ofHaemophilus Influenzaetype Research Articles

Investigating the candidacy of a capsular polysaccharide-based glycoconjugate as a vaccine to combat Haemophilus influenzae type a disease: A solution for an unmet public health need.

After the introduction of the glycoconjugate vaccine based upon the capsular polysaccharide ofHaemophilus influenzaetype b in the mid 1980s there was a remarkable decrease in the number of invasive cases reported for this organism. Since the 1990s several groups have observed the emergence ofHaemophilus influenzaetype a (Hia), especially in indigenous communities in the northern regions of Canada and Alaska, to a stage where a solution is warranted to prevent further unnecessary deaths due to this pathogen. A glycoconjugate vaccine solution based upon the type a capsular polysaccharide (CPS) was investigated pre-clinically in an effort to illustrate the proof of concept for this approach. In this study we describe the growth of Hia and the isolation, purification and conjugation of the CPS to several carrier proteins. The resulting glycoconjugates were immunised in mice and rabbits provoking sera that facilitated bactericidal killing against all type a strains that we tested. This study has illustrated the pre-clinical proof of concept of a glycoconjugate vaccine based on the CPS of Hia asa solution to this emerging disease.

Impaired haemophilus influenzae type b transplacental antibody transmission and declining antibody avidity through the first year of life represent potential vulnerabilities for HIV-exposed but -uninfected infants.

To determine whether immune function is impaired among HIV-exposed but -uninfected (HEU) infants born to HIV-infected mothers and to identify potential vulnerabilities to vaccine-preventable infection, we characterized the mother-to-infant placental transfer of Haemophilus influenzae type b-specific IgG (Hib-IgG) and its levels and avidity after vaccination in Ugandan HEU infants and in HIV-unexposed U.S. infants. Hib-IgG was measured by enzyme-linked immunosorbent assay in 57 Ugandan HIV-infected mothers prenatally and in their vaccinated HEU infants and 14 HIV-unexposed U.S. infants at birth and 12, 24, and 48 weeks of age. Antibody avidity at birth and 48 weeks of age was determined with 1 M ammonium thiocyanate. A median of 43% of maternal Hib-IgG was transferred to HEU infants. Although its level was lower in HEU infants than in U.S. infants at birth (P < 0.001), Hib-IgG was present at protective levels (>1.0 μg/ml) at birth in 90% of HEU infants and all U.S. infants. HEU infants had robust Hib-IgG responses to a primary vaccination. Although Hib-IgG levels declined from 24 to 48 weeks of age in HEU infants, they were higher than those in U.S. infants (P = 0.002). Antibody avidity, comparable at birth, declined by 48 weeks of age in both populations. Early vaccination of HEU infants may limit an initial vulnerability to Hib disease resulting from impaired transplacental antibody transfer. While initial Hib vaccine responses appeared adequate, the confluence of lower antibody avidity and declining Hib-IgG levels in HEU infants by 12 months support Hib booster vaccination at 1 year. Potential immunologic impairments of HEU infants should be considered in the development of vaccine platforms for populations with high maternal HIV prevalence.

Clinical Significance and Characterization of Haemophilus influenzae Type b Genogroup Isolates from Urine Samples in an Adult Male Population

The occurrence and significance of Haemophilus spp. isolated from the genitourinary tract are not well known. Herein, we describe the clinical significance and characteristics of Haemophilus influenzae type b genogroup strains isolated from genitourinary tract specimens from an adult male veteran patient population and, in particular, their associations with prostatitis and epididymitis.

