Robert S. Munson

Cloning and characterization of the genes encoding the haemolysin of Haemophilus ducreyi

We previously identified a heat‐ and protease‐labile haemolytic activity expressed by Haemophilus ducreyi. In order to characterize the haemolysin at the molecular level, genomic DNA from H. ducreyi was probed with haemolysin genes from other Gram‐negative organisms. The haemolysin genes of Proteus mirabilis hybridized to H. ducreyi DNA suggesting that the haemolysin of H. ducreyi is related to the Proteus/Serratia pore‐forming family of haemolysins. Tn916 mutagenesis was employed to isolate haemolysin‐deficient mutants. Approximately 5000 Tn916 transposon mutants were screened for the loss of haemolytic activity and two mutants were identified. One mutant, designated 35 000‐1, was further characterized. Sequences flanking the Tn916 element in strain 35 000‐1 were employed to identify clones from a λDASHII library of H. ducreyi strain 35 000 DNA. A 13 kb insert from one lambda clone was selected for further study. This 13 kb fragment was able to both confer haemolytic activity to Escherichia coli and complement the haemolysin deficiency in strain 35 000‐1. The haemolysin gene cluster was cloned from this 13 kb insert and two genes, designated hhdA and hhdB, were identified. The derived amino acid sequence of these genes demonstrated homology to the haemolysin and activation/secretion proteins of P. mirabilis and Serratia marcescens.

Immunogenicity of Haemophilus influenzae type b polysaccharide--diphtheria toxoid conjugate vaccine in adults.

The capsular polysaccharide of Haemophilus influenzae type b is a poor immunogen in human infants. In an attempt to enhance immunogenicity, this polysaccharide was covalently coupled to diphtheria toxoid and the conjugate tested as a vaccine in adult volunteers. Two injections of PRP-D vaccine were given, separated by one month. The anti-PRP antibody responses in this group were compared with those in a group receiving a comparable dose (20 micrograms) of conventional PRP vaccine. Both vaccines were well tolerated. A single injection of PRP-D was significantly more immunogenic than PRP, eliciting higher serum concentrations of total anti-PRP antibody 1 month later (geo means of 248 and 62 micrograms/ml, respectively; P less than 0.001). In addition, higher concentrations of IgG anti-PRP antibody were observed in the PRP-D group (P less than 0.001). One month after reinjection of vaccine, subjects receiving PRP-D showed a small but significant decline in total antibody (P = 0.03), whereas the serum antibody concentrations in the group that received PRP remained unchanged. At 12 months, the antibody concentrations of the two groups were not significantly different. Bactericidal activity and passive protection activity (infant rat model) were tested in pooled sera from the three highest and three lowest responders in each vaccine group; both PRP and PRP-D vaccines induced biologically active anti-PRP antibody. Thus PRP-D was found to elicit biologically active serum antibody and to be more immunogenic in adults than PRP vaccine; however, the duration of higher concentrations of antibody was transient.

Siblings of patients with Haemophilus meningitis have impaired anticapsular antibody responses to Haemophilus vaccine.

Siblings of patients with type b Haemophilus influenzae meningitis are at increased risk of developing Haemophilus disease. We immunized 26 healthy siblings and 25 control subjects using a vaccine containing the type b polysaccharide capsule (10 micrograms PRP) and pertussis vaccine (4 opacity units) (Lederle Laboratories) to determine whether siblings of patients with Haemophilus meningitis had an impaired antibody response to PRP. Using two intramuscular injections one month apart, we found that the siblings had a lower response to PRP. One month after the second injection, 12 of 24 of the siblings had serum concentrations of anticapsular (PRP) antibody thought to be sufficient to confer protection against Haemophilus disease (greater than or equal to 300 ng/ml), compared with 19 of 24 of the control children tested (50% vs 79%, P = 0.035 by chi-square analysis). In comparison with the normal controls, the siblings produced significantly less IgG anti-PRP antibody but similar amounts of IgM. The impaired responsiveness to PRP was most evident among the 16 children born after their sibling had meningitis and who were not known to have been exposed to type b Haemophilus infection previously. These data indicate that siblings of some patients with type b Haemophilus meningitis have reduced ability to form IgG anti-PRP antibody, which may be associated with increased susceptibility to Haemophilus disease.

Diversity of the outer membrane protein P2 gene from major clones of Haemophilus influenzae type b

In previous studies, it has been demonstrated that outer membrane protein P2 from Haemophilus influenzae type b has porin activity and that antibody directed against P2 is protective in an infant rat bacteraemic model.

Construction of chimaeric genes for mapping a surface‐exposed epitope on the pilus of non‐typable Haemophilus influenzae strain M37

A murine monoclonal antibody (mAb), designated 3H12, reacts with a surface‐exposed conformational epitope on the pilus of non‐typable Haemophilus influenzae strain M37. This antibody does not recognize the related pilus from H. influenzae type b, strain MinnA. Although mAb 3H12 does not recognize strain M37 pilin on Western blots, mAb 3H12 recognizes the recombinant M37 pilin protein expressed by Escherichia coli. In order to map the epitope recognized by mAb 3H12, we constructed a series of chimaeric genes. The chimaeric genes were expressed in E. coli and the chimaeric proteins characterized with respect to their reactivity with mAb 3H12. Residues between 37 and 100 of the M37 pilin protein are essential for the expression of the mAb 3H12 epitope. Residues in the carboxyl half of the M37 protein enhance the reactivity of mAb 3H12 when expressed in the presence of residues 37–100. Construction of chimaeric genes may provide a general methodology for mapping of conformational epitopes expressed by one of a related pair of proteins.

