 48% (P less than 0.005), IgA in 52% (P less than 0.005), and secretory IgA in 18% (P less than 0.005). The titer (log2) of NT H. influenzae-specific IgG antibody (mean +/- standard error, 8.2 +/- 0.1) exceeded the titers of IgM (3.4 +/- 0.1), IgA (3.7 +/- 0.1), and secretory IgA (1.2 +/- 0.3). NT H. influenzae-specific antibody was detected exclusively in MEFs of individuals who possessed homologous serum antibody. Although antibody titers in MEF declined over time, serum antibody titers remained stable. These data suggest that immunity to NT H. influenzae in the middle ear, in part, reflects systemic immunity. Whereas local antibody disappears after resolution of the infection, systemic antibody persists.     

A mutation in the sap operon attenuates survival of nontypeable Haemophilus influenzae in a chinchilla model of otitis media

Bacteria have evolved strategies to resist killing by antimicrobial peptides (APs), important effectors of innate immunity. The sap (sensitivity to antimicrobial peptides) operon confers resistance to AP-mediated killing of Salmonella. We have recently shown that sapA gene expression is upregulated in the middle ear in a chinchilla model of nontypeable Haemophilus influenzae (NTHI)-induced otitis media. Based on these findings, we constructed an NTHI strain containing a Lux reporter plasmid driven by the sapA promoter and demonstrated early yet transient expression of the sap operon within sites of the chinchilla upper airway upon infection. We hypothesized that the sap operon products mediate NTHI resistance to APs. In order to test this hypothesis, we constructed a nonpolar mutation in the sapA gene of NTHI strain 86-028NP, a low-passage-number clinical isolate. The sapA mutant was approximately eightfold more sensitive than the parent strain to killing by recombinant chinchilla beta-defensin 1. We then assessed the ability of this mutant to both colonize and cause otitis media in chinchillas. The sapA mutant was significantly attenuated compared to the parent strain in its ability to survive in both the nasopharynx and the middle ear of the chinchilla. In addition, the mutant was impaired in its ability to compete with the parent strain in a dual-strain challenge model of infection. Our results indicate that the products of the sap operon are important for resisting the activity of APs and may regulate, in part, the balance between normal carriage and disease caused by NTHI.     

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.     

Role of fimbriae expressed by nontypeable Haemophilus influenzae in pathogenesis of and protection against otitis media and relatedness of the fimbrin subunit to outer membrane protein A.

Nontypeable Haemophilus influenzae is a primary pathogen in both acute otitis media (OM) and chronic OM, yet the pathogenesis of this disease is not fully understood. Although fimbriae have been observed on all clinical OM isolates examined to date, their role in pathogenesis remains unclear. Therefore, the gene which codes for the fimbrial subunit protein (fimbrin) in nontypeable H. influenzae 1128 was isolated, cloned, and sequenced. The nucleotide sequence of the fimbrin gene was found to contain an open reading frame of 1,077 bp which would encode a mature fimbrin protein consisting of 338 amino acid with a calculated molecular mass of 36.4 kDa. The translated amino acid sequence was found to be homologous with various OmpA proteins of other gram-negative bacteria, and algorithmic analysis predicted that this protein is organized as a coiled coil. To directly test whether fimbriae are involved in pathogenesis, the fimbrin gene was disrupted, and the biological consequences of disruption were absence of both expression of the fimbrial appendage and the specific immunogold labeling thereof with antisera directed against isolated fimbrial protein, reduced adherence to human oropharyngeal cells in vitro, augmented clearance from the tympanum post-transbullar inoculation, and significantly reduced induction of OM post-intranasal inoculation in a chinchilla model compared with the fimbriated parent strain. We additionally find that either passive immunization or active immunization against isolated fimbrial protein confers partial protection against transbullar challenge. A Western blot (immunoblot) indicated a degree of serological relatedness among fimbrin proteins of 15 nontypeable and type b isolates. These data suggest that fimbrin could be useful as a component of a vaccine to protect against OM.     

Monoclonal anti-LPS inner core antibodies protect against experimental hematogenous Haemophilus influenzae type b meningitis

This study tested the protective activity of antibodies to the LPS core of Haemophilus influenzae (Borrelli et al., Infect. Immun. 1995;63: 3683-92) in a hematogenous meningitis model. Meningitis was established by intraperitoneal inoculation of infant rats with H. influenzae type b (Hib). The severity of infection was determined by daily assessment of mortality, symptoms of disease and weight changes. Mortality occurred rapidly after infection with 10(5)cfu/rat and most animals died within 24 h. At a lower infection dose (10(4)cfu/rat) the rats survived, but developed symptoms of disease such as tremor, hypothermia, lethargy and anorexia within 12-72 h post challenge. Surviving animals showed decreased weight gain. Bacteremia was detected by daily blood-cultures in 10/10 rats and cleared 6 days after inoculation. The monoclonal anti-LPS antibody MAHI 3 was used in passive protection studies. MAHI 3 increased the survival in the high inoculum group (10(5)cfu/rat) from 10-17% in control animals to 60-90%. At the lower inoculum concentration (10(4)cfu/rat) MAHI 3 treatment reduced the symptoms and blood counts. Intraperitoneal injection of MAHI 3 was more effective than intranasal injection as shown by the effect on bacteremia. We conclude that anti-LPS antibodies can protect against mortality caused by hematogenous Hib infections in infant rats.