Impact ofHaemophilus influenzaetype b vaccination on the incidence of invasiveHaemophilus influenzaedisease in France, 15 years after its introduction

We assessed the impact of Haemophilus influenzae type b (Hib) vaccination, introduced in France in early 1993, on the incidence of invasive Haemophilus influenzae (Hi) disease up to 2008.The incidence of Hi meningitis fell from 0·9/100000 in 1991–1992 to 0·09/100 000 in 1996–2008,with a marked decline (96%) in children aged <5 years, including infants aged <3 months, from 12 to 0·4 /100 000. The incidence of invasive Hi disease also decreased in children aged <15 years from 6 to 0·7 /100 000, remained stable in the 15–64 years age group at about 0·5/100 000,and increased slightly from 2·0 to 2·4 /100 000 in persons aged >64 years. No emergence of non-encapsulated or encapsulated non-vaccine serotypes was observed. These findings confirm the major direct impact of Hib vaccination on the incidence of Hi invasive disease in children and the indirect benefit of vaccination for infants too young to be vaccinated.

Risk of Invasive Haemophilus influenzae Type b (Hib) Disease in Adults with Secondary Immunodeficiency in the Post-Hib Vaccine Era

Prior to the introduction of Haemophilus influenzae type b (Hib) conjugate vaccines, invasive Hib disease affected almost exclusively children. According to some recent studies, in the postvaccine era, adults, the elderly, and immunocompromised persons can be affected more often than children. As the production of type-specific anti-capsular polysaccharide antibodies is the major defense mechanism against Hib, individuals with defects in humoral immune responses have high susceptibility to infections caused by Hib. We hypothesized that nonvaccinated adults with chronic conditions causing immunosuppression may lack protective antibody to Hib. We assessed serum anti-Hib IgG levels and bactericidal activity in 59 patients with chronic renal failure, 30 patients with type 2 diabetes mellitus, 28 patients with chronic obstructive pulmonary disease (COPD), and 20 patients with multiple myeloma compared to 32 healthy controls of similar age. Considering antibody at >0.15 μg/ml as the protective correlate in unvaccinated individuals, we detected subprotective Hib antibody levels in 29% of chronic renal failure, 20% of diabetes, 14% of COPD, and 55% of myeloma patients compared to 3% of healthy controls. Additionally, 70% of myeloma and 58% of chronic renal failure patients did not have detectable serum bactericidal activity against Hib. Among individuals with severe diseases causing secondary immunodeficiency, patients with multiple myeloma and chronic renal failure are at an increased risk of invasive Hib disease. Considering that Hib continues to circulate in the population, this study provides a rationale for the immunization of some adult patients with secondary immunodeficiency with the pediatric Hib vaccine to achieve protective immunity.

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.

Genetic Characterization of the Capsulation Locus of Haemophilus influenzae Serotype e

The capsulation (cap) locus of Haemophilus influenzae type e (Hie) was characterized and sequenced. No IS1016 element was found to flank the locus. The 18.2-kb locus included 14 open reading frames (ORFs), which were grouped into three functional regions. Eight new ORFs (named ecs1 to ecs8) were identified in the Hie capsule-specific region II.

Immunogenicity of a Fourth Dose of Haemophilus influenzae Type b (Hib) Conjugate Vaccine and Antibody Persistence in Young Children from the United Kingdom Who Were Primed with Acellular or Whole-Cell Pertussis Component-Containing Hib Combinations in Infancy

In response to the rising incidence of Haemophilus influenzae type b (Hib) disease in the United Kingdom, a national campaign to give a booster dose of single-antigen Hib conjugate vaccine to children aged 6 months to 4 years was undertaken in 2003. Children (n = 386) eligible for Hib vaccine in the campaign were recruited. Hib antibody concentrations were measured before boost and at 1 month, 6 months, 1 year, and 2 years after boost and were analyzed according to children's ages at booster dose and whether a Hib combination vaccine containing acellular pertussis (aP) or whole-cell pertussis (wP) components was given in infancy. The geometric mean antibody concentrations (GMCs) before the booster declined as the time since primary immunization increased (P < 0.001), and GMCs were threefold higher in recipients of wP-Hib than aP-Hib combination vaccines (P < 0.001). GMCs 1 month after the booster increased with age (P < 0.001) as follows: 6 to 11 months; 30 microg/ml (95% confidence interval [CI], 22 to 40); 12 to 17 months, 68 microg/ml (95% CI, 38 to 124); and 2 to 4 years, 182 microg/ml (151 to 220), with no difference according to the type of priming vaccine received. Antibody levels declined after the booster, but 2 years later, GMCs were more than 1.0 microg/ml for all age groups. By extrapolating data for the decline in antibody levels, we found the GMCs 4 years after boosting were predicted to be 0.6, 1.4, and 2.6 microg/ml for those boosted at 6 to 11 months, 12 to 17 months, and 2 to 4 years, respectively, with levels of at least 0.15 microg/ml in about 90% of individuals. A booster dose of Hib vaccine given after the first year of life should provide long-lasting protection.

