Production of Haemophilus influenzae b Meningitis in Infant Rats by Intraperitoneal Inoculation

Sprague-Dawley rats have a marked age-related susceptibility to Haemophilus influenzae type b that does not correlate with serum bactericidal activity. Eighty percent of 5-day-old animals that survive to 48 h after an intraperitoneal inoculation of a mean lethal dose of bacteria have histologically documented meningitis. Animals surviving the inoculations as infants manifest cerebral dysfunction as adults. This model should facilitate experimental study of bacterial meningitis.     

Immunological memory: the role of B cells in long-term protection against invasive bacterial pathogens

Dominic F. Kelly, MRCP; Andrew J. Pollard, PhD; E. Richard Moxon, PhD

JAMA. 2005;294:3019-3023.

Protein-polysaccharide conjugate vaccines that protect against Haemophilus influenzae type b (Hib), serogroup C Neisseria meningitidis, and multiple capsular serotypes of Streptococcus pneumoniae have had a major impact on invasive bacterial disease in childhood when incorporated into routine infant immunization schedules. However, effectiveness data from the United Kingdom suggest that primary infant immunization alone may not be associated with long-term protection. Both immunological priming and antibody persistence are important aspects of long-term protection induced by these vaccines. An improved understanding of the immunobiology of the B-cell response to these vaccines may direct development of immunization strategies that provide sustained protection.

     

Identification of a chromosomal locus for expression of lipopolysaccharide epitopes in Haemophilus influenzae.

Lipopolysaccharide (LPS) is a major virulence determinant of Haemophilus influenzae. The organism is able to display an extensive repertoire of different LPS structures through the loss and acquisition of multiple oligosaccharide epitopes in various combinations. This marked heterogeneity of LPS molecules has complicated the analysis of the structure of LPS and its role in pathogenesis. A genomic library was screened for the ability to transform H. influenzae to express novel LPS epitopes defined by reactivity with oligosaccharide specific monoclonal antibodies. A chromosomal locus, lic-1, involved in expression of at least three different epitopes (recognized by monoclonal antibodies 4C4, 12D9, and 6A2), was identified on a 5.6-kilobase restriction endonuclease fragment. Transformation of H. influenzae with subclones from within lic-1 was used to generate a series of isogenic and phenotypic variants. All transformants displayed phase variation for their newly acquired epitopes. Altered binding specificities of LPS with monoclonal antibodies correlated with changes in sugar compositional analysis. The expression of two epitopes was eliminated by introduction of site-specific mutations in lic-1, confirming the role of lic-1 in oligosaccharide biosynthesis.     

Insertion sequence IS1016 and absence of Haemophilus capsulation genes in the Brazilian purpuric fever clone of Haemophilus influenzae biogroup aegyptius.

Brazilian purpuric fever (BPF) strains of Haemophilus influenzae biogroup aegyptius form a clone of organisms distinct from more innocuous, conjunctivitis-associated isolates. There has been controversy over whether the virulence of BPF strains might derive from the presence of a polysaccharide capsule analogous to that found in conventional invasive H. influenzae, a controversy fuelled by the observation (G. M. Carlone, L. Gorelkin, L. L. Gheesling, A. L. Erwin, S. K. Hoiseth, M. H. O. Mulks, S. P. Connor, R. S. Weyant, J. Myrick, L. Rubin, R. S. Mumford III, E. H. White, R. J. Arko, B. Swaminathan, L. M. Graves, L. W. Mayer, M. K. Robinson, S. P. Caudill, and the Brazilian Purpuric Fever Study Group, J. Clin, Microbiol. 27:609-614, 1989) that a capsulation DNA probe from H. influenzae type b hybridized uniquely to BPF strains. In this work, the basis for this hybridization has been established as the possession by BPF strains, but not by non-BPF strains, of the Haemophilus insertion element IS1016. Although IS1016 is associated with the capsulation locus in some Haemophilus spp., a Southern hybridization study suggests that in BPF strains there are no capsulation genes.     

Participation of complement in the nonimmune host defense against experimental Haemophilus influenzae type b septicemia and meningitis.

This study was undertaken to determine whether the terminal complement components (C3-9) are involved in the nonimmune host defense against Haemophilus influenzae type b septicemia and meningitis. Using cobra venom factor, infant rats were depleted of C3 and C5. After intranasal challenge with H. influenzae type b, the complement-depleted rats developed a greater incidence and magnitude of bacteremia and a higher mortality rate. In contrast to the effects on bacteremia, complement depletion did not directly influence either the occurrence of meningitis or bacterial multiplication within the cerebrospinal fluid. These experiments provide evidence that the complement system may be an important mechanism of natural immunity to H. influenzae type b.     

PCR for capsular typing of Haemophilus influenzae.

A PCR method for the unequivocal assignment of Haemophilus influenzae capsular type (types a to f) was developed. PCR primers were designed from capsule type-specific DNA sequences cloned from the capsular gene cluster of each of the six capsular types. PCR product was amplified only from the capsular type for which the primers were designed. Product was confirmed by using either an internal oligonucleotide or restriction endonuclease digestion. A total of 172 H. influenzae strains of known capsular type (determined genetically) comprising all capsular types and noncapsulate strains were tested by PCR capsular typing. In all cases the PCR capsular type corresponded to the capsular genotype determined by restriction fragment length polymorphism analysis of the cap region. When used in conjunction with PCR primers derived from the capsular gene bexA, capsulate, noncapsulate, and capsule-deficient type b mutant strains could be differentiated. PCR capsular typing overcomes the problems of cross-reaction and autoagglutination associated with the serotyping of H. influenzae strains. The rapid and unequivocal capsular typing method that is described will be particularly important for typing invasive H. influenzae strains isolated from recipients of H. influenzae type b vaccine.     

Development, characterization, and functional activity of a panel of specific monoclonal antibodies to inner core lipopolysaccharide epitopes in Neisseria meningitidis

A panel of six murine monoclonal antibodies (MAbs) recognizing inner core lipopolysaccharide (LPS) epitopes of Neisseria meningitidis was prepared and characterized in order to determine the diversity of inner core LPS glycoforms among disease and carrier isolates. Two of these MAbs, L2-16 (immunoglobulin G2b [IgG2b]) and LPT3-1 (IgG2a), together with a third, previously described MAb, L3B5 (IgG3), showed reactivity, either individually or in combination, with all except 3 of 143 disease and carriage isolates (125 of 126 strains from blood, cerebrospinal fluid, or skin biopsy samples and 15 of 17 from nasopharyngeal cultures). MAbs L3B5, L2-16, and LPT3-1 were further characterized in an indirect immunofluorescence assay. All three MAbs bound to the bacterial cell surface, findings that correlated strongly with whole-cell enzyme-linked immunosorbent assay and immunodot blots. However, in contrast to our findings with L3B5, cell surface binding of L2-16 or LPT 3-1 did not correlate with functional activity as determined by bactericidal or infant rat passive protection assays against wild-type N. meningitidis strains. These findings are provocative with respect to the requirements for protective activity of antibodies and the development of inner core LPS vaccines against invasive meningococcal disease.     

Procalcitonin and C-reactive protein in severe 2009 H1N1 influenza infection

Purpose  

To examine whether, in an adult intensive care unit (ICU), procalcitonin or C-reactive protein (CRP) levels discriminated between 2009 H1N1 influenza infection and community-acquired pneumonia of bacterial origin.