Standardization and a multilaboratory comparison of Neisseria meningitidis serogroup A and C serum bactericidal assays. The Multilaboratory Study Group.

A standardized serum bactericidal assay (SBA) is required to evaluate the functional activity of antibody produced in response to Neisseria meningitidis serogroup A and C vaccines. We evaluated assay parameters (assay buffer, target strains, growth of target cells, target cell number, complement source and concentration, and methods for growth of surviving bacteria) which may affect the reproducibility of SBA titers. The various assay parameters and specificity of anticapsular antibody to five serogroup A strains (A1, ATCC 13077, F8238, F9205, and F7485) and four serogroup C strains (C11, G7880, G8050, and 1002-90) were evaluated with Centers for Disease Control and Prevention meningococcal quality control sera. The critical assay parameters for the reproducible measurement of SBA titers were found to include the target strain, assay incubation time, and complement. The resulting standardized SBA was used by 10 laboratories to measure functional anticapsular antibody against serogroup A strains F8238 and serogroup C strain C11. In the multilaboratory study, SBA titers were measured in duplicate for 14 pairs of sera (seven adults and seven children) before and after immunization with a quadrivalent polysaccharide (A, C, Y, and W-135) vaccine. The standardized SBA was reliable in all laboratories regardless of experience in performing SBAs. For most sera, intralaboratory reproducibility was +/- 1 dilution; interlaboratory reproducibility was +/- 2 dilutions. The correlation between median titers (interlaboratory) and enzyme-linked immunosorbent assay total antibody concentrations was high for both serogroup A (r = 0.86; P < 0.001; slope = 0.5) and serogroup C (n = 0.86; P < 0.001; slope = 0.7). The specified assay, which includes the critical parameters of target strain, incubation time, and complement source, will facilitate interlaboratory comparisons of the functional antibody produced in response to current or developing serogroup A and C meningococcal vaccines.

Removal of lipopolysaccharide from acellular Bordetella pertussis vaccine by detergent treatment.

Protective antigen was extracted from Bordetella pertussis cells with 1.0 M NaCl and precipitated with ammonium sulfate, 20-40% saturation (designated fraction 15A-1B). The protective antigen was purified further by detergent (Emulphogene BC720) treatment and adsorption to aluminum hydroxide gel (designated fraction 15A-108A). Compared with B. pertussis vaccine and fraction 15A-1B, fraction 15A-108A retained protective activity as assessed by the mouse protection test, but had reduced protein and markedly reduced endotoxin content. Fraction 15A-108A also had reduced leukocytosis-promoting, histamine sensitizing splenomegaly-inducing, and adjuvant activities. Emulphogene treatment provided a relatively simple method for removing endotoxin from a potential acellular B. pertussis vaccine.

Nicotinic acid transport in Escherichia coli.

The uptake of nicotinic acid by Escherichia coli is dependent on the presence of the enzyme nicotinic acid phosphoribosyl transferase and a source of energy. Glucose concentrations between 0.1 and 0.5%, a temperature of 46 degrees C and an external concentration of 2.5 X 10(-5) were optimal conditions for nicotinic acid uptake. Saturation kinetics occur with a Km of 1.75 microM and a Vmax of 0.116 nmoles/min/mg dry weight. The intracellular molarity of the accumulated pyridine compounds is 44-fold that of the initial concentration. Inhibitors of respiration and anaerobiosis do not significantly inhibit uptake rate. However, an inhibitor of glycolysis, uncouplers of ATP production and sodium arsenate reduce vitamin transport. A mutant defective in ATPase does not accumulate exogenously supplied nicotinic acid when lactate is used as an energy source, although L-proline, the transport of which is independent of ATP production, is accumulated.

Immunomodulation by Bordetella pertussis: antiviral effects.

Treatment of mice by intraperitoneal inoculation of pertussis vaccine or lipopolysaccharide extracted from B. pertussis will effect resistance to rabies virus, encephalomyocarditis virus, Semliki Forest virus, and Herpes simplex virus. Our previous observations indicated that treatment of C3H/HeN (+/nu) and BDF1 mice with pertussis vaccine injected i.p. five days prior to a mouse adenovirus lethal dose i.p. challenge elicited resistance to clinical disease and death. Susceptibility returned to a portion of the test population 35 days after pertussis vaccine treatment. The pertussis vaccine induced resistance developed in athymic (nude) mice also; however, the population succumbed to infection 35 days later. Titration of pertussis vaccine with respect to induction of resistance indicated the median effective dose (ED50) was approximately 25 micrograms dry weight. This report describes the antiviral activity of acellular components extracted from pertussis vaccine. Extraction of B. pertussis cells with 1.0M NaCl and ammonium sulfate fractionation (20-40% saturation) of the extract resulted in an acellular preparation that induced resistance to lethal dose mouse adenovirus infection. The resistance inducing activity was retained after treatment of the extract with detergent (GAF Emulphogene BC 720) to remove lipopolysaccharide and adsorption to alum gel. Comparison of endotoxin content of pertussis vaccine acellular fractions, polysaccharide fraction and purified lipopolysaccharide suggested that endotoxin probably plays a role in the induction of resistance. The endotoxin content of a Emulphogene-treated preparation that protected 80% of a test population was 39 ng. The lipopolysaccharide extracted from Escherichia coli, Vibrio cholerae, Salmonella typhimurium, and Salmonella minnesota did not induce a resistant state seven days after administration; however, lipopolysaccharide extracted from B. pertussis induced a resistant state.(ABSTRACT TRUNCATED AT 250 WORDS)

Transposed LEU2 gene of Saccharomyces cerevisiae is regulated normally.

The repression of beta-isopropylmalate dehydrogenase, the LEU2 gene product, by leucine and leucine plus threonine was unaffected by the transposition of LEU2 from its original locus on chromosome III to a new locus within the ribosomal deoxyribonucleic acid gene cluster on chromosome XII. Since the expression of the LEU2 gene is probably controlled at a pretranslational level, we conclude that the recombinant plasmid used for transformation carries regulatory information in addition to LEU2 structural information.

Azaserine resistance in Escherichia coli: chromosomal location of multiple genes.

Resistance to azaserine in Escherichia coli is the result of mutations in at least three different loci. All spontaneously arising azaserine-resistant mutants harbor a lesion in the aroP gene. However, a lesion in this gene is not solely responsible for resistance. All spontaneously arising intermediate-level azaserine-resistant mutants also harbor a lesion in a gene designated azaA, which lies near min 43 on the chromosome. High-level resistant mutants harbor lesions in the aroP and azaA genes and in a third gene designated azaB, which lies near min 69 on the chromosome. Transport studies demonstrate that mutants harboring lesions in the azaA gene are not defective in the transport of the aromatic amino acids, but that mutants which harbor lesions in the azaB gene are defective in phenylalanine transport but not in tyrosine or tryptophan transport.