Hib Strains Research Articles

Pilus- and Non-Pilus-Mediated Interactions of Haemophilus infiuenzae Type b with Human Erythrocytes and Human Nasopharyngeal Mucosa

The role of pili of Haemophilus influenzae type b (Hib) in binding to human erythrocytes and in colonization and invasion of human nasopharyngeal (NP) organ cultures has been evaluated. Hib strains 1009 and 1007, NP and cerebrospinal fluid isolates from the same child with Hib meningitis, were studied. Strain 1009 was piliated (P+), produced pilin of approximately 24 kDa, and was hemadsorption-positive (HA+); strain 1007 was nonpiliated (P-), did not produce pilin, and was hemadsorption-negative (HA-). The rate of transition from one hemadsorption phenotype to the other in broth cultures and NP organ culture supernatants was 3 x 10(-4) per bacterium per generation for HA+ to HA- and 7 x 10(-4) per bacterium per generation for HA- to HA+. Growth in human NP organ culture supernatants of the P+HA+ strain was greater than that of the P-HA- strain at 6 and 12 h after infection. No difference was noted when the strains were grown in nutrient broth. Strain 1009 (P+HA+) attached in large clusters to cellular debris and nonciliated cells, a phenomenon never noted with strain 1007 (P-HA-). NP organ cultures infected with strain 1007 showed greater mucosal invasion than those infected with the 1009 strain. While P+HA+ and P-HA- Hib both attached to NP mucosa, P+HA+ strains may have a selective advantage in mucosal colonization but P-HA- strains may be more invasive.

Structural and antigenic conservation of the P2 porin protein among strains of Haemophilus influenzae type b

The P2 porin protein is the most abundant protein in the outer membrane of Haemophilus influenzae type b (Hib). Biochemical and immunochemical techniques were used to characterize the P2 proteins from a number of different Hib strains. P2 proteins from Hib outer membrane vesicles were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose for in situ tryptic digestion. Solid-phase tryptic digests of P2 from eight Hib strains were resolved by high-pressure liquid chromatography and shown to be similar if not identical. Radioimmunoprecipitation analysis involving Hib cells (containing intrinsically radiolabeled proteins or lipooligosaccharide) and Western blot (immunoblot) analysis were used to identify two P2-specific murine monoclonal antibodies (MAbs). These MAbs were shown to be reactive with 120 Hib strains tested in a colony blot radioimmunoassay. One of these MAbs bound to a surface-exposed P2 epitope that was antibody accessible on all Hib strains tested; the other MAb was directed against a P2 epitope that either was not exposed on the cell surface or was otherwise inaccessible to antibody.

Adherence Kinetics of Haemophilus influenzae Type b to Eucaryotic Cells

Haemophilus influenzae type b (Hib) adhere in vitro to buccal epithelial cells (BEC) via pili (fimbriae). In vivo studies have conflicting lines of evidence concerning the role of pili in adherence. This study characterizes the kinetics of Hib binding to BEC and compares pilus versus nonpilus adherence. Adherence was assessed using radioactive, immunofluorescence, and electron microscopic techniques. Paired Hib strains were obtained from three children with Hib meningitis; piliated Hib were isolated from the nasopharynx and nonpiliated isolates from the cerebrospinal fluid. Piliated strains adhered avidly to BEC after a 1-h incubation, whereas nonpiliated strains adhered poorly (p = 0.002). Kinetic analysis revealed increased adherence to BEC by piliated and nonpiliated strains with time, with maximum adherence occurring between 24-36 h. Immunofluorescence and electron microscopic assays visually confirmed the radioactive adherence findings. These results characterize the kinetics of Hib adherence to BEC. BEC incubated with nonpiliated Hib for 30 h were found to be coated with piliated strains, suggesting the induction of pilus production or the selection of a piliated subpopulation.

Bacteriologic epidemiology of Hemophilus influenzae type b strains causing invasive infections in Finland.

