Relationship between serum bactericidal activity and serogroup-specific immunoglobulin G concentration for adults, toddlers, and infants immunized with Neisseria meningitidis serogroup C vaccines.

A new meningococcal group C-CRM(197) conjugate vaccine (MnCC; Meningitec) has been evaluated in multiple clinical trials in the United States and most recently has been approved for routine administration in the United Kingdom. Meningococcal serogroup C (MnC)-specific immunoglobulin G (IgG) antibodies in pre- and postimmunization sera obtained from healthy U.S. adults, toddlers, and infants were quantitated by enzyme-linked immunosorbent assay (ELISA) and by an antibody-dependent, complement-mediated serum bactericidal assay (SBA). Serogroup-specific IgG antibody (micrograms per milliliter) in adults immunized either with the quadrivalent polysaccharide (A, C, Y, and W-135) vaccine or with MnCC showed a strong correlation (r = 0.848 and 0.934, respectively) by linear regression analysis with SBA. Sera from infants immunized with the MnCC (n = 30) and an age-matched unimmunized control group (n = 15) were also analyzed. Linear regression analysis of serum bactericidal and IgG ELISA data from sera obtained at 2 months of age (preimmunization) showed no correlation; however, a high degree of correlation was observed at time points after two (r = 0.877) and three (r = 0.951) immunizations, where significant rises in anti-MnC polysaccharide antibodies occurred relative to the age-matched control group. Infants previously primed with 3 doses of MnCC were given a booster dose of conjugate vaccine at 12 to 15 months of age. The correlation coefficient of ELISA to SBA for combined pre- and postbooster data was r = 0.836 (n = 48 pairs). In conclusion, increases in serum bactericidal activity in immunized adult, toddler, and infant populations were found to correlate very well with increases in serogroup-specific IgG concentrations, whereas the correlation between these two assays in nonimmunized 2-month-old infants was poor. Characterizing the relationship between these methods is important for understanding the significance of antigen-specific antibody concentrations relative to vaccine performance and protection from disease.

Isolation and characterization of the Haemophilus influenzae tolQ, tolR, tolA and tolB genes.

The tolQ, R, A and B genes have been isolated from the DNA of Haemophilus influenzae and sequenced. The deduced amino acid (aa) sequence of the H. influenzae TolQ, TolR, TolA and TolB show 67, 63, 41 and 62% identity with Escherichia coli TolQRAB proteins, respectively. These four proteins are involved in transport of colicins and phages across the cell envelope. The translational stop codon of TolB (the last gene in the cluster) is 23 bases upstream of the start codon of the P6 lipoprotein gene. Primer extension and Northern blot analysis revealed that the start of the P6 transcript is within the tolB gene. Nucleotide sequence (nt) analysis of the entire tolQRABP6 region shows a transcriptional terminator immediately downstream of the P6 gene. The tolQRABP6 gene cluster of H. influenzae may thus constitute an operon.

Storage reservoirs of hemin and inorganic iron in Yersinia pestis.

It is established that a high-frequency chromosomal deletion of ca. 100 kb accounts for the loss of properties making up the pigmented phenotype (Pgm+) of wild-type Yersinia pestis. These determinants are known to include virulence by peripheral routes of injection, sensitivity to the bacteriocin pesticin, adsorption of exogenous hemin or Congo red at 26 degrees C, and growth in iron-sequestered medium at 37 degrees C. We have now identified the outer membrane as the primary site of exogenous hemin storage in Pgm+ cells grown at 26 degrees C. Significant outer membrane storage of hemin did not occur in Pgm- mutants or in Pgm+ cells cultivated at 37 degrees C. However, both Pgm+ and Pgm- organisms grown at 37 degrees C contained a periplasmic reservoir of hemin, which may be associated with a temperature-dependent ca. 70-kDa peptide recently equated with antigen 5. At 37 degrees C, Pgm+ and Pgm- yersiniae also utilized a cytoplasmic ca. 19-kDa bacterioferritin-like peptide for deposition of inorganic iron. Incorporation of [55Fe]hemin into pools at 37 degrees C was not significantly inhibited by competition with excess unlabeled Fe3+. However, excess unlabeled hemin modestly competed with incorporation of label from 55FeCl3. This relative independence of storage pools observed at 37 degrees C is consistent with physiological linkage to in vivo acquisition and transport of Fe3+ from ferritin and of hemin from hemoglobin, myoglobin, or hemopexin.