Comparison of the structure of the genes for outer membrane proteins p1 and p2 of Haemophilus influenzae type b

Size and antigenic heterogeneity have been recognized in both outer membrane protein P1 and outer membrane protein P2 of Haemophilus influenzae type b. To determine the molecular basis for these differences, we have cloned and sequenced the structural genes for OMPs P1 and P2 from prototype isolates with the OMP subtypes 1H, 3L and 6U. The nucleotide and derived amino acid sequences of the P1 genes are characterized by three variable regions dispersed between highly conserved regions. The nucleic acid and derived amino acid sequences of the P2 genes are also highly conserved. The P2 genes from OMP subtype 1H and 3L isolates are identical. The sequence of the 6U gene differs by 13 nucleotides, resulting in 10 amino acid changes.

1070 SERUM ANTIBODY RESPONSES TO LIPOPOLYSACCHARIDE (LPS) IN CHILDREN WITH HAEMOPHILUS INFLUENZAE TYPE B (HIB) MENINGITIS

We employed ELISA to measure antibody response to LPS in paired sera from 24 patients with Hib meningitis (mean age 13.8 mos). The results were compared to serum titers of 24 control children (mean age 15.2 mos). LPS was prepared from 12 CSF isolates and analyzed by SDS-PAGE. Size heterogeneity was observed, but all molecules were low molecular weight, indicating the absence of long O-antigen chains. At least 4 serologically-defined determinants were observed. Three LPS preparations containing all identified determinants were used for measurement of antibody. In acute sera, 12 of 24 meningitis patients had IgM titers ≥1:250, and 12 of 24 had IgG titers ≥1:1000. These titers were not significantly different in control children (8 of 24, and 12 of 24, respectively). Young children with meningitis responded as well to LPS as older children: 11 of 17 patients <12 months had ≥4-fold increases in IgG and/or IgM antibody compared to 4 of 7 patients ≥12 months. In contrast, there was a highly significant inverse relationship between antibody concentration in acute sera and ability to respond to LPS. 10 of 12 patients with initial IgG titers <1:1000 had ≥4-fold increases in IgG antibody compared to only 1 of 12 with acute titers ≥1:1000 (p <.001). Of 8 infants that failed to show serum anticapsular antibody responses (<70 ng/ml), 5 had >4-fold increases in LPS antibody. Thus human infants are more responsive to some Hib LPS determinants than to the type b capsule but the data do not allow distinction between protective and nonprotective antibody.

461 SUBTYPING OF HAEMOPHILUS INFLUENZAE TYPE B BY OUTER MEMBRANE PROTEIN PROFILES: APPLICATION IN DAY CARE CENTER OUTBREAKS

Haemophilus influenzae type b (Hib) may be subclassified into at least 11 different subtypes based on distinctive and stable outer membrane protein profiles as resolved by SDS-PAGE. We compared the subtypes of Hib isolates obtained from index patients, and their respective day care center (DCC) contacts, in 5 centers with 13 cases of invasive disease and 398 contacts. Subtyping was performed on 10 of the 13 isolates from index patients, and 67 of the 77 nasopharyngeal (NP) isolates from contacts. With one exception, invasive isolates from cases within the same center had homologous subtypes (the exception was in a DCC with 2 cases separated by 2 months). 55 of 67 NP isolates (82%) from contacts had subtypes homologous to their respective index isolates. Strains with heterologous subtypes probably represent background carriage (3.4% of all contacts). In the DCC with 2 cases caused by different strains, 23/67 contacts were colonized with the first strain, and 9/67 with the second strain. Carriage in many of the contacts persisted for 1-6 months, and in each child the subtype remained constant (up to 9 positive cultures). NP isolates from 4 household contacts of DCC carriers had homologous subtypes to those found in their respective DCC contacts. Subtyping of Hib by outer membrane protein profiles is an important epidemiologic tool, and provides a method for distinguishing between epidemic and nonepidemic Hib strains.

987 COMPARISON OF OUTER MEMBRANE PROTEINS OF TYPE B AND NONTYPE B HAEMOPHILUS INFLUENZAE

We have proposed a subclassification scheme for H. influenzae type b (Hib) based upon distinctive and reproducible strain differences in the SDS-polyacrylamide gel electrophoresis patterns of outer membrane (OM) proteins. Of 49 invasive isolates from patients hospitalized in St. Louis (StL), 92% could be assigned to 1 of 5 subtypes (1L, 1H, 2L, 2H, 3L). In the present study, we used this system to subtype 86 invasive isolates from patients hospitalized in 12 other states. The results were compared to the subtypes in StL, and to the OM protein patterns of nontype b Haemophilus isolates from middle ear (14 isolates), or blood/CSF (4 isolates). The overall distribution of Hib subtypes in other areas of the U.S. was similar to St.L (p>.35). However, there were possible regional differences: Isolates from New Orleans had a higher proportion of 3L strains than the rest of the U.S.,6/15 (40%) compared to 16/120 (13%), p<.01. Also, 3 of 11 apparently unrelated type b isolates from Denver had identical OM protein patterns not identified in 124 other isolates (p<10−8). Nontype b isolates (middle ear or invasive) had OM protein patterns different from the common type b patterns, and exhibited much greater variability. Thus, type b isolates causing invasive infections in the U.S. appear to be derived from a small number of distinctive clones. Nontype b isolates are genetically distinct from type b strains, and are more heterogenous. These findings may have important epidemiologic and immunologic implications.