Consecutive Hemophilus influenzae type b (Hib) isolates (333 total) from children with invasive disease in Finland in 1985-1986 were analyzed. All belonged to the common genetic clusters described in the USA and Europe. However, detailed typing demonstrated some characteristics unique to Hib strains in Finland. Of the isolates, 86% belonged to one of four distinct patterns according to the combination of outer membrane protein subtype, biotype, and lipopolysaccharide serotype: 1-I-1 (25%), 1-II-9 (8%), and 1c-I-1 (18%). Pattern 1-II-9 has not been previously reported; it was most commonly found in the most densely populated area of Finland and among children cared for outside the home. Multilocus enzyme electrophoresis revealed that 87% of isolates with the pattern 1c-I-1 belonged to the electrophoretic type 21.8, which is seldom recovered from patients with invasive Hib disease in other countries.

Antigenic evidence for simultaneous expression of two different lipooligosaccharides by some strains of Haemophilus influenzae type b.

Isolates of Haemophilus influenzae type b (Hib) can be divided into three antigenic groups based on their reactivities with a set of two monoclonal antibodies (MAbs) directed against epitopes in the oligosaccharide region of Hib lipooligosaccharide (LOS) (P. A. Gulig, C. C. Patrick, L. Hermanstorfer, G. H. McCracken, Jr., and E. J. Hansen, Infect. Immun. 55:513-520, 1987). Approximately 24% of Hib strains react with both of these LOS-specific MAbs. Immunoprecipitation experiments involving several of these strains indicated that the epitopes recognized by these MAbs resided in two different LOS molecules, both of which were synthesized by these particular Hib strains. In addition, Western blot (immunoblot) analysis of proteinase K-treated cell extracts of these strains that had been subjected to sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis revealed two different LOS staining patterns when they were probed independently with the two MAbs. Colony blot radioimmunoassay of hundreds of colonies of one of these Hib strains showed that each colony bound both MAbs. Immune electron microscopy confirmed that individual cells of this same Hib strain expressed both types of LOS molecule at the same time. An antibody accessibility radioimmunoassay was used to show that different Hib strains of this type varied in the relative amounts of each of the two MAbs that they could bind to their cell surfaces. These findings indicate that some Hib strains can synthesize two antigenically distinct LOS molecules simultaneously.

Expression of the heat-modifiable major outer membrane protein of Haemophilus influenzae type b is unrelated to virulence

The heat-modifiable major outer membrane protein (P1) of Haemophilus influenzae type b (Hib) has been shown to be both exposed on the cell surface and capable of inducing the synthesis of antibodies protective against experimental Hib disease. Chemical mutagenesis of a recombinant plasmid containing the Hib gene encoding P1 resulted in inactivation of P1 expression by this plasmid. The mutated P1 gene was transformed into Hib to obtain an isogenic mutant lacking only the ability to synthesize this surface protein. In addition, the P1 gene was inserted into a plasmid shuttle vector and used to construct a recombinant Hib strain that overexpressed the P1 protein. Lack of P1 expression did not affect the ability of Hib to grow in vitro. Neither the absence nor the overproduction of P1 affected expression of capsular polysaccharide and lipooligosaccharide by Hib. The P1-negative mutant and the P1-overexpressing strain were both as susceptible to the bactericidal activity of pooled normal human serum as was the wild-type parent strain, while the P1-negative mutant was as resistant to the bactericidal activity of normal infant rat serum as was the wild-type parent strain. The P1-negative mutant was no less virulent than was the wild-type parent strain in an animal model system, such that both the numbers of animals infected by this mutant and the mean magnitudes of the resultant bacteremias were essentially identical to those obtained with challenge by the wild-type parent strain. Similarly, overexpression of P1 did not detectably affect the virulence of Hib. These data indicate that this protective protein antigen plays no detectable role in the expression of virulence by Hib, as assessed in an animal model system.