Molecular analysis of the P2 porin protein of nontypeable Haemophilus influenzae.

The P2 porin protein is the most abundant outer membrane protein (OMP) of nontypeable Haemophilus influenzae (NTHI) and shows extensive antigenic heterogeneity among strains. To study the molecular basis of this heterogeneity, the DNA sequences of the genes encoding the P2 proteins of three unrelated strains of NTHI were determined, and restriction fragment length polymorphisms around the P2 genes of 35 strains were analyzed. The deduced amino acid sequences of the P2 genes from the three strains of NTHI revealed four major (12 to 35 amino acids long) and several smaller (2 to 7 amino acids) hypervariable regions in each protein. The major variations occurred in identical portions of the genes, and these regions showed a high antigenic index and surface exposure probability in computer modeling analysis. Differences in the molecular mass of the P2 protein correlate with differences in the size of the variable region in each strain. Oligonucleotide primers suitable for amplification of the P2 genes by polymerase chain reaction were developed. Restriction fragment length polymorphism analysis showed marked heterogeneity in and around the ompP2 locus of 35 NTHI strains. These results contrast with the high degree of conservation of the P2 genes in H. influenzae type b strains. We conclude that the molecular mass and antigenic heterogeneity of the P2 molecule of NTHI is due to variations in gene sequence that are clustered primarily in four large hypervariable regions of the gene.

Neonatal, urogenital isolates of biotype 4 nontypeable Haemophilus influenzae express a variant P6 outer membrane protein molecule.

The P6 outer membrane protein is a highly conserved molecule which is present on the surface of all strains of Haemophilus influenzae. Sixty strains of nontypeable H. influenzae which caused invasive disease or colonized the female urogenital tract were studied with monoclonal antibodies 7F3 and 4G4, which recognize different surface-exposed epitopes on the P6 molecule. All 60 strains expressed the epitope recognized by 4G4, whereas 47 of 60 strains expressed the epitope recognized by antibody 7F3. The 7F3-nonreactive strains were all biotype 4 and were recovered from the blood of neonates or postpartum women or from the female urogenital tract. The P6 genes from two 7F3-nonreactive strains were cloned, and the nucleotide sequences were determined. Analysis of amino acid sequences, immunoassays with synthetic peptides, and site-directed mutation of the P6 gene indicate that the epitope recognized by antibody 7F3 is conformational and that the sequence Asp-Ile-Thr is critical in maintaining the conformation of the epitope. We conclude that the unusually virulent clone family of biotype 4 strains of nontypeable H. influenzae express a variant P6 molecule which has an alteration in a highly conserved surface-exposed epitope.

Outer membrane protein P6 of Haemophilus influenzae binds to its own gene.

Outer membrane protein P6 is an important antigen expressed on the surface of all strains of Haemophilus influenzae. The predicted amino acid sequence of P6 contains a region of alpha helices that shares sequence identity with a family of helix-turn-helix DNA-binding proteins. A search for sequence-specific binding sites that resemble an operator region within the gene revealed a sequence with striking homology to the consensus operator sequence for lambda Cro and repressor. To test the hypothesis that P6 binds its own gene, purified P6 on nitrocellulose was probed with plasmid DNA containing the P6 gene. P6 bound the P6 gene in this Southwestern blot assay. To further test the observation, gel shift analysis was performed. Gel shift assays using a P6-specific monoclonal antibody demonstrated that P6 in crude cell extracts binds to the region of the gene containing the putative binding site. Competition with a synthetic oligonucleotide corresponding to the putative binding site inhibited binding of P6 to the P6 gene on nitrocellulose and in the gel shift assay. In addition, this oligonucleotide bound directly to P6 on nitrocellulose. Finally, DNase footprinting confirmed that P6 bound specifically to the same region of the P6 gene. These results indicate that P6 binds to a sequence-specific site within its own gene, suggesting that P6 regulates its own expression. This represents the first example of a Gram-negative outer membrane protein binding to its own gene and has potentially important implications as a mechanism for regulation of expression of outer membrane antigens.

Molecular conservation of the P6 outer membrane protein among strains of Haemophilus influenzae: analysis of antigenic determinants, gene sequences, and restriction fragment length polymorphisms.

