Induction of meningeal inflammation by outer membrane vesicles of Haemophilus influenzae type b

Haemophilus influenzae type b (Hib) lipooligosaccharide (LOS) induces meningitis in an established rabbit model of experimental meningitis. To evaluate the inflammatory ability of Hib LOS in its native state as part of outer membrane of Hib, the CSF inflammatory response to Hib outer membrane vesicles (OMV) was compared with that produced by Hib LOS. A significant dose-dependent meningeal inflammatory response, as evidence by increased concentrations of white blood cells and protein and lactate concentration in CSF, was found after intracisternal injection of Hib OMV containing 0.02-200 ng of LOS. On a LOS weight basis, purified LOS and LOS in OMV did not differ significantly with regard to the CSF inflammatory response. Preincubation of Hib OMV with monoclonal antibodies directed against cell surface-exposed epitopes on Hib LOS did not affect the inflammatory ability of OMV, whereas preincubation of OMV with polymyxin B significantly reduced both pleocytosis and lactate concentration. Thus, Hib OMV represents a relevant nonreplicating vehicle in which Hib LOS might interact with CNS tissues to produce meningitis.     

The other siblings: Respiratory infections caused by Moraxella catarrhalis and Haemophilus influenzae

Respiratory infections remain substantial causes of morbidity and mortality globally. In this paper, two substantial players in bacterial-associated respiratory disease are assessed as to their respective roles in children and adults and in the developed and developing world. Moraxella catarrhalis, although initially thought to be a nonpathogen, continues to emerge as a cause of upper respiratory disease in children and pneumonia in adults. No vaccine is currently available to prevent M. catarrhalis infection. Haemophilus in.uenzae type b, originally thought to be the cause of in.uenza, has now been limited epidemiologically in the developed world due to an effective immunization but it continues to be a major player in the developing world. Nonencapsulated strains of H. influenzae still remain as significant causes of respiratory infections in the developing world especially in exacerbation of chronic obstructive lung disease. Finally, and in brief, the spectrum of Brazilian purpuric fever due to a specific biotype of H. influenzae is discussed.     

Identification of a specific epitope of Haemophilus influenzae on a 16,600-dalton outer membrane protein

A mouse monoclonal antibody that recognizes an epitope on a 16,600-dalton outer membrane protein was developed to nontypable Haemophilus influenzae. This epitope was present on all 115 isolates of H. influenzae tested, including typable and nontypable strains. Screening of 89 strains of other bacteria demonstrated that this epitope is a highly specific marker for H. influenzae because the epitope was absent in virtually all other bacterial species tested. Western blot assays were performed with two normal human serum samples and convalescent-phase serum from an adult with bacteremia due to nontypable H. influenzae. Antibody to the 16,600-dalton outer membrane protein was present in all three human serum samples.     

Changes in outer membrane proteins of nontypable Haemophilus influenzae in patients with chronic obstructive pulmonary disease

Five individual colonies of Haemophilus influenzae were isolated from each of one to three cultures of sputum collected from 18 patients with chronic obstructive pulmonary disease (COPD). The isolates were studied to investigate whether the major outer membrane proteins (MOMPs) changed during persistence. The relationship between isolates was analyzed by fingerprinting their chromosomal DNA. The fingerprints of eight strains (isolated from eight patients) with various MOMP compositions were different, whereas fingerprints of isolates with identical MOMP compositions were indistinguishable. In 12 patients, two or more strains with different MOMP compositions were found; one strain was isolated from the sputum samples of each of the six remaining patients. In seven of the 12 patients, strains with different MOMPs but with indistinguishable fingerprints were found. The differences were found in proteins b,c (five patients) and d (five patients). In patients with COPD, the MOMPs of H. influenzae are subject to changes that may enable this bacterium to escape immunological defense mechanisms.     