Meningitis Due to Haemophilus influenzae Type b Resistant to Ampicillin and Chloramphenicol

Invasive disease due to Haemophilus influenzae type b (Hib) resistant to chloramphenicol and ampicillin is rare in the United States. Review of the literature reveals that all previously reported cases occurred in children with meningitis. These children were treated initially with ampicillin and chloramphenicol and had complicated courses characterized by delayed sterilization of the cerebrospinal fluid. The present report describes an infant who developed meningitis due to ampicillin- and chloramphenicol-resistant Hib. The patient received cefotaxime from the onset of therapy and had an uncomplicated course. The presence of Hib strains resistant to chloramphenicol and ampicillin should be considered in patients with meningitis due to Hib who respond poorly to treatment with these two drugs. Furthermore, the in vitro susceptibility of all Hib isolates to chloramphenicol (as well as to other antimicrobial agents) should be evaluated routinely. If the incidence of such resistant organisms increases, a change will be warranted in the commonly recommended combination of ampicillin and chloramphenicol as empiric therapy for bacterial meningitis in pediatric patients.

Role of cerebrospinal fluid pleocytosis and Haemophilus influenzae type b capsule on blood brain barrier permeability during experimental meningitis in the rat.

The influence of leukocytes and Haemophilus influenzae type b (Hib) capsule on blood brain barrier permeability (BBBP) to circulating 125I-albumin in normal and leukopenic rats was assessed after intracisternal inoculation of encapsulated (Rd-/b+/02) or unencapsulated (Rd-/b-/02) isogenic strains of Hib. Both normal and leukopenic animals had increased BBBP 18 h after inoculation, with normal rats demonstrating significantly increased BBBP after challenge with the encapsulated strain. Despite cerebrospinal fluid (CSF) pleocytosis in normal rats, CSF bacterial concentrations were not lower. Normal rats cleared unencapsulated Rd-/b-/02 more effectively than leukopenic rats, with BBBP correlating with CSF bacterial density and not leukocyte concentrations. Challenge with heat-killed Rd-/b+/02 resulted in increased BBBP in both normal and leukopenic rats, with greater BBBP at higher bacterial concentrations. The data suggest: (a) significant increases in BBBP occur in the near absence of CSF leukocytes; (b) CSF leukocytes can augment changes in BBBP; (c) type b capsule inhibits host clearance mechanisms within the CSF; and (d) BBBP appears to correlate with bacterial concentrations within the CSF.

Heterohybridomas secreting human monoclonal antibodies against Haemophilus influenzae type b.

Seven human monoclonal antibodies (HmAb) directed against outer membrane antigens of Haemophilus influenzae type b (Hib) were produced by fusing Sp2/HPT heteromyeloma cells with human tonsillar lymphocytes sensitized in vitro for 6 days. The heterohybridomas were maintained in culture for at least one year and secreted, when cultured in Dulbecco's modified Eagle's medium without fetal calf serum, between 1 and 15 micrograms/10(6) cells/ml/24 h. All of the HmAb were IgGs except HiH-12 which is an IgM. Antibodies directed against the lipopolysaccharide and proteins of apparent molecular masses of 43, 37 and 27 kDa were identified by immunoblotting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of outer membrane. Binding radioimmunoassay with live bacteria showed that five out of seven HmAb adsorbed to cell surface-exposed antigenic determinants. HmAb HiH-6, HiH-7 and HiH-10 reacted with a surface-accessible determinant on the 43-kDa outer membrane protein. In a dot enzyme immunoassay, these HmAb recognized 103 out of 111 Hib strains isolated worldwide. The strains were selected to represent the most common genotypic variations among Hib. None of these HmAb reacted with other bacterial species tested. These HmAb may serve to study the bacterial surface antigens implicated in the human humoral response and protection to Hib infections.

Functional activities of human antibody induced by the capsular polysaccharide or polysaccharide-conjugate vaccines against Haemophilus influenzae type b

