Probing the ability of the coat and vertex protein of the membrane-containing bacteriophage PRD1 to display a meningococcal epitope

Bacteriophage PRD1 is an icosahedral dsDNA virus with a diameter of 740 A and an outer protein shell composed of 720 copies of major coat protein P3. Spike complexes at the vertices are composed of a pentameric base (protein P31) and a spike structure (proteins P5 and P2) where the N-terminal region of the trimeric P5 is associated with the base and the C-terminal region of P5 is associated with receptor-binding protein P2. The functionality of proteins P3 and P5 was investigated using insertions and deletions. It was observed that P3 did not tolerate changes whereas P5 tolerated changes much more freely. These properties support the hypothesis that viruses have core structures and functions, which remain stable over time, as well as other elements, responsible for host interactions, which are evolutionally more fluid. The insertional probe used was the apex of exposed loop 4 of group B meningococcal outer membrane protein PorA, a medically important subunit vaccine candidate. It was demonstrated that the epitope could be displayed on the virus surface as part of spike protein P5.     

Effect of Gm allotypes on IgG2 antibody responses and IgG2 concentrations in children and adults

Earlier studies have suggested that in adults the n-positive allele of the human IgG2 gene is more productive than the n-negative allele. This superiority was seen to be manifested in IgG2 antibody responses to polysaccharides, in the higher serum concentration of total IgG2 in the n/n than in -/- individuals, and in the higher concentration of n-positive than n-negative IgG2 in heterozygotes. The present study shows that in 1- or 2-year-old children, the concentration of IgG2 was independent of allotype G2m(n), and both alleles of a heterozygote contributed an average of one-half of the total IgG2. On the other hand, the superiority of the n-positive allele was also seen in young children in IgG2 antibody responses induced by the Haemophilus influenzae type b polysaccharide (Hib). The effect of allotype n on antibody responses was evident only when the immunogen was the Hib polysaccharide. When the immunogen was a conjugate of Hib and diphtheria toxoid, the IgG2 antibody responses of n-positive and n-negative vaccinated individuals were almost equal, both in adults and in children.     

Clearance of Chlamydia pneumoniae infection in H-2 class I human leucocyte antigen-A2.1 monochain transgenic mice

CD8+ T cells have been suggested to play an important role in protective immunity against pulmonary Chlamydia pneumoniae infection in mice. Moreover, several classical major histocompatibility complex class I - restricted cytotoxic CD8+ T lymphocytes (CTL) specific for C. pneumoniae- derived peptides have been identified. Here, we studied the outcome of C. pneumoniae infection in human leucocyte antigen (HLA)-A2.1 transgenic mice (HHD mice) that are only able to express a classical human class I molecule (HLA-A2.1). C. pneumoniae infection was self-restricted in HHD mice which were able to develop specific immune responses and a protective immunity against a subsequent rechallenge in a manner comparable to wildtype mice. Furthermore, accumulation of functional and C. pneumoniae-specific T cells to the site of infection was detected after challenge. Antigen processing and HLA-A2.1-dependent presentation was studied by immunizing the HHD mice with chlamydial outer protein N (CopN). Isolation of a peptide-specific CTL line from the CopN-immunized mice suggests that the HLA-A2.1 molecule can support the development of CTL response against a chlamydial protein in mice. These findings suggest that the transgenic mouse model can be used for further characterization of the HLA-A2.1-restricted CD8+ T-cell response during C. pneumoniae infection and for identification of CD8 epitopes from chlamydial antigens.     

Immunity to Chlamydia pneumoniae induced by vaccination with DNA vectors expressing a cytoplasmic protein (Hsp60) or outer membrane proteins (MOMP and Omp2)

Immune responses induced by intramuscular DNA immunization with Chlamydia pneumoniae genes coding for the major outer membrane protein (MOMP), cysteine-rich outer membrane protein 2 (Omp2) or the heat shock protein 60 (Hsp60) were studied. BALB/c mice were vaccinated intramuscularly three times at 3-week intervals and challenged intranasally 2 weeks after the last injection. Immunization with pmomp or phsp60 showed 1.2-1.5 log reduction in the mean lung bacterial counts after the challenge. Specific antibodies were detected only in sera of the mice immunized with pomp2 and phsp60. Although immunization with pomp2 resulted in a strong serum antibody response against Omp2 protein, it failed to protect the mice. Immunization with any of the three vaccines did not reduce the severity of histologically assessed pneumonia, but resulted in significantly higher lymphoid reaction in the lung indicating immunological memory.     