Detection of Haemophilus influenzae by loop-mediated isothermal amplification (LAMP) of the outer membrane protein P6 gene

It is difficult and time-consuming to distinguish Haemophilus influenzae from the genotypically similar Haemophilus parainfluenzae, which is a commensal of the human oral cavity. The novel nucleic acid amplification technique of loop-mediated isothermal amplification (LAMP), which amplifies DNA under isothermal conditions (63 degrees C) with high specificity, efficiency, and rapidity, was evaluated for H. influenzae detection. A H. influenzae-specific LAMP primer set was designed for the outer membrane protein P6 gene. Primer set specificity was validated using 4 Haemophilus spp. and 13 other species. Within 60 min, LAMP detected 100 or more copies of purified DNA with a sensitivity that was 10-fold higher than that of conventional PCR. This method can be used to differentiate H. influenzae from H. parainfluenzae strains. Thus, LAMP may represent a sensitive and reliable means of diagnosing H. influenzae infection.     

Clinical features fail to distinguish respiratory infections caused by Branhamella catarrhalis from those caused by Haemophilus influenzae

Branhamella catarrhalis is being isolated with increasing frequency from patients with symptoms and signs of respiratory tract infection. Records of 77 patients were reviewed to define the spectrum of respiratory illness and to compare clinical and laboratory features with those of respiratory infection due to Haemophilus influenzae. Both B catarrhalis and H influenzae caused respiratory infection predominantly in elderly males with underlying heart or lung disease. There were no clinical or laboratory features aside from sputum Gram stain and culture which differentiated the two groups. Although fewer than one-half of each group received antibiotics, no patient developed progressive respiratory disease.     

Nasopharyngeal carriage of Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and Alloiococcus otitidis in young children in the era of pneumococcal immunization, Taiwan

We applied a multiplex polymerase chain reaction (PCR) and culture to detect Streptococcus pneumoniae and detected 3 other respiratory pathogens--Haemophilus influenzae, Moraxella catarrhalis, and Alloiococcus otitidis--simultaneously by PCR, in the nasopharynx of 386 children aged under 5 y. S. pneumoniae was the most common pathogen carried by children in all age groups, with the rate ranging from 15.8% in children aged 3-4 y to 28.6% in children aged 2-3 y. H. influenzae and M. catarrhalis showed similar carriage rates across all the age groups. Only 2 young children (0.5%) carried A. otitidis. Higher carriage of S. pneumoniae was found in children who had not received the heptavalent pneumococcal conjugate vaccine (PCV7). Cefotaxime non-susceptibility was high (51.4%) in S. pneumoniae nasopharyngeal isolates. Serotype 6B was the most common in fully immunized carriers and also in those who received catch-up immunization. Due to low PCV7 coverage in Taiwan, the carriage of vaccine and non-vaccine serotypes of S. pneumoniae in children remains common.     

Modification of otitis media in chinchillas rechallenged with nontypable Haemophilus influenzae and serological response to outer membrane antigens

Otitis media was produced in chinchillas by right-sided intrabullar inoculation with nontypable Haemophilus influenzae, and susceptibility to reinfection was investigated. After resolution of initial right-sided infection, animals underwent ipsilateral or contralateral intrabullar rechallenge with the same strain. After ipsilateral rechallenge right ears were completely protected against reinfection; previously uninfected left ears were similarly protected on contralateral rechallenge. Previously infected ears remained fully susceptible to infection with a heterologous strain of nontypable H. influenzae. Using an enzyme-linked immunosorbent assay, we measured the serological response to outer membrane protein and lipopolysaccharide antigens during initial infection. A greater than or equal to 10-fold rise in titer of antibody to homologous outer membrane proteins was observed in all 11 animals tested. Most animals exhibited a minimal serological response to lipopolysaccharide. Thus experimental otitis media due to nontypable H. influenzae induces strain-specific protective immunity and a concomitant serological response to outer membrane proteins.     

