L. van Alphen

DETECTION OF HAEMOPHILUS INFLUENZAE IN CEREBROSPINAL FLUIDS BY POLYMERASE CHAIN REACTION DNA AMPLIFICATION

Surmmary Two primer sets were chosen for the detection of Haemophilus influenzae in cerebrospinal fluid by polymerase chain reaction (PCR) DNA amplification. One primer set was selected from sequences encoding a capsulation-associated protein and reacted with target DNA from all 15 capsulate H. influenzae strains (all serotypes) examined. The other primer set was selected from the DNA sequence of a gene encoding for outer-membrane protein P6 and reacted with the 15 capsulate and 10 non-capsulate strains of H. influenzae tested. This primer set also reacted with the closely related species H. haemolyticus and H. aegyptius, and with two of nine H. parainfluenzae strains. In reconstruction experiments, PCR DNA amplification was able to detect as few as five H. influenzae cells when 40 cycles of amplification were used. Two hundred cerebrospinal fluid (CSF) samples collected consecutively from patients suffering from meningitis were investigated by PCR; 40 were culture-positive for H. influenzae and 39 of these were also clearly positive in the PCR test with both primer sets. Contamination occurred to some extent with 40 cycles of amplification but was completely eliminated when the number of cycles was reduced to 35. We conclude that the two primer sets are appropriate for the detection of H. influenzae by PCR, each having its own specificity. When these two primer sets are used, PCR is a technique of equivalent sensitivity to culture for the detection of H. influenzae in CSF.

Antibody avidity and immunoglobulin G isotype distribution following immunization with a monovalent meningococcal B outer membrane vesicle vaccine.

ABSTRACT The avidity maturation and immunoglobulin G (IgG) isotype distribution of antibodies after vaccination with a meningococcal B outer membrane vesicle (OMV) vaccine were evaluated as indicators of protective immunity. Pre- and postvaccination sera from 134 healthy toddlers (ages, 2 to 3 years) immunized with a monovalent meningococcal B OMV (serosubtype P1.7-2,4) vaccine adsorbed with AlPO4 or Al(OH)3 were analyzed by enzyme-linked immunosorbent assay (ELISA) methods. The children were vaccinated three times with intervals of 3 to 6 weeks between vaccinations or twice with an interval of 6 to 10 weeks between vaccinations. A booster was given after 20 to 40 weeks. The avidity index (AI) of antibodies increased significantly during the primary series of vaccinations and after the booster was given. No differences in AIs were found when the results obtained with the two vaccination schedules or with the two adjuvants were compared. After vaccination, IgG1 was the predominant IgG isotype, followed by IgG3. No IgG2 or IgG4 was detected. There was a strong correlation between serum bactericidal activity (SBA) and ELISA titers (r = 0.85 [P < 0.0001] for total IgG, r = 0.83 for IgG1 [P < 0.0001], r = 0.82 for IgG3 [P < 0.0001], and r = 0.84 [P < 0.0001] for the avidity titer). When two subgroups with similar anti-OMV IgG levels were compared before and after the booster vaccination, the higher AI after the booster vaccination was associated with significantly increased SBA. We concluded that avidity maturation occurs after vaccination with a monovalent meningococcal B OMV vaccine, especially after boosting, as indicated by a significant increase in the AI. Vaccination with the monovalent OMV vaccine induced mainly IgG1 and IgG3 isotypes, which are considered to be most important for protection against meningococcal disease. An increase in the AI of antibodies is associated with increased SBA, independent of the level of specific IgG and the IgG isotype distribution. Measuring the AI and IgG isotype distribution of antibodies after vaccination can be a supplementary method for predicting protective immunity for evaluation in future phase III trials with meningococcal serogroup B vaccines.

HLA-B27 as a relative risk factor in ankylosing enthesopathy in transgenic mice.

HLA-B27 is a risk factor for several human diseases through a mechanism that is not yet understood. This article describes a naturally occurring joint disease in laboratory mice, ANKENT. ANKENT begins with mild inflammation and culminates in irreversible stiffening of the ankle and/or tarsal joints in one or both hind paws. The macroscopic and histologic features of ANKENT, its relationship to age, gender, and environment, and some immunologic aspects are considered. With respect to genetics, it is demonstrated that an HLA-B27 transgene is a relative risk factor for ANKENT. Its impact depends on the H-2 haplotype, reaching a relative risk value of 9.4 for C57Bl/10, H-2b males (p < 0.025). Several features of ANKENT are reminiscent of human AS: joint pathology, age and gender distribution, the presence of non-MHC as well as MHC risk factors (including HLA-B27), and the suspicion that environmental factors are involved.

