Purification and immunogenicity of genetically obtained pneumolysin toxoids and their conjugation to Streptococcus pneumoniae type 19F polysaccharide.

As part of an ongoing study concerned with improving human vaccines against Streptococcus pneumoniae, the genes for two defined pneumolysin (PL) toxoids (pneumolysoids), Pd-A (PL with a Cys----Gly substitution at amino acid 428) and Pd-B (PL with a Trp----Phe substitution at position 433), were inserted into the high-expression vector pKK233-2 in Escherichia coli and the pneumolysoids were purified. Groups of mice which had been immunized with either Pd-A, Pd-B, or native PL purified from S. pneumoniae were then challenged either intranasally or intraperitoneally with virulent pneumococci. Mice in all immunized groups survived significantly longer than sham-immunized controls. Both pneumolysoids were more effective than PL as protective immunogens. Pneumolysoid Pd-B was conjugated covalently with pneumococcal type 19F capsular polysaccharide (19F PS), and the immunogenicities of both the protein and the PS moieties of the conjugate in mice were determined. Significant anti-PL titers were obtained, and the immunogenicity of the 19F PS moiety was markedly enhanced compared with that of unconjugated PS. Conjugation also appears to have converted the 19F PS into an antigen capable of inducing a booster effect. These results support the notion that the efficacy of human, PS-based antipneumococcal vaccines might be improved by supplementation with pneumolysoid in the form of a covalent pneumolysoid-PS conjugate.

Pneumococcal polysaccharides complexed with C3d bind to human B lymphocytes via complement receptor type 2.

The immunoregulatory function of the complement system has been the focus of many investigations. In particular, fragments of complement factor C3 have been shown to play a role in B-lymphocyte activation and proliferation, lymphokine production, and the generation of in vitro antibody production. Purified pneumococcal polysaccharides (PS) can induce direct activation of C3 via the alternative pathway. Using sera of C1q-deficient patients and healthy subjects, we demonstrated that C3d, a split product of C3 that is generated after degradation of iC3b, can be bound to PS antigens. The binding of C3d to PS can occur in the absence of specific antibodies. Subsequently, we showed that PS complexed with C3d can be recognized by complement receptor type 2 that is expressed on B cells. Treatment of B cells with a monoclonal antibody recognizing the C3d-binding site of complement receptor type 2 reduces the binding of PS-C3d to the cells. In addition, we showed that PS4 complexed with C3d exerted an increased immunogenicity compared with free PS4. Our results show that the complement system plays a role in the activation of PS-specific B cells, carrying membrane receptors for C3d. Consequently, the complement system plays a regulatory role in the antibody response to T-cell-independent type 2 antigens such as PS.

A 43-kilodalton pneumococcal surface protein, PspA: isolation, protective abilities, and structural analysis of the amino-terminal sequence.

PspA is an antigenically variable surface protein of Streptococcus pneumoniae that appears to be essential for full pneumococcal virulence. In addition, monoclonal antibodies to PspA protect mice against infection with specific strains of pneumococci virulent for mice. In this study, we have isolated the 43-kDa N-terminal half of the native 84-kDa PspA and determined the sequence of the first 45 amino acids. This sequence, the first obtained for a pneumococcal surface protein, is consistent with that of an amphiphatic coiled-coil alpha helix with a 7-residue periodicity common to fibrous proteins such as tropomyosin and streptococcal M protein. The 7-residue periodicity begins with residue 8 and extends throughout the remaining sequence for nearly 11 turns of the helix. Mice immunized with this purified PspA segment were protected from fatal pneumococcal challenge, thus demonstrating that those PspA epitopes eliciting protection were present in the N-terminal half of the molecule.

Control of components of bacterial polysaccharide vaccines by physical methods.

Analysis of polysaccharide components of meningococcal- and pneumococcal vaccines was carried out by proton n.m.r. spectroscopy and gas chromatography. The meningococcal polysaccharides were of high purity but showed differences in degree and position of O-acetylation between manufacturers. The level of contamination of the pneumococcal polysaccharides by C-substance was quantified. These methods provide an alternative to immunological methods for determining serotype and purity.

Temperate bacteriophages of Streptococcus pneumoniae that contain protein covalently linked to the 5' ends of their DNA.