How do caregivers know when to take their child for immunizations?

BackgroundChildhood vaccinations help reduce and eliminate many causes of morbidity and mortality among children. The objective of this study was to compare 4:3:1:3:3 (4+ doses of diphtheria and tetanus toxoids and pertussis vaccine, 3+ doses of poliovirus vaccine, 1+ doses of measles-containing vaccine, 3+ doses of Haemophilus influenzae type b vaccine, and 3+ doses of hepatitis B vaccine) coverage among children whose caregivers learned by different methods when their child's most recent immunization was needed.MethodsBetween July 2001 and December 2002, a portion of households receiving the National Immunization Survey were asked how they knew when to take the child in for his/her most recent immunization. Responses were post-coded into several categories: 'Doctor/nurse reminder at previous immunization visit', 'Shot card/record', 'Reminder/recall', and 'Other'. Respondents could give more than one answer. Children who did not receive any vaccines, had ≤ 1 visits for vaccinations, or whose caregiver did not provide an answer to the question were excluded from analyses. Chi-square analyses were used to compare 4:3:1:3:3 coverage among 19–35 month old children.ResultsChildren whose caregivers indicated that a doctor/nurse told them at a previous immunization visit when to return for the next immunization had significantly greater 4:3:1:3:3 coverage than those who did not choose the response (77.2% vs. 70.1%, p < 0.01). However, no significant difference in coverage was found between households that did/did not indicate that reminder/recalls (71.0% vs. 75.5%, p = 0.24) helped them remember when to take their child for their most recent immunization visit; only borderline significance was found between those that did/did not choose shot cards (70.6% vs. 76.2%, p = 0.07).ConclusionA doctor or nurse's reminder during an immunization visit of the next scheduled immunization visit effectively encourages caregivers to bring children in for immunizations, providing an inexpensive and easy way to effectively increase immunization coverage.

Chemical structure, conjugation, and cross-reactivity of Bacillus pumilus Sh18 cell wall polysaccharide.

Bacillus pumilus strain Sh18 cell wall polysaccharide (CWP), cross-reactive with the capsular polysaccharide of Haemophilus influenzae type b, was purified and its chemical structure was elucidated using fast atom bombardment mass spectrometry, nuclear magnetic resonance techniques, and sugar-specific degradation procedures. Two major structures, 1,5-poly(ribitol phosphate) and 1,3-poly(glycerol phosphate), with the latter partially substituted by 2-acetamido-2-deoxy-alpha-galactopyranose (13%) and 2-acetamido-2-deoxy-alpha-glucopyranose (6%) on position O-2, were found. A minor component was established to be a polymer of -->3-O-(2-acetamido-2-deoxy-beta-glucopyranosyl)-1-->4-ribitol-1-OPO3-->. The ratios of the three components were 56, 34, and 10 mol%, respectively. The Sh18 CWP was covalently bound to carrier proteins, and the immunogenicity of the resulting conjugates was evaluated in mice. Two methods of conjugation were compared: (i) binding of 1-cyano-4-dimethylaminopyridinium tetrafluoroborate-activated hydroxyl groups of the CWP to adipic acid dihydrazide (ADH)-derivatized protein, and (ii) binding of the carbodiimide-activated terminal phosphate group of the CWP to ADH-derivatized protein. The conjugate-induced antibodies reacted in an enzyme-linked immunosorbent assay with the homologous polysaccharide and with a number of other bacterial polysaccharides containing ribitol and glycerol phosphates, including H. influenzae types a and b and strains of Staphylococcus aureus and Staphylococcus epidermidis.

