Reconstitution of a porin-deficient mutant of Haemophilus influenzae type b with a porin gene from nontypeable H. influenzae.

The major outer membrane protein (OmpP2) of nontypeable Haemophilus influenzae (NTHI) has been shown to vary markedly with respect to both size and the presence of specific surface-exposed epitopes among strains of this unencapsulated pathogen. In contrast, the OmpP2 proteins of H. influenzae type b (Hib) strains are well conserved at the level of primary protein structure and have in common several surface-exposed antigenic determinants that have not been detected in NTHI strains. The availability of an isogenic, avirulent Hib ompP2 mutant made it possible to investigate whether an NTHI OmpP2 protein could function properly in the Hib outer membrane. A plasmid shuttle vector (pGJB103) was used to clone the ompP2 gene from NTHI TN106 into a recombination-deficient H. influenzae strain in which expression of the NTHI OmpP2 protein was detected by means of an NTHI TN106 OmpP2-specific monoclonal antibody. The amino acid sequence of this NTHI OmpP2 protein, as deduced from the nucleotide sequence of the NTHI TN106 ompP2 gene, was determined to be 83% identical to that of the Hib OmpP2 protein. Transformation of this cloned NTHI ompP2 gene into the Hib ompP2 mutant yielded a Hib transformant strain that expressed the NTHI OmpP2 protein. Expression of this NTHI OmpP2 protein allowed the Hib ompP2 mutant, which normally grows poorly in vitro, to grow in a manner indistinguishable from that of the wild-type Hib strain. More importantly, the introduction of this functional NTHI ompP2 gene into the avirulent Hib ompP2 mutant restored the virulence of this strain to wild-type levels. These results indicate that an NTHI OmpP2 protein can be expressed and function properly in the Hib outer membrane.     

Structural and serological relatedness of Haemophilus influenzae type b pili.

The structural and serological relatedness of the pilus proteins of several isolates of Haemophilus influenzae type b cultured from patients with invasive disease and from different anatomic sites within the same patient was examined. Epithelial cell-adherent variants of 25 nonadherent parent isolates were obtained by selection for organisms that adhered to human erythrocytes. Outer membrane protein analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of an additional 24- to 24.5-kilodalton protein among all adherent variants but absent from all nonadherent parent isolates. Polyclonal rabbit antiserum against the intact native pilus protein of H. influenzae M43 cross-reacted with 20 of 25 adherent H. influenzae in both immunodot and slide-agglutination assays. No differences in reactivity among isolates cultured from more than one anatomic site in the same patient were noted. Anti-M43 pilus antiserum had bactericidal activity against both the homologous strain and a heterologous strain that demonstrated serologic identity in the immunodot and slide agglutination assays. The adherence of these strains to human epithelial cells in vitro was inhibited by Fab fragments purified from the antipilus antiserum. These data indicate that a remarkable degree of homogeneity in pilin subunit size exists among the pili of H. influenzae type b and that major antigenic determinants are shared among most of these pili. Also, antibodies directed against H. influenzae pilus proteins may be able to contribute to host defenses through serum bactericidal activity and by blocking the adherence of this bacterium to host epithelial cells.     

Conserved and nonconserved epitopes among Haemophilus influenzae type b pili.

We investigated the binding of antibodies raised against four different Haemophilus influenzae type b (Hib) plus antigen preparations to the native pili and denatured pilins of 21 Hib isolates. Antibodies against live piliated Hib M43p+, adsorbed with a nonpiliated variant to remove nonpilus antibodies, bound to 18 of the 21 piliated Hib isolates in immunodot assays but failed to recognize the denatured pilins from any of the strains in Western immunoblot assays. Similarly, antibodies against purified native pili of strain E1ap+ bound to 11 of 21 piliated strains in immunodot assays but to only 2 of 21 piliated strains in Western blot assays. The native pili of all 21 strains were recognized by one or both of the antisera. These observations suggest that the immunodominant epitopes of native Hib pili are dependent on conformation and are moderately conserved. In contrast, antibodies against denatured M43p+ pilin or against a peptide derived from amino acids 5 through 17 of M43p+ pilin failed to bind to native pili from any of the 21 piliated isolates on immunodot assay. However, both sera recognized the denatured pilins from all the piliated strains on Western blot assay. These data indicate that the immunodominant epitopes of denatured pilins are highly conserved among different strains of Hib but are unavailable on intact pili for antibody binding.     

