In vitro human antibody production to the Haemophilus influenzae type b capsular polysaccharide

In vitro production of human antibody to the Haemophilus influenzae type b capsular polysaccharide (PRP) and to tetanus toxoid (TT) and diphtheria toxoid was measured in culture supernatants of peripheral blood mononuclear cells and by enumeration of antibody secreting cells (AbSC) in an enzyme-linked immunosorbent-plaquing assay. Normal adult peripheral blood mononuclear cells stimulated with Epstein-Barr virus secreted anti-PRP antibody with a frequency of 1/552 to 1/1190 relative to total Ig secreting cells; the frequency of AbSC to tetanus toxoid (TT) was 7.5 times higher (p less than 0.05). These frequencies did not change significantly after in vivo immunization, although the isotype distribution shifted toward increased IgG for TT and increased IgG and IgA for PRP. At 8 days postimmunization, spontaneous AbSC to PRP and TT were detected; frequencies for total anti-TT AbSC again being higher than anti-PRP, but there were significantly more IgA plaques among anti-PRP AbSC. Spontaneous AbSC were suppressed in culture by pokeweed mitogen and enhanced by cyclosporine. Three wk after in vivo immunization with PRP and TT, in vitro stimulation with pokeweed mitogen, Staphylococcus aureus Cowan 1 bacteria, or antigen induced anti-TT but not anti-PRP in vitro antibody secretion, although Epstein-Barr virus induced both. These data suggest that PRP, a polysaccharide, and TT, a protein, differ in their requirements for in vitro activation with antigen and mitogens.     

Functional activity of different IgG subclass antibodies against type b capsular polysaccharide of Haemophilus influenzae

The biologic activity of different human IgG subclass antibodies directed against the Haemophilus influenzae type b (Hib) capsular polysaccharide (PRP) was compared by using an in vitro complement-mediated bactericidal assay and an in vivo passive protection assay in infant rats. An IgG pool was made by Sephacryl S-300 chromatography of sera from adults immunized with PRP vaccine. An IgG2 subclass fraction was prepared by column immunoabsorption of the IgG pool with anti-IgG1 monoclonal antibody. An IgG1 subclass fraction was eluted from the affinity matrix. IgG1, IgG2, IgG3, and IgG4 concentrations in the fractions were measured by solid-phase competitive radioimmunoassays, and anti-PRP antibody was measured by a modified Farr assay. Each fraction was greater than 90% pure IgG2 or IgG1, respectively. There were no significant differences in the minimal anti-PRP antibody concentrations required to kill 50% of Hib cells in vitro (IgG, 0.22; IgG1, 0.21; and IgG2, 0.42 microgram/ml). Similarly, equivalent amounts of anti-PRP antibody of the IgG1 or IgG2 fractions protected against bacteremia (IgG1, 0.12; IgG2, 0.24 microgram per rat). IgG absorbed to remove anti-PRP antibody was neither bactericidal nor protective. Thus IgG1 and IgG2 anti-PRP antibody have equivalent functional activities against Hib as determined by these biologic assays.     

Recognition of riboflavin and the capsular polysaccharide of Haemophilus influenzae type b by antibodies generated to the haptenic epitope D-ribitol

D-Ribitol, a five–carbon sugar alcohol, is an important metabolite in the pentose phosphate pathway; it is an integral part of riboflavin (vitamin B2) and cell wall polysaccharides in most Gram-positive and a few Gram-negative bacteria. Antibodies specific to D-ribitol were generated in New Zealand white rabbits by using reductively aminated D-ribose-BSA conjugate as the immunogen. MALDI-TOF and amino group analyses of ribitol-BSA conjugate following 120 h reaction showed ~27–30 mol of ribitol conjugated per mole BSA. The presence of sugar alcohol in the conjugates was also confirmed by an increase in molecular mass and a positive periodic acid–Schiff staining in SDS-PAGE. Caprylic acid precipitation of rabbit serum followed by hapten affinity chromatography on ribitol–KLH–Sepharose CL-6B resulted in pure ribitol–specific antibodies (~45–50 μg/mL). The affinity constant of ribitol antibodies was found to be 2.9?×?10⁷ M^?1 by non-competitive ELISA. Ribitol antibodies showed 100 % specificity towards ribitol, ~800 % cross–reactivity towards riboflavin, 10–15 % cross–reactivity with sorbitol, xylitol and mannitol, and 5–7 % cross–reactivity with L-arabinitol and meso-erythritol. The specificity of antibody to ribitol was further confirmed by its low cross-reactivity (0.4 %) with lumichrome. Antibodies to D-ribitol recognized the purified capsular polysaccharide of Haemophilus influenzae type b, which could be specifically inhibited by ribitol. In conclusion, antibodies specific to D-ribitol have been generated and characterized, which have potential applications in the detection of free riboflavin and ribitol in biological samples, as well as identification of cell-surface macromolecules containing ribitol.     