Infections caused by Haemophilus influenzae are a major worldwide health problem. In particular, nontypeable strains of H. influenzae are a common cause of otitis media in infants and children. A vaccine to prevent these infections would result in the prevention of substantial morbidity and cost savings. A problem in identifying an appropriate vaccine antigen has been the enormous antigenic heterogeneity among nontypeable strains of H. influenzae. The present study was undertaken to characterize the conservation of the P6 outer membrane protein (approximately 16,000 daltons) among strains of H. influenzae. A total of 20 type b strains and 20 nontypeable strains of diverse geographic and clinical origins was studied. Three approaches were taken. (i) Antigenic determinants recognized by monoclonal and polyclonal antibodies were present on P6 in all 40 strains tested. The molecular weight of P6 was identical in all strains. (ii) Comparison of the DNA sequences of the P6 genes from three epidemiologically and serologically unrelated strains demonstrated 100% homology at the amino acid level and 97 to 99% homology at the nucleotide level. (iii) Restriction fragment length polymorphism analysis demonstrated that the P6 gene and flanking sequences were highly conserved among all strains. These three independent series of experiments indicated that the P6 protein is highly conserved among strains of H. influenzae. P6 should receive serious consideration for inclusion in a vaccine to prevent infections caused by nontypeable H. influenzae.

Outer membrane peptides of Yersinia pestis mediating siderophore-independent assimilation of iron.

It is established that wild-type cells of Yersinia pestis absorb exogenous hemin or Congo red and thus grow as pigmented colonies at 26 degrees C on media containing these chromatophores (Pgm+). Pgm+ isolated are known to possess a siderophore-independent mechanism of iron-transport (required for growth in iron-deficient medium) which is absent in avirulent Pgm- mutants. Production of the bacteriocin pesticin and linked invasins (Pst+) is an additional defined virulence factor of yersiniae; mutation of Pgm+,Pst- organisms to pesticin-resistance (Pstr) results in concomitant conversion to Pgm-. In this study, autoradiograms of two-dimensional gels of [35S]methionine-labeled outer membranes from Pgm- mutants were compared to those of the Pgm+,Pst+ or Pgm+,Pst- parent. An apparently single predominant peptide present in these preparations (greater than 10% of total membrane protein) existed as a family of iron-modifiable 17.9-kDa molecules focusing down to isoelectric points of about 4.6 and up to 5.89. Expression of eight detectable Pst(+)-specific peptides was not significantly influenced by exogenous iron. Pgm+ yersiniae constitutively produced pigmentation-specific peptide F and five iron-repressible peptides termed IrpA to IrpE. Typical spontaneous mutation to Pgm- resulted in loss of peptide F and IrpB-E. A rare Pgm+,Pstr mutant, selected on Congo red agar containing pesticin, also lost IrpB-E but retained peptide F. This isolate, like Pgm- mutants, failed to grow in iron-deficient medium. Regardless of phenotype, all yersiniae utilized hemin, hemopexin, myoglobin, hemoglobin, and ferritin, but not transferrin or lactoferrin, as sole sources of iron.

Resistance to pesticin, storage of iron, and invasion of HeLa cells by Yersiniae.

The independent abilities of Yersinia pestis to absorb exogenous pigments including hemin and Congo red (Pgm+) and to produce the bacteriocin pesticin with genetically linked invasive enzymes (Pst+) are established virulence factors of the species. Pst- Pgm+ strains of Y. pestis are sensitive to pesticin (Psts), and mutation of these isolates to pesticin resistance (Pstr) is known to result in concomitant conversion to Pgm-. Wild-type cells of Yersinia pseudotuberculosis and Yersinia enterocolitica are Pgm- but may be Psts; mutation of the latter to Pstr also results in avirulence. In this study, typical Pgm- mutants of Y. pestis exhibited a dramatic nutritional requirement at 37 degrees C but not 26 degrees C for iron which could be fulfilled by either Fe3+ or hemin. Iron privation of Pgm- yersiniae resulted in formation of osmotically stable spheroplasts similar to those previously observed after exposure of Psts bacteria to pesticin. At 37 degrees C, Pgm+ organisms rapidly overgrew initially predominant Pgm- populations in iron-deficient medium. However, Pgm-isolates could undergo a second mutation that permitted successful competition with Pgm+ cells in this environment. The mutation to Pstr in Y. pseudotuberculosis and Y. enterocolitica did not promote a similar requirement for iron but rather prevented these organisms from penetrating HeLa cells. The ability to invade these nonprofessional phagocytes was not shared by Pgm+ or Pgm- cells of Y. pestis.