Occult bacteremia with nontypeable Haemophilus influenzae.

A 2-year-old boy had occult bacteremia with nontypeable Haemophilus influenzae 6 weeks after receiving H. influenzae type b polysaccharide vaccine. Evaluation of his host defense was normal. As determined by outer membrane protein electrophoresis and Southern hybridization analysis, this strain was not related to type b strains. Its virulence in rats was similar to that of another nontypeable strain and less than that of a type b strain.     

Long PCR-ribotyping of nontypeable Haemophilus influenzae.

PCR-ribotyping, a new typing method based on long PCR, has been developed for nontypeable Haemophilus influenzae (NTHi). Ribosomal operons of NTHi were amplified by long PCR and were found to be highly polymorphic for internal HaeIII sites. The technique was applied to 49 isolates previously subjected to conventional ribotyping, and the two methods showed a high level of concordance for serial isolates from individual subjects. PCR-ribotyping provides a powerful new typing tool for strain characterization in epidemiological investigations of NTHi.     

Haemophilus influenzae infections in the H. influenzae type b conjugate vaccine era

The widespread use of Haemophilus influenzae type b (Hib) conjugate vaccines has nearly eradicated invasive Hib disease where the vaccines are used. This success was accompanied by a shift in capsular serotypes of invasive H. influenzae disease, with nontypeable strains replacing type b strains as the most common bloodstream isolate, but there is no convincing evidence of a true increase in the incidence of non-serotype b invasive infections. H. influenzae causes predominantly mucosal infections. The introduction of vaccines for otitis media and global shifts in antimicrobial susceptibility emphasize the importance of continued surveillance of H. influenzae colonization and disease patterns.     

Analysis of invasive Haemophilus influenzae infections after extensive vaccination against H. influenzae type b

Little clinical and microbiological information is available about invasive Haemophilus influenzae infection after widespread vaccination against H. influenzae type b (Hib). We conducted an active community surveillance study on invasive H. influenzae during a 2-year period in a community of more than 5 million people after vaccination against Hib in children was introduced. The median incidence was 16.3 cases/100000 persons per year in children less than 1-year-old and 4.41 cases/100000 persons in children less than <5 years old. The highest incidence in adults was observed in patients greater than 70 years old. Clinical diagnoses included bacteremia, pneumonia, and meningitis. Of the H. influenzae-infected patients, 74.3% had underlying predisposing conditions, including impaired immunity and respiratory diseases. A total of 73.6% of the isolates were nontypeable and 16.5, 6.6, and 3.3% were types b, f, and e, respectively. Infections due to capsulated strains b, e, and f were evenly distributed between children and adults. Ampicillin and cotrimoxazole resistance occurred at frequencies of 24.2 and 48.4%, respectively. Antibiotic resistance was more prevalent in capsulated than in noncapsulated H. influenzae. Invasive isolates were highly resistant to antibiotics that were used infrequently in the community. Nontypeable H. influenzae were genetically much more heterogeneous than capsulated strains. Capsule-deficient mutants (b(-)) were not detected. Plasmid carriage was linked to antibiotic resistance and capsulated strains. Over the study period, the incidence of invasive H. influenzae infections, either encapsulated or not, did not increase. In the post-Hib vaccination era, most invasive infections were due to noncapsulated strains and occurred in the extreme ages of life in patients with predisposing conditions.     