We have characterized three temperate bacteriophages of pneumococcus (HB-3, HB-623, and HB-746). Although all the phages belong to the same family, the polypeptide composition of the virions and the DNA restriction endonuclease analysis of their DNAs revealed differences among the three phages. The genomes of these bacteriophages have been isolated as DNA-protein complexes. The protein is specifically associated with the two 5' termini of the DNA as shown by experiments carried out with exonucleases. The protein bound to the DNA in the three phages studied, iodinated in vitro with 125I, has a molecular weight of 23,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment of the complexes with chaotropic agents suggested that the protein is covalently bound to the 5' termini of the DNA. Comparative pulsed-field gel electrophoresis analysis and Southern hybridization of the SmaI restriction fragments of DNAs from one lysogenic bacteria and its parental strain revealed that the prophage genome was integrated in the host chromosome.

[The detection of streptococcal cell-wall proteins that form complexes with human macroglobulins]. / Vyiavlenie belkov kletochnoi stenki streptokokkov, obrazuiushchikh kompleksy s makroglobulinami cheloveka.

The composition of the extracts of the cultures of individual streptococcal strains, studied by immunoblotting techniques, has been shown to contain proteins with a molecular weight of 70-80 KD. These proteins have pronounced affinity to human macroglobulins: alpha-macroglobulin, alpha-glycoprotein associated with pregnancy and protein A. The significance of this phenomenon on the cellular and somatic levels is discussed.

Contraimunoeletroforese no diagnóstico etiológico das meningites bacterianas / Counterimmunoelectrophoresis on etiological diagnosis of bacterial meningitis

Contraimunoeletroforese (CIE) tem sido utilizada para o diagnóstico etiológico de meningites bacterianas, através da detecçäo de antígenos polisacarídicos capsulares no líquido cefalorraquiano. O material é composto por 340 exames de amostras de LCR de 292 pacientes, 171 do sexo masculino e 121 do sexo feminino com idade entre 4 dias e 74 anos. Sendo 91 pacientes com perturbaçöes neurológicas diversas e 146 pacientes com diversas formas de meningites sépticas nas quais o agente etiológico foi identificado pelo exame bacterioscópico direto e provas culturais e 55 casos de meningites sépticas em que o agente etiológico näo foi identificado; em 25 pacientes foram feitos exames em 2 amostras; em 10, em 3 amostras e em 1, em 4 amostras. A primeira amostra foi sempre colhida antes da antibioticoterapia e as seguintes após o início da terapêutica. Este material foi dividido em grupos, para se verificar a especificidade e a sensibilidade da contraimunoeletroforese para Haemophilus influenzae tipo B, Neisseria meningitidis A, B e C e Streptococcus pneumoniae (83 sorotipos). A especificidade da contraimunoeletroforese para o Haemophilus influenzae é de 98, 49 e a sensibilidade é de 94,8%. Para o Neisseria meningitidis a especificidade éde 98,58% e a sensibilidade é de 44,4% e, para o Streptococcus pneumoniae a especificidade éde 93,55% e sensibilidade é de 41,1%.

Covalent linkage between the capsular polysaccharide and the cell wall peptidoglycan of Streptococcus pneumoniae revealed by immunochemical methods.

The attachment of capsular polysaccharide to Streptococcus pneumoniae was examined using monoclonal and polyclonal antibodies. Among the strains examined, the capsular polysaccharide of types 2, 4, 6A, 6B, 7F, 8, 14, 19F and 23F was bound to the pneumococci whereas that of a type 3 strain was not. Sequential treatment with 2% SDS at 100 degrees C, pronase, and EDTA did not dissociate the capsular polysaccharide from the pneumococci. Treatment of the cells with mutanolysin, a muramidase that degrades the cell wall peptidoglycan of pneumococci and other streptococci, released both the capsular and the cell wall C-polysaccharide (C-Ps). Type 6A capsular polysaccharide released from cell walls by mutanolysin treatment, was fractionated by high performance liquid chromatography and examined by immunoelectrophoresis. It was found to be bound to both the C-Ps and the peptidoglycan. The bond between the capsular polysaccharide and the peptidoglycan has not yet been identified but is probably covalent, as the two components could not be dissociated after boiling in SDS. Based on our studies with type 6A, we propose that capsular polysaccharide and C-Ps of the pneumococcus are linked to the peptidoglycan at different sites and, thereby, indirectly to each other. Studies in mice showed that the peptidoglycan enhanced the serum antibody response to C-Ps but not to type 6A polysaccharide.