AWARE Ceftaroline Surveillance Program (2008-2010): Trends in Resistance Patterns Among Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis in the United States

Ceftaroline fosamil, the prodrug form of the active metabolite ceftaroline, is a new broad-spectrum parenteral cephalosporin with antibacterial activity against the prevalent respiratory pathogens Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and Staphylococcus aureus. Bacterial resistance surveillance (5330 isolates) was conducted in the United States between 2008 and 2010 to assess the in vitro activity of ceftaroline and comparator antibacterial agents against invasive respiratory isolates of S. pneumoniae (3329 isolates), H. influenzae (1545 isolates), and M. catarrhalis (456 isolates). All organisms were cultured from patient infections in 71 US hospital laboratories and were submitted to a central reference monitor for broth microdilution testing by Clinical and Laboratory Standards Institute reference methods. Against S. pneumoniae, ceftaroline inhibited 98.7% of strains at the susceptible breakpoint of ≤0.25?μg/mL (50% minimum inhibitory concentration [MIC(50)], 0.01?μg/mL; 90% MIC [MIC(90)], 0.12?μg/mL) and was 16-fold more active than ceftriaxone (MIC(90), 2?μg/mL). Among 70 ceftriaxone-resistant pneumococcal isolates, all were inhibited by ≤0.5?μg/mL of ceftaroline. Haemophilus influenzae (MIC(50), ≤0.008?μg/mL; MIC(90), 0.015?μg/mL) and M. catarrhalis (MIC(50), 0.06?μg/mL; MIC(90), 0.12?μg/mL) were very susceptible to ceftaroline regardless of β-lactamase production. Whereas the high-level of activity of ceftaroline was maintained against S. pneumoniae and H. influenzae from 2008 through 2010, increased rates of nonsusceptibility were observed for amoxicillin/clavulanate, erythromycin, and levofloxacin among S. pneumoniae and for trimethoprim/sulfamethoxazole and azithromycin among H. influenzae. In summary, ceftaroline resistance surveillance (Assessing Worldwide Antimicrobial Resistance Evaluation [AWARE] Program) in the United States (2008-2010) documented in vitro sustained potency and spectrum against Gram-positive and Gram-negative pathogens known to cause community-acquired bacterial pneumonia.     

Sequence stability of the gene encoding outer membrane protein P2 of nontypeable Haemophilus influenzae in the human respiratory tract

Nontypeable Haemophilus influenzae (NTHI) is an important cause of lower respiratory tract infections in patients with chronic obstructive pulmonary disease. Recent findings suggest that the major outer membrane protein P2 should be reconsidered as a vaccine candidate for NTHI. A P2-based vaccine would require a relative degree of sequence stability of the gene encoding P2 (ompP2) during colonization. To characterize the sequence stability of ompP2 during colonization of the human respiratory tract, ompP2 genes from 13 sets of isolates that persisted in patients with chronic obstructive pulmonary disease (mean colonization, 7 months) were sequenced. In 9 sets of isolates, ompP2 did not change. Sequence changes were noted in 4 sets of isolates. Most of these changes occurred within areas of repetitive DNA, suggesting that this type of DNA has a role in antigenic variation of P2. The sequence of ompP2 is relatively stable during persistence of NTHI in the human host.     

Distribution and excretion of capsular antigen after immunization with Haemophilus influenzae type b polysaccharide-Neisseria meningitidis outer membrane protein conjugate vaccine

The occurrence of Haemophilus influenzae type b capsular polysaccharide antigenemia and antigenuria following immunization was studied in 48 healthy 2-month-old infants. Each received a conjugate vaccine consisting of H. influenzae type b capsular polysaccharide covalently linked to Neisseria meningitidis serotype b outer membrane protein at 2 and 4 months of age. Infants were alternated at enrollment for collection of blood and urine after either the first or second dose. Specimens were obtained "early" (2-3 days) after immunization and "late" (7 days) after immunization and assayed for antigen. Antigen was detected in the serum of 3 (6%) of 48 infants, uniformly in the "early" specimen obtained after the first dose of vaccine. Antigenuria occurred in 37 (80%) of 46 infants; for greater than or equal to 7 days in 12 (26%). Antigenuria was frequent after administration of the vaccine but antigenemia was not. These data should be considered in the evaluation of an infant with suspected H. influenzae type b invasive disease.     