Characterization of Haemophilus influenzae type b fimbriae.

We confirmed that the fimbriae of Haemophilus influenzae type b conferred hemagglutinating activity (HA) towards human erythrocytes, and erythrocytes of certain other species. Most (17/25) cerebrospinal fluid isolates lacked detectable HA on direct testing, but selective enrichment for fimbriation (f+) indicated that 22 of 25 strains could produce these surface structures. HA was unchanged from pH 4.5 to 9.5 and was not inhibited by mannose or certain other simple sugars. The HA titer of a suspension of three f+ strains was slightly decreased at 50 degrees C; HA was lost by heating at 60 degrees C for 3 min. Growth on a variety of solid and liquid media and under differing degrees of oxygenation did not change the HA titer of a suspension of three f+ strains. Fimbriation was not lost on repeated subculture. Wild-type fimbriated strains, and those derived by transformation, did not contain detectable plasmid DNA. Transformation of a strain lacking fimbriae to f+ was associated with the appearance of an outer membrane protein of 24 kilodaltons. This protein was purified from one strain to homogeneity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis by selective detergent solubilization and ammonium sulfate fractionation. Colonization capacity was equivalent with an isogenic untypable strain lacking or possessing fimbriae. Fimbriae of type b H. influenzae possess characteristics similar to those structures on other gram-negative bacteria; their role in cell physiology or pathogenesis of invasive disease is unknown.     

Haemophilus influenzae type b antigenuria in children.

Detection of Haemophilus influenzae type b (HIb) antigenuria by latex agglutination has been shown to be sensitive, specific, and rapid. In children, antigenuria persisted for a mean duration of 10 days and a maximum of 18 days. Antigenuria was demonstrated in 25 of 30 patients with HIb infection but not in 62 with other types of infection. In five children, antigenuria confirmed the diagnosis in the absence of bacteriological confirmation. In five other children, antigenuria was not found, but in this group the antigen was detected in another body fluid or HIb was recovered.     

Haemophilus influenzae type b conjugate vaccines

Haemophilus influenzae type b (Hib) is one of the leading causes of invasive bacterial infection in young children worldwide. During childhood, acquisition of antibody directed against the polysaccharide capsule of the organism, presumably as a result of asymptomatic carriage, confers protection and disease is much less common after the age of 4 years. Like other polysaccharides, the polyribosyl ribitol phosphate (PRP) of the Hib capsule is a T-independent antigen and not immunogenic when administered as a vaccine in infancy. Because the highest rates of disease occur in the first 2 years of life, efficacious Hib vaccines have been designed by covalently linking the PRP capsule to a carrier protein that recruits T-cell help for the polysaccharide immune response and induces anti-PRP antibody production even in the first 6 months of life. Introduction of Hib protein-polysaccharide conjugate vaccines into many industrialized countries over the past 15 years has resulted in the virtual elimination of invasive Hib disease. However, despite the success of the vaccine programme several factors may interfere with the effectiveness of the vaccine in the routine programme, as observed in the UK recently. Such factors may include interference with other concomitant vaccines, waning immunity in the absence of booster doses of vaccine, and reduced natural boosting as a result of decreased transmission of the organism. However, the burden of disease remains highest in resource-poor countries and urgent efforts are needed to provide the benefits of this vaccine for children living in regions where it cannot be used for economic and logistical reasons.     

Clonality of multidrug-resistant nontypeable strains of Haemophilus influenzae.

The genetic structure of a population of multidrug-resistant nontypeable (unencapsulated) Haemophilus influenzae strains isolated at a hospital in Barcelona, Spain, was investigated by using multilocus enzyme electrophoresis to determine the allelic variation in 15 structural loci. In our study we have also included some antimicrobial agent-susceptible strains isolated at the same hospital. All enzymes were polymorphic for two to eight electromorphs, and the analysis revealed 43 distinct electrophoretic types among the 44 isolates. The mean genetic diversity of the entire population was 0.55. Multilocus linkage disequilibrium analysis of the isolates revealed a strong association between alleles, suggesting little possibility of recombination. Furthermore, the dendrogram and the allele mismatch distribution are typical of a population with no extensive genetic mixing.     