Detection of Haemophilus influenzae type b by real-time PCR.

A real-time PCR assay targeting the capsulation locus of Haemophilus influenzae type b (Hib) was developed. The linear detection range was from 1 to 10(6) microorganisms per reaction mixture. No H. influenzae other than Hib or any other control bacteria typically found in the upper respiratory tract was detected.

Differences in genetic and transcriptional organization of the glpTQ operons between Haemophilus influenzae type b and nontypeable strains.

The glpTQ operon of Haemophilus influenzae type b (Hib) and nontypeable H. influenzae (NTHi) strains is highly conserved, except for a 1.4-kb glpTQ intergenic region that was found in most Hib strains. The presence of this intergenic region results in divergent glpTQ transcriptional profiles for Hib and NTHi where Hib strains appear to have evolved an alternative promoter for glpQ expression. Based on the intergenic region's low G+C content, we speculate that this DNA fragment was acquired by lateral transfer.

Role of surface-exposed loops of Haemophilus influenzae protein P2 in the mitogen-activated protein kinase cascade.

The outer membrane of gram-negative bacteria contains several proteins, and some of these proteins, the porins, have numerous biological functions in the interaction with the host; porins are involved in the activation of signal transduction pathways and, in particular, in the activation of the Raf/MEK1-MEK2/mitogen-activated protein kinase (MAPK) cascade. The P2 porin is the most abundant outer membrane protein of Haemophilus influenzae type b. A three-dimensional structural model for P2 was constructed based on the crystal structures of Klebsiella pneumoniae OmpK36 and Escherichia coli PhoE and OmpF. The protein was readily assembled into the beta-barrel fold characteristic of porins, despite the low sequence identity with the template proteins. The model provides information on the structural features of P2 and insights relevant for prediction of domains corresponding to surface-exposed loops, which could be involved in the activation of signal transduction pathways. To identify the role of surface-exposed loops, a set of synthetic peptides were synthesized according to the proposed model and were assayed for MEK1-MEK2/MAPK pathway activation. Our results show that synthetic peptides corresponding to surface loops of protein P2 are able to activate the MEK1-MEK2/MAPK pathways like the entire protein, while peptides modeled on internal beta strands are unable to induce significant phosphorylation of the MEK1-MEK2/MAPK pathways. In particular, the peptides corresponding to loops L5 (Lys206 to Gly219), L6B (Ser239 to Lys253), and L7 (Thr280 to Lys287) activate, as the whole protein, essentially JNK and p38.

Structure and function of Hib pili from Haemophilus influenzae type b.

Pathogenic bacteria are specifically adapted to bind to their customary host. Disease is then caused by subsequent colonization and/or invasion of the local environmental niche. Initial binding of Haemophilus influenzae type b to the human nasopharynx is facilitated by Hib pili, filaments expressed on the bacterial surface. With three-dimensional reconstruction of electron micrograph images, we show that Hib pili comprise a helix 70 A in diameter with threefold symmetry. The Hib pilus filament has 3.0 subunits per turn, with each set of three subunits translated 26.9 A along and rotated 53 degrees about the helical axis. Amino acid sequence analysis of pilins from Hib pili and from P-pili expressed on uropathogenic Escherichia coli were used to predict the physical location of the highly variable and immunogenic region of the HifA pilin in the Hib pilus structure. Structural differences between Hib pili and P-pili suggest a difference in the strategies by which bacteria remain bound to their host cells: P-pili were shown to be capable of unwinding to five times their original length (E. Bullitt and L. Makowski, Nature 373:164-167, 1995), while damage to Hib pili occurs by slight shearing of subunits with respect to those further along the helical axis. This capacity to resist unwinding may be important for continued adherence of H. influenzae type b to the nasopharynx, where the three-stranded Hib pilus filaments provide a robust tether to withstand coughs and sneezes.