Immune response to surface protein A of Streptococcus pneumoniae and to high-molecular-weight outer membrane protein A of Moraxella catarrhalis in children with acute otitis media

The immune response was evaluated in 11 children with Streptococcus pneumoniae and in 9 children with Moraxella catarrhalis otitis media. The age of the children had a range of 4-32 months. The mean IgG, IgM, and IgA antibody responses to surface protein A (PspA) of S. pneumoniae in sera from children at the acute and convalescent stages were 4864 versus 5831 ng/mL, P<.05, 1075 versus 3752 ng/mL, P<.05, and 67 versus 93 ng/mL, nonsignificant (NS), respectively. The mean IgG, IgM, and IgA antibody responses to the high-molecular-weight outer membrane protein (UspA) of M. catarrhalis in sera from children at acute and convalescent stages were 710 versus 935 mg/mL, NS; 1895 versus 2646 ng/mL, NS; and 121 versus 204 ng/mL, P<.05, respectively. These data show that PspA and UspA are immunogenic in children after otitis media.     

Efficacy of immunization with outer membrane proteins for induction of pulmonary clearance of nontypeable Haemophilus influenzae in a rat respiratory model

Three strains of nontypeable Haemophilus influenzae namely NTHi-I, NTHi-II and NTHi-III were isolated from the sputum of patients with bronchitis and identified by biochemical, serological and electron microscopy. The polypeptide patterns of isolates were compared and found to have similar sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) polypeptide patterns, although some of the bands were specific in some strains. A similar comparison was made on extracted outer membrane proteins (OMPs) on the above mentioned strains, using Triton X-100 and sodium dodecyle sulphate (SDS). It was found that the polypeptides with molecular weights of 70, 42, 33 and 27 KDa were identified as P1, P2, P4 and P5 respectively. The protein estimation of crude OMPs from the three strains were calculated, and OPM-I prepared from NTHi-I showed the highest amount of protein and was chosen for its immunogenicity in a rat respiratory model. The efficacy of immunization with OMP was determined by enhancement of pulmonary clearance of live bacteria in the rat lung. A significant protective immune response induced by OMP was observed by enhanced respiratory clearance of nontypeable H. influenzae following mucosal immunization.     

Platelet-platelet and platelet-leukocyte interactions induced by outer membrane vesicles from N. meningitidis

A large part of native meningococcal lipopolysaccharide (LPS), i.e., LPS integrated in the outer cell membrane, is released in the form of 'blebs' from surplus outer membrane material. In the present study we investigated the effects of purified outer membrane vesicles (OMVs) on blood platelet-platelet and platelet-leukocyte interactions. Citrated whole blood was stimulated in vitro with equal amounts (on a weight basis) of OMV-integrated LPS, purified LPS (P-LPS) from the same meningococcal strain and purified E. coli -LPS. The samples were analyzed by flow cytometry. Upon OMV stimulation platelet aggregation increased 2.1-fold, platelet degranulation 1.8-fold, (measured as CD62P expression), platelet binding to monocytes 2.6-fold, whereas platelet binding to granulocytes increased 2.8-fold. Also, the fraction of large heteroconjugates, i.e., large CD45-positive cell aggregates increased 15.7-fold compared to control. P-LPS and E. coli -LPS also significantly increased platelet aggregation and heteroconjugate formation but did not influence platelet degranulation and binding of platelets to leukocytes in whole blood. When using platelet-rich plasma (PRP), OMVs increased platelet aggregation 2.1-fold and CD62P expression 1.9-fold. P-LPS and E. coli -LPS also significantly increased platelet aggregation in PRP but did not influence platelet degranulation. None of the LPS preparations induced platelet microvesiculation, either in whole blood or in PRP. Conclusion: Meningococcal-derived OMVs as well as purified meningococcal LPS, contribute to increased platelet-platelet and platelet-leukocyte aggregation and may thus be of great importance in the development of microthrombosis and organ dysfunction related to fulminant meningococcal septicemia.     