Characterization of transferrin binding proteins 1 and 2 in invasive type b and nontypeable strains of Haemophilus influenzae.

Haemophilus influenzae has the ability to obtain iron from human transferrin via two bacterial cell surface transferrin binding proteins, Tbp1 and Tbp2. Although a wide array of strains have been shown to express these receptor proteins, two studies have recently identified a series of isolates which appeared to lack the ability to bind transferrin. Included in this group were the members of a cryptic genospecies of nontypeable biotype IV strains which appear to possess a tropism for female urogenital tissues and are major etiologic agents of neonatal and postpartum bacteremia due to H. influenzae. The present study employed oligonucleotide primers specific for genes encoding the Tbp proteins of a type b biotype I strain of H. influenzae to probe the genomic DNAs of isolates from the previous studies. The tbpA and tbpB genes which encode Tbp1 and Tbp2, respectively, were detected in all of the strains tested either by PCR amplification directly or by Southern hybridization analysis. All of the strains displayed a transferrin binding phenotype, and affinity isolation of receptor proteins with transferrin-conjugated Sepharose recovered Tbp1 and/or Tbp2 from 11 of 14 strains, including 2 of the nontypeable biotype IV strains. In addition, all of the strains were capable of growing on human transferrin specifically, indicating that the mechanism of iron assimilation from transferrin is functional and is not siderophore mediated. These results confirm the presence of tbp genes in all of the invasive H. influenzae isolates characterized to date, suggesting that Tbp-mediated iron acquisition is important in disease which initiates from either the respiratory or urogenital mucosa.     

Expression in Escherichia coli of a high-molecular-weight protective surface antigen found in nontypeable and type b Haemophilus influenzae.

An Escherichia coli clone producing a high-molecular-weight surface antigen of Haemophilus influenzae type b (Hib) was isolated from a library of Hib DNA fragments cloned as lysogens in a lambda replacement vector. The antigen is found in sarcosyl-insoluble outer membrane protein preparations and was produced by all 36 H. influenzae isolates tested. Absorption studies indicated that the antigen is a surface determinant on all isolates tested. Antibodies to the antigen (D15) were found in eight of nine convalescent-phase sera from children with invasive Hib infection. Affinity-purified antibodies prepared against the cloned antigen gave protection against the development of bacteremia in a rat pup model.     

Pathogenesis of bloodstream invasion with Haemophilus influenzae type b

Possible route(s) by which encapsulated bacteria invade the blood from the nasopharynx include (i) the direct invasion of submucosal blood vessels and (ii) clearance via lymphatics to regional nodes followed by bloodstream invasion. These possibilities were investigated in rats after intranasal inoculation with 10(5) Haemophilus influenzae type b. Within 24 h of inoculation, 10 of 42 rats with sterile blood cultures had similar numbers of H. influenzae b recovered from both cervical (local) and periiliac (distant) lymph nodes, which suggested early bacteremic spread. When virtually continuous blood cultures were obtained for 30 min after inoculation with 10(8) H. influenzae b, early transient bacteremia was documented in four of eight rats. Also, we found no significant difference in bacteremia among rats whose cervical lymph nodes had been removed surgically compared with sham-operated rats. These findings favor the hypothesis of a rapid, perhaps direct invasion of pharyngeal blood vessels as an initial determinant of the systemic spread of H. influenzae b.     

A recombinant non-fatty acylated form of the Hi-PAL (P6) protein of Haemophilus influenzae elicits biologically active antibody against both nontypeable and type b H. influenzae.