To determine the in vitro and in vivo activity of human antibody induced by different Haemophilus influenzae type b (Hib) vaccines, pooled human antisera obtained from adults immunized with either polyribosyl ribitol phosphate (PRP) or PRP-conjugated with diphtheria toxoid (PRP-D) vaccines were evaluated for opsonic and protective activity against Hib. In vitro, opsonophagocytic studies revealed that PRP-D induced antisera were approximately 2.5-fold more effective than PRP-induced antisera in supporting neutrophil-mediated killing of an Hib strain. In vivo studies using the infant rat model or Hib disease revealed that the decay of PRP antibody was similar with PRP or PRP-D induced antisera in unchallenged rats. However, in infant rats challenged with Hib, the PRP induced antibody decayed more rapidly than the PRP-D induced antibody as shown by significantly shorter half-life of the former. The protective efficacy was significantly greater with the PRP-D induced antisera than with the PRP induced antisera. This finding was shown by the significantly lower incidence of bacteraemia and the 5-fold lower dose of antibody required for 50% protection against bacteraemia in rats receiving the PRP-D induced antisera. The findings suggest that antibody to PRP induced by PRP-D vaccine is more opsonic and protective against Hib, and also decays more slowly in infant rats challenged with Hib than does antibody induced by PRP vaccine. Further studies are needed to elucidate the reason(s) for this difference in functional activity and in half-life of PRP antibody induced by PRP or by PRP-D vaccines.

Haemophilus influenzae type b lipooligosaccharide: stability of expression and association with virulence.

Spontaneous antigenic and phenotypic variations in the lipooligosaccharide (LOS) of two strains of Haemophilus influenzae type b (Hib) were previously shown to be associated with changes in virulence (A. Kimura and E.J. Hansen, Infect. Immun. 51:69-79, 1986). The goal of the present study was to define further the stability of LOS expression by this pathogen and the role of Hib LOS in virulence. Variation in LOS antigenic reactivity, as detected with LOS-specific monoclonal antibodies, was observed in 3 of 30 Hib strains after single-colony passage. When large numbers of individual colonies from seven other Hib strains were screened, however, spontaneous LOS antigenic variation was detected in all of the strains. Antigenic variation was not consistently associated with an altered LOS phenotype, as determined by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis and silver staining of LOS preparations. Changes in the LOS antigenic phenotype were correlated with altered virulence potential in two strains. In these strains, acquisition of reactivity with certain LOS-directed monoclonal antibodies was associated with the synthesis of a higher-molecular-weight LOS, enhanced virulence, and increased resistance to serum killing involving the classical complement pathway.

HAEMOPHILUS INFLUENZAE TYPE B STRAINS OF OUTER MEMBRANE SUBTYPES 1 AND 1c CAUSE DIFFERENT TYPES OF INVASIVE DISEASE

Of 275 consecutive Haemophilus influenzaetype b (Hib) strains isolated from children with invasive disease in Finland in 1985-86, 74% were of the common European outer membrane protein (OMP) subtype 1 and 22% were of OMP subtype 1c, which is usually rare. Strains of subtype 1c caused proportionately more meningitis and less epiglottitis than did strains of subtype 1. Furthermore, children with disease due to strains of subtype 1c were younger than those with disease due to strains of subtype 1. The significant difference in association between subtype and the diagnosis of epiglottitis or meningitis remained even when the strong influence of age was accounted for. This finding may suggest a true difference in the virulence between these subtypes not previously demonstrated for Haemophilus influenzae type b.

Distinct geographic distribution of subtypes of Haemophilus influenzae type b in Western Europe.

The spread within populations and the geographic distribution of Haemophilus influenzae type b (Hib) strains has hardly been investigated [1, 2], mainly because there have been no reliable and discriminating subtyping methods. Biotyping has been used for a long time for typing H. influenzae. Most Hib strains, however, are biotype I [3]. Recently, Barenkamp et al. [4] described 21 subtypes of strains isolated in the United States; his differences between subtypes were based on the outer membrane protein composition as analyzed by SDS-PAGE subtyping. Eighty-nine percent of the strains belong to six subtypes [4, 5]. Isolated strains of the most frequently encountered subtypes were equally distributed over the country, but distinct regional differences were found in the distribution of some of the less-common subtypes [6]. We found that 93% of the Hib strains from patients with meningitis in the Netherlands belong to one subtype (subtype 1) [7]. Strains with this subtype, which is identical to subtype 3L described by Barenkamp [4], were isolated from 15% of the cases in the United States [5]. In addition to SDS-PAGE subtyping, serotyping based on differences in lipopolysaccharides was introduced to further differentiate the Hib strains isolated in the Netherlands [7]. Based on biotyping, SDS-PAGE subtyping, and lipopolysaccharide serotyping, the Dutch strains appeared homogeneous [7]. These strains may be descendents from one ancestral clone [8]. In this paper we show that only a few subtypes of Hib are predominant in Western Europe and that the distribution of these subtypes shows distinct geographic differences. The significance of this finding for the understanding of the endemic spread of Hib disease is discussed.