Xylella fastidiosa outer membrane vesicles modulate plant colonization by blocking attachment to surfaces

Outer membrane vesicles (OMVs) of Gram-negative bacteria have been studied intensively in recent years, primarily in their role in delivering virulence factors and antigens during pathogenesis. However, the near ubiquity of their production suggests that they may play other roles, such as responding to envelope stress or trafficking various cargoes to prevent dilution or degradation by other bacterial species. Here we show that OMVs produced by Xylella fastidiosa, a xylem-colonizing plant pathogenic bacterium, block its interaction with various surfaces such as the walls of xylem vessels in host plants. The release of OMVs was suppressed by the diffusible signal factor-dependent quorum-sensing system, and a X. fastidiosa ΔrpfF mutant in which quorum signaling was disrupted was both much more virulent to plants and less adhesive to glass and plant surfaces than the WT strain. The higher virulence of the ΔrpfF mutant was associated with fivefold higher numbers of OMVs recovered from xylem sap of infected plants. The frequency of attachment of X. fastidiosa to xylem vessels was 20-fold lower in the presence of OMVs than in their absence. OMV production thus is a strategy used by X. fastidiosa cells to adjust attachment to surfaces in its transition from adhesive cells capable of insect transmission to an “exploratory” lifestyle for systemic spread within the plant host which would be hindered by attachment. OMV production may contribute to the movement of other bacteria in porous environments by similarly reducing their contact with environmental constituents.Many important plant diseases such as Pierce disease of grapes and citrus variegated chlorosis (CVC) are associated with the xylem-limited bacteria Xylella fastidiosa (1). Infected plants exhibit progressive leaf scorching or other foliar symptoms consistent with the water stress that is associated with the occlusion of large numbers of xylem vessels by bacterial cells or by tyloses that are induced by the presence of bacteria within vessels (2–4). The virulence of X. fastidiosa is associated with its ability to migrate widely and proliferate within xylem vessels after its spatially limited introduction by infested sharpshooter vectors during feeding (5). Disease symptoms may be largely an inadvertent effect caused by successful colonization that causes interference with xylem sap flow (6). Cells of X. fastidiosa colonize specific areas of the foreguts of insect vectors, where they multiply and form a biofilm, being firmly attached to the foregut cuticular lining to endure the high fluid flow during sap feeding (7, 8). This turbulent environment may lead to occasional detachment of cells, allowing pathogen inoculation into plants (9). Thus, insect colonization and transmission of X. fastidiosa depends on its ability to attach to the insect’s foregut.X. fastidiosa uses diffusible signaling factors (XfDSF), a family of related unsaturated fatty acids, to regulate its behavior in a cell density-dependent manner (10, 11). XfDSF-mediated signaling suppresses motility and stimulates the production of cell-surface adhesins, thus increasing cell aggregation, surface attachment, and biofilm formation (10, 12–14). A ΔrpfF mutant of X. fastidiosa, blocked in the production of XfDSF, is hypervirulent to grapevine but is impaired in insect colonization and transmission (11, 12, 15). The accumulation of diffusible signaling factors (DSF) with increasing cell concentration increases the adhesiveness of the cells, presumably better to enable their acquisition by insect vectors, but reduces their ability to move and multiply within plants. These observations support the hypothesis that XfDSF signaling is used in a context-dependent lifestyle switch that enables a subset of the bacterial cells in a plant to become more adhesive, and thus able to be acquired by insects, by inducing a phenotype incompatible with the movement of the more solitary cells throughout the plant (6).A recent study (16) indicated that an extracellular factor produced by X. fastidiosa attenuated its ability to adhere to surfaces. This extracellular factor, produced by the WT strain and in greater amounts by the ΔrpfF mutant during both plant colonization and growth in broth culture, suppressed transmission by insect vectors and blocked adhesion of X. fastidiosa cells to various surfaces.Although the nature of this antiadhesive extracellular factor was unclear, it seemed likely that it could be one or more of the surface components overreleased by the ΔrpfF mutant, perhaps related to outer membrane (OM) proteins, because at least 11 OM protein-encoding genes are up-regulated in the ΔrpfF mutant as compared with the WT strain (14). X. fastidiosa OM proteins such as hemagglutinin-like proteins and the autotransporter XatA have been shown to be localized not only in the OM but also extracellularly, both as soluble proteins or in outer membrane vesicles (OMVs) (17, 18).OMVs are spheroid particles ranging in size from ca. 20 to 250 nm that are produced through the blebbing and pinching off of portions of the OM from all Gram-negative bacteria investigated to date (19–22). OMVs contain integral OM proteins embedded within the glycerophospholipid-LPS double layer along with OM-anchored lipoproteins and entrapped soluble periplasmic proteins (20, 22). OMVs from certain bacteria contain a variety of virulence factors and antigens and thus participate in pathogenesis (23–26). Other functions assigned to OMVs include modulating the immune response (27), trafficking of degradative enzymes against competing bacterial species (28, 29), delivering cargoes to benefit complex microbial communities (30), serving as a response to envelope stress (22, 31), and perhaps even acting as decoys for predators such as viruses (32).In this study, we demonstrate that X. fastidiosa is a particularly active producer of OMVs that, in turn, are an extracellular antiadhesive factor in this species. Using immunoassays against XadA1, an OM protein found to be solely present in the OM and in OMVs, nanoparticle-tracking analysis (NTA), and fluorescence and electron microscopy, we show that the production of OMVs is suppressed by DSF-mediated quorum sensing in X. fastidiosa both in vitro and within the host plant. We propose that secretion of OMVs by X. fastidiosa cells participates in a strategy to adjust its adhesiveness to surfaces when transitioning from a biofilm-forming stage capable of being vectored to a nonadhesive “exploratory” lifestyle for spreading in xylem vessels. Such a novel function of OMVs might contribute to the behavior of other species in which such planktonic/sessile transitions are necessary.     