Cross-Reactivity of Antibodies against PorA after Vaccination with a Meningococcal B Outer Membrane Vesicle Vaccine

ABSTRACT The cross-reactivity of PorA-specific antibodies induced by a monovalent P1.7-2,4 (MonoMen) and/or a hexavalent (HexaMen) meningococcal B outer membrane vesicle vaccine (OMV) in toddlers and school children was studied by serum bactericidal assays (SBA). First, isogenic vaccine strains and PorA-identical patient isolates were compared as a target in SBA, to ensure that the vaccine strains are representative for patient isolates. Geometric mean titers (GMTs) in SBA against patient isolates with subtypes P1.5-2,10 and P1.5-1,2-2 after vaccination with HexaMen were generally lower than those against vaccine strains with the same subtype, although the percentage of vaccine responders (≥4-fold increase in SBA after vaccination) was not affected. Using various P1.7-2,4 patient isolates, GMTs as well as the number of vaccine responders were higher than for the P1.7-2,4 vaccine strain, indicating that the use of the P1.7-2,4 vaccine strain may have underestimated the immunogenicity of this subtype in HexaMen. Secondly, the cross-reactivity of antibodies induced by MonoMen and HexaMen was studied using several patient isolates that differed from the vaccine subtypes by having minor antigenic variants of one variable region (VR), by having a completely different VR or by having a different combination of VRs. MonoMen induced P1.4-specific antibodies that were cross-reactive with P1.4 variants P1.4-1 and P1.4-3. HexaMen induced a broader cross-reactive antibody response against various patient isolates with one VR identical to a vaccine subtype or a combination of VRs included in HexaMen. Cross-reactivity, measured by a fourfold increase in SBA after vaccination, against these strains ranged from 23 to 92% depending on the subtype of the tested strain and was directed against both VR1 and VR2. The extended cross-reactivity of vaccinee sera induced by HexaMen against antigenic variants has important favorable implications for meningococcal B OMV vaccine coverage.

GENOMIC DNA-FINGERPRINTING OF CLINICAL HAEMOPHILUS-INFLUENZAE ISOLATES BY POLYMERASE CHAIN-REACTION AMPLIFICATION - COMPARISON WITH MAJOR OUTER-MEMBRANE PROTEIN AND RESTRICTION-FRAGMENT-LENGTH-POLYMORPHISM ANALYSIS

Non-capsulate strains of Haemophilus influenzae were genotyped by analysis of variable DNA segments obtained by amplification of genomic DNA with the polymerase chain reaction (PCR fingerprinting). Discrete fragments of 100-2000 bp were obtained. The reproducibility of the procedure was assessed by comparing: (i) the fingerprints of 16 colonies of a single H. influenzae strain; (ii) isolates obtained from individual sputum samples (a total of 57 H. influenzae isolates from three cystic fibrosis patients); and (iii) 17 isolates collected during an outbreak of H. influenzae infection in a local pulmonary rehabilitation centre. The discriminatory power of the method was demonstrated by showing that the PCR fingerprints of eight unrelated H. influenzae strains from sputum samples of patients with chronic obstructive pulmonary disease (COPD) and 32 strains from cystic fibrosis patients were all different. These 40 isolates also differed with respect to their restriction fragment length polymorphisms (RFLP) and major outer-membrane protein (MOMP) composition. Twelve MOMP antigenic strain variants from sputum samples of five COPD patients had identical PCR fingerprints and RFLPs. It was concluded that PCR fingerprinting is a reliable and reproducible method for genotyping non-capsulate strains of H. influenzae. The discriminatory power of PCR fingerprinting was similar to that of RFLP analysis, but the results of PCR fingerprinting were easier to interpret.

Characterization of Adherence of Nontypeable Haemophilus influenzae to Human Epithelial Cells

ABSTRACT The adherence of 58 nontypeable Haemophilus influenzaeisolates obtained from patients with otitis media or chronic obstructive pulmonary disease (COPD) and obtained from the throats of healthy individuals to Chang and NCI-H292 epithelial cells was compared. Otitis media isolates, but not COPD isolates, adhered significantly more to both cell lines than did throat isolates. Since high-molecular-weight (HMW) proteins are major adhesins of nontypeableH. influenzae, the isolates were screened for HMW protein expression by Western blotting with two polyclonal sera and PCR withhmw-specific primers. Twenty-three of the 32 adhering isolates (72%) and only 1 of the 26 nonadherent strains were HMW protein or hmw gene positive. Among the 32 isolates adhering to either cell line, 5 different adherence patterns were distinguished based on the inhibiting effect of dextran sulfate. UsingH. influenzae strain 12 expressing two well-defined HMW proteins (HMW1 and HMW2) and its isogenic mutants as a reference, we observed HMW1-like adherence to both cell lines for 16 of the 32 adherent isolates. Four others showed HMW2-like adherence to NCI-H292. Of the three other patterns of adherence, one probably also involved HMW protein. Screening of the isolates with six HMW-specific monoclonal antibodies in a whole-cell enzyme-linked immunosorbent assay showed that the HMW proteins of COPD isolates and carrier isolates were more distinct from the HMW proteins from H. influenzae strain 12 than those from otitis media isolates. Characterization of the HMW protein of a COPD isolate by adherence and DNA sequence analysis showed that despite large sequence diversity in the hmwA gene, probably resulting in the antigenic differences, the HMW protein mediated the HMW2-like adherence of this strain.