Interaction of fimbriae of Haemophilus influenzae type B with heparin-binding extracellular matrix proteins.

The interaction of the fimbriae of Haemophilus influenzae type b (Hib) with two heparin-binding extracellular matrix proteins, human fibronectin (Fn) and heparin-binding growth-associated molecule (HB-GAM) from mouse, were studied. The fimbriated Hib strain 770235 fim+, as well as the recombinant strain E. coli HB101(pMH140), which expressed Hib fimbriae, adhered strongly to Fn and HB-GAM immobilized on glass. Purified Hib fimbriae bound to Fn and HB-GAM, and within the Fn molecule, the binding was localized to the N-terminal 30,000-molecular-weight (30K) and 40K fragments, which contain heparin-binding domains I and II, respectively. Fimbrial binding to Fn, HB-GAM, and the 30K and the 40K fragments was inhibited by high concentrations of heparin. The results show that fimbriae of Hib interact with heparin-binding extracellular matrix proteins. The nonfimbriated Hib strain 770235 fim- exhibited a low level of adherence to Fn but did not react with HB-GAM, indicating that Hib strains also possess a fimbria-independent mechanism to interact with Fn.

Pulsed-field gel electrophoresis used to investigate genetic diversity of Haemophilus influenzae type b isolates in Australia shows differences between Aboriginal and non-Aboriginal isolates.

We used pulsed-field gel electrophoresis to study the epidemiology and population structure of Haemophilus influenzae type b. DNAs from 187 isolates recovered between 1985 and 1993 from Aboriginal children (n = 76), non-Aboriginal children (n = 106), and non-Aboriginal adults (n = 5) in urban and rural regions across Australia were digested with the SmaI restriction endonuclease. Patterns of 13 to 17 well-resolved fragments (size range, approximately 8 to 500 kb) defining 67 restriction fragment length polymorphism (RFLP) types were found. Two types predominated. One type (n = 37) accounted for 35 (46%) of the isolates from Aboriginals and 2 (2%) of the isolates from non-Aboriginals, and the other type (n = 41) accounted for 2 (3%) of the isolates from Aboriginals and 39 (35%) of the isolates from non-Aboriginals. Clustering revealed seven groups at a genetic distance of approximately 50% similarity in a tree-like dendrogram. They included two highly divergent groups representing 50 (66%) isolates from Aboriginals and 6 (5%) isolates from non-Aboriginals and another genetically distinct group representing 7 (9%) isolates from Aboriginals and 81 (73%) isolates from non-Aboriginals. The results showed a heterogeneous clonal population structure, with the isolates of two types accounting for 42% of the sample. There was no association between RFLP type and the diagnosis of meningitis or epiglottitis, age, sex, date of collection, or geographic location, but there was a strong association between the origin of isolates from Aboriginal children and RFLP type F2a and the origin of isolates from non-Aboriginal children and RFLP type A8b. The methodology discriminated well among the isolates (D = 0.91) and will be useful for the monitoring of postvaccine isolates of H. influenzae type b.

A Simple and Rapid Method for Measuring Unconjugated Capsular Polysaccharide (PRP) ofHaemophilus influenzaetype b in PRP-tetanus toxoid conjugate vaccine

The authors developed a simple rapid method for quantitation of free capsular polysaccharide ofHaemophilus influenzaetype b (polyribosyl ribitol phosphate, PRP) in PRP-tetanus toxoid conjugate vaccine based on acid precipitation of tetanus toxoid (TT). Acid hydrolysis of PRP during the assay was not detected. The conditions used in the assay did not precipitate unconjugated PRP or adipic acid dihydrazide derivatized PRP. The method was highly reliable, reproducible and sensitive. The accuracy of the assay was confirmed by spiking known amounts of unconjugated PRP to PRP-TT conjugate preparations. A PRP-TT preparation, incubated at 37°C for 6 months showing most of the PRP as unconjugated (87% determined by this method), was not immunogenic in mice for the PRP component even after two injections. In contrast, the same preparation held at 4°C for 20 months, showing 17% unconjugated PRP, induced IgG antibodies to PRP which were boosted after second injection. Therefore, this method is very useful to evaluate the stability of PRP-TT conjugate vaccine. The assay may be useful for characterizing other polysaccharide-protein conjugate vaccines.