Platelet-platelet and platelet-leukocyte interactions induced by outer membrane vesicles from N. meningitidis

A large part of native meningococcal lipopolysaccharide (LPS), i.e., LPS integrated in the outer cell membrane, is released in the form of 'blebs' from surplus outer membrane material. In the present study we investigated the effects of purified outer membrane vesicles (OMVs) on blood platelet-platelet and platelet-leukocyte interactions. Citrated whole blood was stimulated in vitro with equal amounts (on a weight basis) of OMV-integrated LPS, purified LPS (P-LPS) from the same meningococcal strain and purified E. coli-LPS. The samples were analyzed by flow cytometry. Upon OMV stimulation platelet aggregation increased 2.1-fold, platelet degranulation 1.8-fold, (measured as CD62P expression), platelet binding to monocytes 2.6-fold, whereas platelet binding to granulocytes increased 2.8-fold. Also, the fraction of large heteroconjugates, i.e., large CD45-positive cell aggregates increased 15.7-fold compared to control. P-LPS and E. coli-LPS also significantly increased platelet aggregation and heteroconjugate formation but did not influence platelet degranulation and binding of platelets to leukocytes in whole blood. When using platelet-rich plasma (PRP), OMVs increased platelet aggregation 2.1-fold and CD62P expression 1.9-fold. P-LPS and E. coli-LPS also significantly increased platelet aggregation in PRP but did not influence platelet degranulation. None of the LPS preparations induced platelet microvesiculation, either in whole blood or in PRP. CONCLUSION: Meningococcal-derived OMVs as well as purified meningococcal LPS, contribute to increased platelet-platelet and platelet-leukocyte aggregation and may thus be of great importance in the development of microthrombosis and organ dysfunction related to fulminant meningococcal septicemia.     

Glutathione import in Haemophilus influenzae Rd is primed by the periplasmic heme-binding protein HbpA

Glutathione (GSH) is a vital intracellular cysteine-containing tripeptide across all kingdoms of life and assumes a plethora of cellular roles. Such pleiotropic behavior relies on a finely tuned spatiotemporal distribution of glutathione and its conjugates, which is not only controlled by synthesis and breakdown, but also by transport. Here, we show that import of glutathione in the obligate human pathogen Haemophilus influenzae, a glutathione auxotrophe, is mediated by the ATP-binding cassette (ABC)-like dipeptide transporter DppBCDF, which is primed for glutathione transport by a dedicated periplasmic-binding protein (PBP). We have identified the periplasmic lipoprotein HbpA, a protein hitherto implicated in heme acquisition, as the cognate PBP that specifically binds reduced (GSH) and oxidized glutathione (GSSG) forms of glutathione with physiologically relevant affinity, while it exhibits marginal binding to hemin. Dissection of the ligand preferences of HbpA showed that HbpA does not recognize bulky glutathione S conjugates or glutathione derivatives with C-terminal modifications, consistent with the need for selective import of useful forms of glutathione and the concomitant exclusion of potentially toxic glutathione adducts. Structural studies of the highly homologous HbpA from Haemophilus parasuis in complex with GSSG have revealed the structural basis of the proposed novel function for HbpA-like proteins, thus allowing a delineation of highly conserved structure-sequence fingerprints for the entire family of HbpA proteins. Taken together, our studies unmask the main physiological role of HbpA and establish a paradigm for glutathione import in bacteria. Accordingly, we propose a name change for HbpA to glutathione-binding protein A.     