Structural studies of the capsular polysaccharide from Streptococcus pneumoniae type 7A.

Application of methylation analysis, specific degradations, and n.m.r. spectroscopy to the capsular polysaccharide elaborated by Streptococcus pneumoniae type 7A indicates a hexasaccharide repeating-unit with the structure (Formula; see text).

Molecular size characterization of bacterial capsular polysaccharide vaccines by high performance liquid chromatography.

Bacterial capsular polysaccharides are major virulence factors and some are used as vaccinal antigens. Their molecular size is an important physicochemical criterion which correlates with immunogenicity. This article describes a new application of high performance liquid chromatography (HPLC), based on molecular sieving, for such an evaluation. This HPLC method is rapid, accurate, reproducible, requires only very low amounts of product and presents good correlation with conventional gel permeation chromatography.

Relationship of high tissue concentrations of azithromycin to bactericidal activity and efficacy in vivo.

Measurement of killing kinetics of azithromycin against strains of Streptococcus pneumoniae and Klebsiella pneumoniae in vitro showed that it had a limited bactericidal activity (greater than 90% kill) for the first eight hours of incubation, but developed complete bactericidal activity (greater than 99.9% kill) by 24 h incubation. Since high and sustained tissue levels of azithromycin occur in animals and humans, it was proposed that it might produce a bactericidal effect in vivo. This was demonstrated in a lung infection model in mice, designed to mimic the in-vitro killing studies. A 25 mg/kg dose of azithromycin given 24 h before intranasal challenge reduced the recoverable Str. pneumoniae population by greater than 99.9%, in comparison with untreated controls. Erythromycin did not produce a bactericidal effect at 100 mg/kg, and roxithromycin only reduced the viable count by 96%, at a dose of 50 mg/kg. Against a K. pneumoniae lung infection, a 50 mg/kg dose of azithromycin reduced the bacterial count by 99%. The bactericidal effect was correlated with lung tissue concentrations of azithromycin. In a proliferating Escherichia coli paper disc infection model, extravascular fluid concentrations of azithromycin were correlated with a 99.9% reduction in bacterial count, while corresponding serum concentrations were always less than the MIC. Dosing with azithromycin eradicated Haemophilus influenzae from the bulla (middle ear) of gerbils, as was not the case with erythromycin and roxithromycin. This effect was correlated with the antibiotic concentration in bulla lavage.

Meningitis due to penicillin-resistant Streptococcus pneumoniae in adults.

A 33-year-old woman with quiescent systemic lupus erythematosus developed meningitis due to a penicillin-resistant strain of Streptococcus pneumoniae. After an initial failure of penicillin therapy, the patient responded to cefotaxime. The meningitis was complicated by total neurosensory hearing loss. Howell-Jolly bodies noted on peripheral blood smear led to a radionuclide spleen scan, which documented functional asplenia. This woman is the second patient in the United States with meningitis caused by a strain of S. pneumoniae moderately resistant to penicillin G (minimal inhibitory concentration, 1.0 microgram/mL). The resistant isolate displayed resistance to penicillin that was not of enzymatic origin but rather was due to an alteration of penicillin-binding proteins. This experience illustrates the importance of testing the in vitro susceptibility of the pneumococcus to penicillin G.

Variation in the molecular weight of PspA (pneumococcal surface protein A) among Streptococcus pneumoniae.

Pneumococcal surface protein A (PspA) has been shown to be a virulence factor of pneumococci and to elicit protective anti-pneumococcal antibodies in mice. PspAs from different pneumococcal isolates have been shown to exhibit antigenic variability. In previous studies with three strains, two different apparent molecular weights of PspA were observed. In this report we have studied the variation in molecular weight of PspA from 43 pneumococcal strains reactive with anti-PspA monoclonal antibodies, Xi64 and/or Xi126. The relative molecular mass (Mr) of the major PspA band ranged from 67 k to 99 k in the different strains. Variations in Mr of PspA were observed even within strains of the same capsular type. The molecular size of PspA from strain Rx1 was not affected by treatment with a variety of chemical, enzymatic, and physical procedures, suggesting that the differences in Mr of PspA among different strains, was not due to uncontrolled variations in PspA preparation. The Mr of PspA of a given strain was found to be stable both in vivo and in vitro. As a result variations in the Mr of PspA from clinical isolates, should allow discrimination between strains within a given capsular type in epidemiologic studies.