Conservation of epitopes in the oligosaccharide portion of the lipooligosaccharide of Haemophilus influenzae type b

The antigenic characteristics of the lipooligosaccharide (LOS) of Haemophilus influenzae type b (Hib) were examined in strains obtained over an extended period of time. These Hib strains were isolated from patients with systemic Hib disease in Dallas, Tex., over a 20-year period and in New York City between 1941 and 1956. The antigenic characteristics of the LOS of these Hib strains were examined by using a set of four murine monoclonal antibodies directed against epitopes present in the oligosaccharide portion of the LOS molecule. The same basic set of LOS antigenic determinants that is expressed by recent Hib isolates was also found to be present in this collection of Hib strains spanning a 40-year period. Some variation with time was detected in the distribution of the systemic disease isolates among four Hib LOS antigenic groups; however, only 2 of 188 Hib isolates failed to react with a set of two LOS-specific monoclonal antibodies. Therefore, little variation has occurred among Hib strains with regard to the LOS epitopes defined by these monoclonal antibodies over a considerable period of time.

Antigenic and phenotypic variations of Haemophilus influenzae type b lipopolysaccharide and their relationship to virulence.

Haemophilus influenzae type b (Hib) strains NO100 and COL10 were found to produce bacteremia in infant rats at a much lower frequency than other Hib strains previously tested. These relatively avirulent strains were the only Hib strains among 200 clinical isolates examined to date which failed to react with two Hib lipopolysaccharide (LPS)-specific monoclonal antibodies (MAbs). LPS analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that strains NO100 and COL10 possessed LPS which migrated faster than the LPS of Hib strains that reacted with one of the two or with both of these MAbs. These observations suggested that the relative lack of virulence of strains NO100 and COL10 might be related to their unusual LPS phenotype. To determine whether alteration of LPS structure would affect the virulence of these strains, we identified and isolated isogenic LPS antigenic variants of strains NO100 and COL10 using the LPS-specific MAbs 4C4 and 5G8 in a colony blot radioimmunoassay. Antigenic variation of LPS was found to occur spontaneously in these two strains at a relatively high frequency in terms of both acquisition and loss of MAb reactivity (ca. 0.2 to 16.7%). LPS antigenic variants of strains NO100 and COL10 reactive with both MAbs 4C4 and 5G8 (4C4+ 5G8+) were more virulent in the infant rat model than their respective 4C4- 5G8- parental strains (P less than 0.01). An antigenic variant of COL10 reactive with only MAb 4C4 (4C4+ 5G8-) was also significantly more virulent than its 4C4- 5G8- parent. These LPS antigenic variants with increased virulence synthesized altered LPS molecules which possessed apparent molecular weights higher than those of the LPS of the parental strains. Increased resistance of strain NO100 to the bactericidal activity of normal infant rat serum was associated with changes in LPS structure, while strain COL10 and its LPS variants were all uniformly resistant to serum bactericidal activity. Our results demonstrate that (i) spontaneous antigenic and phenotypic variation of LPS occurs at a relatively high frequency in some strains of Hib; (ii) the higher-molecular-weight type of LPS is associated with the full expression of Hib virulence; (iii) LPS phenotype may not correlate with Hib serum resistance; and (iv) serum resistance of Hib is not an accurate indicator of virulence.

Coprecipitation of lipopolysaccharide and the 39,000-molecular-weight major outer membrane protein of Haemophilus influenzae type b by lipopolysaccharide-directed monoclonal antibody.