Evidence for covalent attachment of phospholipid to the capsular polysaccharide of Haemophilus influenzae type b.

Cells of Haemophilus influenzae type b were grown in a liquid medium containing [3H]palmitate or [14C]ribose or both for two generations of exponential growth. Radiolabeled type-specific capsular polysaccharide, polyribosyl ribitol phosphate (PRP), was purified from the culture supernatant by Cetavlon precipitation, ethanol fractionation, and hydroxylapatite and Sepharose 4B chromatography. The doubly labeled ( [3H]palmitate and [14C]ribose) PRP preparation was found to coelute in a single peak from a Sepharose 4B column, suggesting that both precursors were incorporated into the purified PRP. A singly labeled ( [3H]palmitate) purified PRP preparation was found to be quantitatively immune precipitated by human serum containing antibody against PRP. The radioactivity of this preparation could not be dissociated from PRP by treatment with chloroform-methanol, 6 M urea, sodium dodecyl sulfate, or Zwittergent. Only after acid, alkaline, or phospholipase A2 treatment of PRP labeled with [3H]palmitate or [3H]palmitate and [14C]ribose followed by chloroform-methanol extraction could most of the 3H-radioactivity be recovered in the organic phase. The chloroform-soluble acid-hydrolyzed or phospholipase A2-treated product was identified as palmitic acid after thin-layer chromatography. These results strongly suggest that a phospholipid moiety is covalently associated with the H. influenzae type b polysaccharide PRP.     

Restricted Ig H chain V gene usage in the human antibody response to Haemophilus influenzae type b capsular polysaccharide

The mechanisms that govern the content of the human antibody repertoire are poorly understood. To investigate the antibody response to a clinically relevant Ag, we have produced heterohybridomas secreting human antibodies directed against the capsular polysaccharide of Haemophilus influenzae type b (Hib PS). Immune lymphocytes were harvested 7 days after immunization with either of two vaccine formulations, a plain polysaccharide vaccine (Hib PS) or a polysaccharide-protein conjugate of Hib PS and diphtheria toxoid (Hib PS-D). H chain V region gene nucleic acid sequences were determined for five stable hybridomas. All use members of the VHIII gene family and are 83% to 98% homologous to two candidate germ-line sequences. A variety of D and JH segments are used. Thus the Ig H chain repertoire appears to be restricted to a limited group of VHIII family members. The previously reported expression of homologous sequences in the human fetal repertoire suggests that the inability of young children to respond to this Ag is not caused by deficiencies of these important elements early in development. The restricted use of VHIII gene segments suggests that this gene family plays a pivotal role in the immune response to this important childhood pathogen.     

Recognition of flavin mononucleotide,Haemophilus influenzae type b and its capsular polysaccharide vaccines by antibodies specific to D-ribitol-5-phosphate