Immunogenicity and reactogenicity of Haemophilus influenzae type b-meningococcus group B outer membrane protein conjugate vaccine in children 2-60 months of age.

Haemophilus influenzae b - Neisseria meningitidis group B outer membrane protein conjugate vaccine (Hib-OMP) was given to 571 children 2-60 months of age. Two doses of Hib-OMP were given, 2 months apart, to 2-11 month old infants, and a single dose to children 12-60 month old. Sera were obtained from a subset of vaccinees at each immunization, and at follow-up 1 month and 1 year after immunization. Geometric mean antibody concentration (micrograms/ml) before and after full immunization were respectively 0.111 and 3.549 for 2-3 month old, 0.108 and 5.048 for 4-5 month old, 0.082 and 6.933 for 6-11 month old; they were 0.103 and 3.500 for 12-17 month old, 0.167 and 7.791 for 18-23 month old and 0.243 and 12.781 for children greater than or equal to 24 months. Detectable antibody (greater than or equal to 0.125 micrograms/ml) failed to develop in 2/399 (0.5%) after primary immunization, and 12/252 (4.8%) lost detectable antibody 1 year later. Six of these 12 infants were less than 12 month old. The vaccine was immunogenic as early as 3-5 months of age. The need for booster immunization needs to be assessed.     

Horizontal transfer of the gene encoding outer membrane protein P2 of nontypeable Haemophilus influenzae,in a patient with chronic obstructive pulmonary disease

An adult with chronic obstructive pulmonary disease was monitored prospectively for 2 years. Nontypeable Haemophilus influenzae was isolated from sputum cultures at 22 of 23 monthly clinic visits. Analysis of the isolates, by pulsed-field gel electrophoresis (PFGE), revealed that the patient was colonized by 3 different strains during the 2-year period. The gene encoding outer-membrane protein (OMP) P2, ompP2, was amplified from sputum samples and selected strains obtained from this patient. Analysis of the ompP2 sequences, in combination with the PFGE patterns, indicated that ompP2 horizontal transfer between 2 strains occurred in the respiratory tract, between clinic visits 13 and 14. Observation of ompP2 horizontal transfer in the human respiratory tract has important implications for both the understanding of ompP2 diversity among strains and the future design of OMP P2-based vaccines.     

Serum anticapsular antibody response in the first week after immunization of adults and infants with the Haemophilus influenzae type b-Neisseria meningitidis outer membrane protein complex conjugate vaccine

Eight healthy adults and 48 infants 2 and 4 months old were immunized with Haemophilus influenzae type b-Neisseria meningitidis outer membrane protein complex conjugate vaccine (PRP-OMP) to evaluate antibody kinetics in the first days after immunization. Five adults (63%) had some decrease in antibody, although the geometric mean did not decrease significantly. With one exception, the nadir occurred on postimmunization day 3. Seven had an antibody increase by day 7. Of the children, 6 (75%) of 8 and 17 (77%) of 23 had a decrease in antibody in serum obtained on day 2-3 after the first or second dose, respectively, the magnitude of which directly correlated with the preimmunization antibody concentration. However, the geometric mean did not decrease significantly. Within 1 week of immunization, 85% of infants had an increase in antibody, significantly greater after the second dose than after the first. A high concentration of maternally derived antibody before immunization correlated negatively with antibody response. Thus, a transient decrease in antibody occurs in most adults and infants 2-3 days after immunization with PRP-OMP followed by a prompt increase by day 7.