The major outer membrane protein of Haemophilus influenzae type b (Hib) with an apparent molecular weight of 39,000 (39K) was purified from three different Hib strains and was shown to be free from detectable contamination with other proteins. However, these purified 39K protein preparations were found to contain Hib lipopolysaccharide (LPS). Immunization of rats with these 39K protein preparations resulted in the production of antisera containing both 39K protein-directed and LPS-directed antibodies, as determined by Western blot analysis. The reactivity pattern of the LPS-directed serum antibodies with different Hib strains was identical to the reactivity of these Hib strains with a set of monoclonal antibodies (mabs) previously shown to immunoprecipitate the 39K protein in a radioimmunoprecipitation (RIP) system. Examination of the antigenic specificities of the 39K protein-immunoprecipitating mabs by using Western blot analysis showed that these mabs were actually directed against Hib LPS. RIP analysis of 125I-labeled Hib cells and 32P-labeled Hib cells revealed that the 39K protein and LPS existed as a complex in a RIP system, which resulted in the coprecipitation of both antigens by LPS-directed mabs. The interaction between LPS and the 39K protein was highly selective for this protein and did not involve other outer membrane proteins. The LPS/39K protein complex could be reconstituted by mixing purified LPS and purified 39K protein; it could also be reconstituted with 39K protein from one Hib strain and LPS from another Hib strain. These findings have necessitated the reinterpretation of previous studies involving the 39K protein-immunoprecipitating mabs. Of primary importance is the fact that the demonstrated immunoprotective ability of a 39K protein-immunoprecipitating mab (E. J. Hansen, S. M. Robertson, P. A. Gulig, C. F. Frisch, and E. J. Haanes, Lancet i:366-368, 1982) must now be regarded as evidence that antibody directed against Hib LPS can be protective against experimental Hib disease.

1175 ENHANCED SURVIVAL OF INFANT RATS WITH OVERWHELMING HAEMOPHILUS INFLUENZAE TYPE B (Hib)SEPSIS BY POLYMYXIN B (PB) - ENDOTOXIN (ETX) INTERACTION

5-6 day old Sprague-Dawley rats were Infected Ip with 105-106 cfu of Hib strain Eagan. Ampmpicillin (Amp) (0.5 mg/kg & 3 q3hrs) given 3 hrs after infection was curative in all animals, but falled to have any effect upon survival if initiated 12 hrs later, even at a 500 mg/kg dose (47 and 45 animals, 19 controls). At this 12 hour stage an ineffective antimicrobial dose of PB (.0125mg/kg × 3 q3hrs) alone (n=33) or In combination with 500 mg/kg Amp (n=27) achieved a significant increase in survival at 17 hrs (p<.008) and 20 hrs (p<.006> and in combination at 36 hrs (p<.009) after infection. Bacterial quantitation by plate dilution and ETX activity in whole blood by a chromogenic limulus lysate assay were performed simultaneously at 30 and 300 minutes after initiation of treatment and in controls. Amp significantly reduced bacterial counts in survivors at both times (p<.025 and p<.004)compared to controls or PB treated animals but increased the activity of free endotoxin at 30 minutes (p<.004) compared to untreated controls. At all times Amp treated animals had greater ETX actlvlty/cfu (p<,006 and p<,005) when compared to surviving control or PB treated animals (50 animals). In vitro ETX release from Hib grown in broth was increased after addition of 100 μg/ml of Amp compared to 8 μg/ml PB or controls. These experiments indicate that Polymyxin B may modify the action or release of endotoxin in this model.