[Rapid determination of bactericidal kinetics by evaluating intracellular adenosine-triphosphate in bioluminescence]. / Détermination rapide de la vitesse de bactéricidie par évaluation de l'adénosine-triphosphate intracellulaire en bioluminescence.

Killing kinetics measurement is usually time-consuming and tedious. Bioluminescent adenosine-triphosphate (ATP) assay, after intracellular nucleotide release by bacterial lysis, selects very quickly normal from antibiotic-modified and dead bacteria. Two simultaneous assays are performed with more and less strong lysis reagents (nucleotide releasing bacterial NRB, nucleotide releasing somatic NRS, Lumac). Bioluminescence produced in a luciferine - luciferase system is measured with Biocounter M 2010 luminometer. Differential values of two assays reflect the intracellular ATP fraction of strongest bacteria in tested cultures. Killing curves of some beta lactamines (aminopenicillin and cephalosporins) were studied with active Escherichia coli and Streptococcus pneumoniae cultures. Bactericidal action was seen within few hours, and similar variations of intracellular ATP fraction and numbers of colony-forming units obtained by reference method were observed. This method, well-suited to large series of assays and very rapid (intracellular ATP assay within one minute), performs detailed killing kinetics in real time.

G.L.C.-M.S. of N-(1-deoxyalditol-1-yl)octadecylamine derivatives in the analysis of methanolysates of neoglycolipids obtained by reductive amination.

Hydrophobic conjugates of a series of aldoses have been prepared by reductive amination with octadecylamine and sodium cyanoborohydride, as model compounds for the analysis of reductively aminated oligosaccharides derived from capsular polysaccharides of Streptococcus pneumoniae. In the context of the methanolysis procedure for sugar analysis, g.l.c. and g.l.c.-m.s. (e.i.-mode) studies were carried out on the N-(1-deoxyalditol-1-yl)octadecylamine derivatives obtained after treatment with methanolic HCl, and subsequent N-acetylation and trimethylsilylation.

Variation in penicillin-binding protein patterns of penicillin-resistant clinical isolates of pneumococci.

A large number of pneumococcal isolates (over 80 strains) from a variety of geographic locales and representing a spectrum of resistance levels from a penicillin MIC of 0.003 microgram/ml up to an MIC of 16 micrograms/ml were analyzed for their penicillin-binding protein (PBP) patterns. With a few exceptions, the great majority of strains with penicillin MICs up to about 0.05 microgram/ml contained the same set of five PBPs with molecular sizes typical of those of susceptible pneumococci. In strains with penicillin MICs of about 0.1 microgram/ml and up, virtually all isolates showed two common features: (i) all isolates showed loss of PBP 1A (98 kilodaltons) with or without a parallel appearance of a "new" PBP that ranged in molecular size between 96 and 97 kilodaltons; and (ii) in strains with penicillin MICs of 0.5 microgram/ml or more, PBP 2B could not be detected on the fluorograms even with very high concentrations of radioactive penicillin. Beyond these two common features, resistant strains with similar penicillin MICs showed a surprising variety of PBP profiles (i.e., in the number and molecular sizes of PBPs), each characteristic of a given isolate. We suggest that in pneumococci remodeling of critical PBPs in more than one way may result in comparable levels of penicillin resistance.

Application of high-resolution n.m.r. spectroscopy to the elucidation of the structure of the specific capsular polysaccharide of Streptococcus pneumoniae type 7F.

The specific capsular polysaccharide of Streptococcus pneumoniae type 7F (American type 51) is a high-molecular-weight neutral polymer composed of 2-acetamido-2-deoxy-D-galactose, 2-acetamido-2-deoxy-D-glucose, D-glucose, D-galactose, L-rhamnose, and 2-O-acetyl-L-rhamnose residues. N.m.r. spectroscopy (1H and 13C), in conjunction with composition and methylation analyses, and periodate oxidation data, showed the polysaccharide to be a branched polymer with a repeating heptasaccharide unit having the following structure. (formula; see text)