An approximately 15,000-dalton outer membrane lipoprotein of Haemophilus influenzae, the Hi-PAL (P6) protein, has been shown to elicit bactericidal and protective antibodies against both type b and nontypeable H. influenzae strains and is a vaccine candidate for these organisms. To determine whether the lipid modification of this protein is required for immunogenicity or the elicitation of biologically active antibodies, a genetic fusion was constructed that contains the sequence of mature Hi-PAL fused to the polylinker region of pUC19. The protein expressed by this clone does not contain detectable lipid and was purified to homogeneity. This recombinant fusion protein, rPAL, elicited a strong immune response when injected into rabbits, and the antiserum reacted well with native Hi-PAL. The antiserum was bactericidal against a number of clinical nontypeable strains, duplicating the activity of anti-Hi-PAL. The anti-rPAL antiserum was also protective against type b bacteremia in the infant rat model. These results demonstrate that purified rPAL elicits antibodies with biological activities that are similar to those of anti-Hi-PAL antibodies. Thus, the lipid component of Hi-PAL is not required for either immunogenicity or elicitation of biologically active antibodies.     

Comparative virulence of Haemophilus influenzae with a type b or type d capsule.

To determine the importance of specific capsule type in the pathogenesis of invasive Haemophilus influenzae disease, we compared the virulence of type b and type d strains isolated from different children with the virulence of transformation-derived type b and type d organisms. In addition, the unencapsulated derivative of these strains was also examined. Virulence was assessed by determining the ability of the strains to produce bacteremia with intranasal or subcutaneous inoculation. Unencapsulated derivatives were unable to cause bacteremia by any route; all type b strains (whether natural or derived by transformation), a natural type d, and a type d derived by transformation were able to produce bacteremia with similar frequency (42 to 62%) when 10(7) colony-forming units was given intranasally. Subcutaneous inoculation of 10(3) colony-forming units of strains with the type b capsule produced bacteremia at a greater frequency than did the strains with the type d capsule (P less than 0.002). The type d isolate was more virulent than a mutagenized derivative of the strain. We conclude that the type b strains are more virulent than type d when inoculated subcutaneously.     

The gene encoding protein D (hpd) is highly conserved among Haemophilus influenzae type b and nontypeable strains.

The molecular conservation of a surface-exposed lipoprotein, protein D, of Haemophilus influenzae was studied by cloning and sequencing of the gene encoding protein D from three encapsulated type b strains and three nontypeable strains of H. influenzae. These nucleotide sequences were analyzed with previously reported sequences from one type b strain and one nontypeable strain. The nucleotide sequences and the deduced amino acid sequences for protein D were highly conserved. The deduced amino acid sequence (364 amino acids) of protein D from six strains differed only in two amino acids near the C-terminal end. The remaining two strains, one type b and one nontypeable, differed from the consensus sequence in 7 amino acids each. Protein D is 64 and 36% identical and 77 and 56% similar to the glycerophosphodiester phosphodiesterases (GlpQ) of Escherichia coli and Bacillus subtilis.     

Reactivity of Haemophilus influenzae type b anti-pili antibodies

The reactivity of anti-pilus antibodies to native and denatured Haemophilus influenzae b (Hib) pili was studied using rabbit serum prepared against piliated H. influenzae b strain M43 (p+) and adsorbed with its non-piliated variant, strain M42 (p-). The specificity of the adsorbed serum for Hib pili was documented by immunogold electron microscopy and by immunoprecipitation, which revealed the 24 kDa pilin band from strain M43 (p+) that was not seen on strain M42 (p-). In immunodot assays, the anti-pilus antibodies reacted with the native pili present on the outer membrane of strain M43 (p+), but on Western blot assay using denatured outer membranes, the anti-pilus antibodies did not react with the 24 kDa pilin subunit. These data demonstrate that the anti-pilus antibodies in the adsorbed serum recognize conformational epitopes that depend on the tertiary or quaternary structure of Hib pili.