Direct and Indirect Methods for Molar-Mass Analysis of Fragments of the Capsular Polysaccharide ofHaemophilus influenzaeType b

Two methods are described for direct molar-mass measurement of low-molar-mass fragments obtained by oxidative cleavage of the capsular polysaccharide ofHaemophilus influenzaetype b. Absolute molar masses were determined by size-exclusion chromatography (SEC) with detection by multiangle laserlight-scattering photometry (MALLS) and differential refractometry (RI). The end-group structure of the polysaccharide fragments allowed the direct measurement of average chain length by quantitative1H NMR, from which molar masses were derived. Variation between the molar masses obtained by the two methods ranged from 5 to 7%. When molar masses estimated by indirect methods were compared to SEC-MALLS/RI data, significant deviations were observed. Analysis by SEC with secondary calibration with dextran standards gave molar masses that exceeded the SEC-MALLS/RI data by as much as 2.5-fold. Molar masses estimated by a combination of colorimetric assays varied from the SEC-MALLS/RI data by as much as 50%. These results demonstrated the applicability and superior accuracy of the direct methods of molar-mass determination of the polysaccharide fragments.

Glycoconjugate Vaccines: What Next?

The principle that infants can be protected from invasive diseases caused by encapsulated organisms has been proved with the introduction of Haemophilus influenzae type b conjugate vaccines. The use of glycoconjugates to implement some of the goals of the Children's Vaccine Initiative requires a clear delineation of the chemical and immunological specifications for optimal vaccines.

Evaluation ofHaemophilus influenzaeType B Conjugate Vaccine (Meningococcal Protein Conjugate) in Canadian Infants

To assess adverse effects and immune responses with a three-dose series of Haemophilus influenzae type b meningococcal protein conjugate (PedvaxHIB or Hib.OMP) vaccine, including any immunological response alterations from concurrent administration with routine vaccines for infants. Randomized, controlled trial with treatment group crossover for dose 3. Two public health units near Vancouver. One hundred and ten healthy infants eight to 14 weeks old were enrolled; 105 completed the study (95%). All participants received two doses of diphtheria-pertussis-tetanus (dpt) vaccine (at two and four months of age) and one dose of measles-mumps-rubella (mmr) vaccine at 12 months. In each instance, Hib.OMP was given either concurrently in another limb or after a delay of two weeks (after dpt) or four weeks (after mmr). Adverse effects, particularly fever and local erythema, were monitored by parents for 72 h after each dose of Hib.OMP vaccine. Five blood samples were taken at prescribed intervals to assess responses to each dose of Hib.OMP and to selected other vaccine antigens. Follow-up was obtained after all 322 doses of Hib.OMP. Local adverse effects were infrequent and mild: 13% had redness, 17% tenderness. Systemic effects in those given Hib.OMP alone included fever in 8%, irritability in 29%. Anti-polyribose-ribitol phosphate (prp) responses to Hib.OMP were not impaired by coadministration with dpt or mmr vaccines, nor were tetanus or diphtheria antitoxin levels or rubella or measles response rates affected. After two doses of Hib.OMP, 92% were seropositive and 64% had greater than 1.0 μg/mL of anti-prp. After three doses, 100% were seropositive and 82% exceeded 1.0 μg/mL. Hib.OMP vaccine was well tolerated, immunogenic and compatible with vaccines routinely given to infants in Canada.