Outbreak of multiresistant non-encapsulated Haemophilus influenzae infections in a pulmonary rehabilitation centre.

15 out of 21 patients admitted to a pulmonary rehabilitation centre were infected with a non-encapsulated strain of Haemophilus influenzae. All isolates showed identical outer membrane protein patterns, harboured a 40 MD plasmid, produced beta-lactamase, and were resistant to amoxycillin, co-trimoxazole, chloramphenicol, and tetracycline. The strain was first isolated from sputum of another 3 patients in the same hospital ward. 2 of them later introduced it into the rehabilitation centre. The strain spread among the other patients over the next 2 months. The absence of a common iatrogenic source of the organism and its slow spread indicate that the most likely means of transmission was person to person.

Functional activity of antibodies against the recombinant OpaJ protein from Neisseria meningitidis.

ABSTRACT The opacity proteins belong to the major outer membrane proteins of the pathogenic Neisseria and are involved in adhesion and invasion. We studied the functional activity of antibodies raised against the OpaJ protein from strain H44/76. Recombinant OpaJ protein was obtained from Escherichia coli in two different ways: cytoplasmic expression in the form of inclusion bodies followed by purification and refolding and cell surface expression followed by isolation of outer membrane complexes (OMCs). Immunization with purified protein and Quillaja saponin A (QuilA) induced high levels of Opa-specific antibodies, whereas the E. coli OMC preparations generally induced lower levels of antibodies. Two chimeric Opa proteins, hybrids between OpaB and OpaJ, were generated to demonstrate that the hypervariable region 2 is immunodominant. Denatured OpaJ with QuilA induced high levels of immunoglobulin G2a (IgG2a) in addition to IgG1, whereas refolded OpaJ with QuilA induced IgG1 exclusively. These sera did not induce significant complement-mediated killing. However, all sera blocked the interaction of OpaJ-expressing bacteria to CEACAM1-transfected cells. In addition, cross-reactive blocking of OpaB-expressing bacteria to both CEACAM1- and CEA-transfected cells was found for all sera. Sera raised against purified OpaJ and against OpaJ-containing meningococcal OMCs also blocked the nonopsonic interaction of Opa-expressing meningococci with human polymorphonuclear leukocytes.

Selection of Recombinant Antibodies Specific for Pathogenic Streptococcus suis by Subtractive Phage Display

ABSTRACT A semisynthetic antibody phage display library was used to select recombinant antibodies directed against surface components of a pathogenic strain of Streptococcus suis serotype 2 and against extracellular factor (EF), a protein known to be exclusively associated with pathogenic S. suis serotype 2 strains. Three distinct monoclonal phage antibodies directed against conformational epitopes of surface protein components of S. suis were selected. In addition, three different monoclonal phage antibodies were isolated that recognized EF. To isolate antibody fragments that recognize epitopes specific for a pathogenic S. suis serotype 2 strain, compared to a nonpathogenic serotype 2 strain, we applied a subtractive selection procedure. With this procedure, only one distinct phage antibody was found, and it was shown to be directed against EF. This demonstrates the selectivity of the applied procedure and confirms that EF is indeed differentially expressed by pathogenic and nonpathogenic strains. It also shows that EF is a very dominant antigen in phage antibody selections.

Differential binding of Haemophilus influenzae to human tissues by fimbriae

The hypothesis was investigated that tissue tropism of Haemophilus influenzae during colonisation and infection is associated with the ability of fimbriate bacteria to bind to the organs and cell types involved. H. influenzae type b with fimbriae (strain 770235f+) bound to several cell types, including ciliated columnar epithelial cells, pneumocytes, ependymal cells, glial cells, connective tissue fibroblasts, synovial cells, antigen-presenting cells, lymphocytes, erythrocytes and endothelial cells. Binding of H. influenzae to kidney, liver and conjunctiva cells was poor. Fimbriae-specific monoclonal antibody (MAb 6HE8) inhibited this binding. Some binding to endothelial cells and macrophages was also observed with non-fimbriate strains. This binding was not inhibited by MAb 6HE8. We conclude that in-vitro binding of fimbriate H. influenzae is mainly to those tissues and cells where H. influenzae is found during colonisation and infection. The data suggest that a shift to the non-fimbriate form is required for bacteria in the bloodstream to escape clearance mechanisms mediated by blood cells.