567 SURVIVAL AND TRANSMISSION OF HAEMOPHILUS INFLUENZAE TYPE B (Hib) IN DAY CARE

Hib is thought to be a fastidious organism. Transmission in institutional settings is assumed to be via large droplet respitory secretions. The role of environmental surfaces and fomites in transmission is unknown. Cultures were obtained from Hib carrier (Hib-C) children and their freshly handled fomites at three day care centers. Hib was recovered from 5/5 cultures of moist nasal secretions (NS), 1/5 cough plates held 10 inches from the mouth, but not from 27 cultures of saliva, hands, cribs and toys of Hib-C. Pooled NS from 3 Hib-C contained 1.6×107 Hib CFU/ ml. Infected NS were smeared onto dry and premoistened washrags (WR) and Kleenex®. Survival of Hib was determined by growth in supplemented overnight BHI broth at intervals 0-5 hr UR and 0-18 hr Kleenex®. Hib in NS were recovered from dry WR for 3 hr, from moistened WR for 5 hr and Kleenex® for 18 hr. A pharyngeal Hib strain was diluted to 6×106 CFU/ml in unsupplemented BHI broth and 50 μl were placed on moisten gauze (MG) and dry gauze (DG) and wax paper (WP). Colony counts were determined for 6-7 days. There was no drop in CFU in broth on gauze or WP until dried (0.5-3 hr). Thereafter dried Hib survived on MG at 102 CFU/ml for 3 days and was still recovered on day 6. Hib was recovered from DG and WP at 102 CFU/ml at seven days. NS of Hib-C contain high concentrations of Hib and support prolonged survival. Use of WR and Kleenex® for more than one child may contribute to transmission of Hib.

A minor high-molecular-weight outer membrane protein of Haemophilus influenzae type b is a protective antigen.

Cell surface-exposed antigenic determinants of several high-molecular-weight outer membrane proteins of Haemophilus influenzae type b (Hib) have been shown to be consistently immunogenic in human infants convalescing from Hib meningitis. A monoclonal antibody (mab), 6G12, directed against one of these cell surface-exposed outer membrane proteins that has an apparent molecular weight of 98,000 (98K) was identified by radioimmunoprecipitation analysis. Of 120 clinical isolates of Hib, 83 were found to possess antigenic determinants which reacted with mab 6G12 in a colony blot-radioimmunoassay procedure, indicating that the antigenic determinant recognized by mab 6G12 is present in the majority of Hib strains. A different radioimmunoassay, which uses whole Hib cells as antigen, confirmed that strains reactive with mab 6G12 in the colony blot-radioimmunoassay procedure possessed a cell surface-exposed and antibody-accessible antigenic determinant recognized by this mab. Hib strains which did not react with mab 6G12 were found to lack a 98K protein. Passive immunization with mab 6G12 reduced the level of bacteremia that developed in infant rats challenged with the homologous Hib strain against which this mab was raised. In contrast, no protection was observed when the challenge strain was one which lacks the antigenic determinant recognized by mab 6G12. Radioimmunoprecipitation analysis of sera from human infants convalescing from Hib meningitis detected an antibody response directed against the 98K protein. The protection against experimental Hib disease provided by antibody to the 98K protein, the immunogenicity of this protein in human infants, and its presence in a majority of Hib strains indicate that the 98K outer membrane protein may have potential for vaccine development.

EVIDENCE FOR COMMON ANTIGENICITY IN THE PILI OF HAEMO PHILUS INFLUENZAE TYPE B (HIB)

Antisera to the pilus of a single strain of Hib (C54) were made by hyperimmunization with a pilus-enriched (p+) variant of C54 followed by absorption against a variant of C54 that was identical in encapsulation, outer membrane protein composition, and lipopolysaccharide subtype but lacked pili (p-). 4 different antisera were prepared (2 in different species of mice and 2 in guinea pigs). All 4 antisera agglutinated (aggl) the homologous C54 p+ but did not aggl C54 p-. C54 causes hemagglutination (HA) of human RBCs and adheres well to human epithelial cells (EC) (4.8 bacteria/EC) whereas the p- variant is HA- and adheres poorly (0.2/EC). Addition of antisera resulted in complete inhibition of HA and >60% reduction in C54 bacterial adherence. 9 strains isolated from CSF, blood, and mucosal surfaces were pilus-enriched and studied for cross-reactivity with the pilus of C54. 5 were HA+ with human RBCs only (like C54 and 80% of all Hib isolates), 2 were HA+ with human and rat RBCs (like 12% of all Hib) and 2 were HA+ with all animal and human RBCs tested (like 8% of all Hib). Despite these HA species differences (the only type of heterogeneity found to date) the 4 antisera aggl all 9 strains when p+ but not when p-. Similarly, the antisera inhibited HA of human RBCs and adherence to EC. Thus, pili from different strains of Hib share antigenic determinant(s), and common antipilus antibodies can produce the biologically relevant effect of inhibiting bacterial adherence.