Chlamydia pneumoniae proteins induce secretion of the 92-kDa gelatinase by human monocyte- derived macrophages

Chlamydia pneumoniae, an intracellular Gram-negative respiratory bacterium, and macrophages are present in inflammatory tissue sites such as atherosclerotic lesions, where abnormal degradation of the extracellular matrix takes place. To evaluate the potential of C pneumoniae for participation in matrix destruction, we studied the effect of this bacterium on the production of 3 matrix-degrading metalloproteinases, 92-kDa gelatinase, interstitial collagenase-1, and stromelysin-1, and their natural inhibitor TIMP-1 (tissue inhibitor of metalloproteinases-1) by human monocyte-derived macrophages differentiated in vitro. Spontaneous production of collagenase and stromelysin by these cells was minimal and was not influenced by C pneumoniae. In contrast, the cells secreted substantial basal quantities of 92-kDa gelatinase, the secretion of which was stimulated (on average, 2.5-fold) by C pneumoniae. C pneumoniae regulated the expression of 92-kDa gelatinase by macrophages at the pretranslational level. Macrophages secreted only small quantities of TIMP-1. The chlamydial proteins Omp2, MOMP, and HSP60 were also found to participate in the induction of 92-kDa gelatinase by C pneumoniae. Denaturation of chlamydial proteins by boiling reduced 92-kDa gelatinase secretion only partially (by 35%), suggesting that the heat-stabile lipopolysaccharide molecules also stimulate secretion of the enzyme. The results show that production of 92-kDa gelatinase by human macrophages is selectively upregulated by C pneumoniae, which suggests that these bacteria, when present in a macrophage-containing inflammatory environment, actively participate in the destruction of the extracellular matrix.     

Cloning of an outer membrane protein of Neisseria meningitidis in Escherichia coli

A gene bank of chromosomal DNA of Neisseria meningitidis group B was constructed in phage lambda EMBL3, and screened by rabbit polyclonal antibodies to major outer membrane (OM) proteins of the meningococcus. Several clones expressing a 28 kDa protein were found. The gene coding for the 28 kDa protein was subcloned into plasmid pUC18 in Escherichia coli. The protein was expressed in E. coli and located in the OM. Rabbit antibodies were raised to the 28 kDa protein purified from E. coli and used to localize the protein in the meningococcus. The antiserum recognized a minor protein of similar electrophoretic mobility in the outer membrane complex (OMC) of the meningococcus. The 28 kDa protein was found to be common to different Neisseria species; it was expressed by both pathogenic and nonpathogenic Neisseria.     

Expression of pertussis toxin subunit S4 as an intracytoplasmic protein in Bacillus subtilis

The expression and secretion of pertussis toxin subunits S1 to S5 in Bacillus subtilis by the aid of a bacillar signal sequence has been reported. While secretion of subunit S1 was high, that of others was low. Ways have now been explored to improve the yield, using S4 as an example. The addition of a protease inhibitor was found to increase the amount of S4 in the culture supernatant, but the final amount was still much below that of S1. However, intracellular expression of S4 gave a high yield (500 mg I⁻¹) and the aggregated protein could easily be isolated in a few simple steps.     

Immune responses to borrelial VlsE IR6 peptide variants

Laboratory confirmation of Lyme borreliosis (LB) relies mainly on the demonstration of anti-borrelial antibodies. In recent studies, a novel VlsE protein IR₆ peptide-based assay has been introduced. Our aim was to evaluate the IR₆ peptides from three Borrelia burgdorferi sensu lato genospecies in the serodiagnosis of European and North American patients. Five VlsE protein IR₆ peptide variants representing sequences from B. burgdorferi sensu stricto, B. garinii, and B. afzelii were used as antigens in both IgG and IgM enzyme-linked immunosorbent assays (ELISA). Serum antibodies of 187 patients at different stages of LB from Europe and the United States were evaluated for serodiagnosis. For comparison samples were tested with one of the commercial IR₆ ELISAs. Three B. afzelii IR₆ variant peptides revealed antibodies that were concordant with each other. B. burgdorferi sensu stricto peptide antibodies mostly paralleled B. afzelii peptide antibodies, and positive values were also obtained in the majority of European sera. For several sera, B. garinii IR₆ peptide antibodies were discordant to B. afzelii peptide antibodies. The commercial IR₆ peptide antibody assay (C6 ELISA) results correlated better with B. burgdorferi sensu stricto IR₆ than with B. garinii IR₆ peptide IgG results, especially in sera from patients with facial palsy. Thus, antibody specificity to IR₆ peptides may vary according to the infecting Borrelia species. In some manifestations of the disease, C6 ELISA may not cover all LB cases. Evidently, the methodological aspects in ELISA design for peptide antibody measurements are important as well as the amino acids sequence of the antigen.