D-Ribitol-5-phosphate (Rbt-5-P) is an important metabolite in the pentose phosphate pathway and an integral part of bacterial cell wall polysaccharides, specifically as polyribosyl ribitol phosphate (PRP) in Haemophilus influenzae type b (Hib). The major objective of this study was to investigate whether an antibody specific to Rbt-5-P can recognize the PRP of Hib. D-Ribose-5-phosphate was reacted with proteins in the presence of sodium cyanoborohydride to obtain Rbt-5-P epitopes; 120 h reaction resulted in conjugation of ~30 and ~17 moles of Rbt-5-P/mole of BSA and OVA, respectively, based on decrease in amino groups, MALDI–TOF analyses, an increase in apparent molecular weight (SDS–PAGE) and glycoprotein staining. Immunization of rabbits with Rbt-5-P–BSA conjugate generated antibodies to Rbt-5-P as demonstrated by dot immunoblot and non-competitive ELISA. Homogeneous Rbt-5-P-specific antibody was purified from Rbt-5-P–BSA antiserum subjected to caprylic acid precipitation followed by hapten–affinity chromatography; its affinity constant is 7.1?×?10⁸ M^?1. Rbt-5-P antibody showed 100 % specificity to Rbt-5-P, ~230 %, 10 % and 3.4 % cross-reactivity to FMN, riboflavin and FAD, respectively; the antibody showed ~4 % cross-reactivity to D-ribitol and <3 % to other sugars/sugar alcohols. Rbt-5-P–specific antibody recognized Hib conjugate vaccines containing PRP which was inhibited specifically by Rbt-5-P, and also detected Hib cell-surface capsular polysaccharides by immunofluorescence. In conclusion, Rbt-5-P–protein conjugate used as an immunogen elicited antibodies binding to an epitope also present in PRP and Hib bacteria. Rbt-5-P–specific antibody has potential applications in the detection and quantification of free/bound Rbt-5-P and FMN as well as immunological recognition of Hib bacteria and its capsular polysaccharide.     

Detection of mucosal and serum antibodies specific for the capsular polysaccharide of Haemophilus influenzae type b by enzyme-linked immunosorbent assay

A sensitive enzyme-linked immunosorbent assay (ELISA) with rough-surfaced glass beads used as the solid phase was developed for detection of IgG, IgM, and IgA antibodies to Haemophilus influenzae type b capsular polysaccharide (HITB-CP). A successful method for indirect coating of glass bead surfaces with HITB-CP, and parameters affecting the specificity and sensitivity of the assay are described. This ELISA system proved to be 100 times as sensitive as the standard indirect fluorescent-antibody assay. The assay was applied to the measurement of antibodies to HITB-CP in serum and nasal secretions and proved to be a useful tool in the evaluation of immunological response to HITB infection.     

Isoelectric focusing of human antibody to the Haemophilus influenzae b capsular polysaccharide: restricted and identical spectrotypes in adults

Serum antibody to the capsular polysaccharide of Haemophilus influenzae b of human adults was analyzed by isoelectric focusing. Restricted antibody spectrotype patterns were commonly observed with as few as one spectrotype in some subjects after immunization with the isolated capsular polysaccharide. Some patterns were as restricted as human hybridoma antibody. There was no correlation of antibody titer and heterogeneity of patterns. The dominant spectrotype persisted unchanged for over 2 yr after immunization, and the pattern detected in preimmunization serum samples persisted unchanged after immunization. Indistinguishable patterns were commonly observed in genetically unrelated adults. Adults immunized with conjugate vaccines, which were composed of oligosaccharides prepared from the capsular polysaccharide that were covalently linked to protein carriers, also produced restricted serum antibody spectrotype patterns. Immunization with the cross-reactive polysaccharide of E. coli K100 induced a spectrotype pattern that was restricted but different from that induced by the H. influenzae b capsular polysaccharide.     

An NMR spectroscopic identity test for the control of the capsular polysaccharide from Haemophilus influenzae type b.

We describe the use of Nuclear Magnetic Resonance (NMR) spectroscopy to control the identity of purified bulk capsular polysaccharide [called poly(ribosylribitolphosphate) or PRP] from Haemophilus influenzae type b (Hib), and derivatised forms, used in the production of Hib polysaccharide-protein conjugate vaccines. We describe the approaches we have developed to validate this test.     

Intrinsic tritium labeling of the capsular polysaccharide antigen of Haemophilus influenzae type B.

The capsular polysaccharide of Haemophilus influenzae type b was intrinsically labeled with tritium by a microculture technique with 6-3H-D-glucose and was isolated in radioantigenically pure form by a combination of selective precipitation and molecular sieve chromatography. Labeling with tritated sugar residues approached one-fourth maximum and produced a specific activity 10-fold that previously described for extrinsic labeling methods. In radioantigen-binding assays for antibody, sensitivity depended on the size of the antigen; preparations were readily made that could detect 0.01 microgram Ab/ml in serum samples of 25 microliter. Stability of the labeled antigen appears limited only by the primary radiodecomposition of tritium.     

The capsular polysaccharide of Haemophilus influenzae type B prevents killing by complement and antibodies against outer membrane protein a

Abstract The ability of antibodies, raised in rabbits against purified outer membrane protein a ( M _r 47 000) of Haemophilus influenzae type b, to promote complement-dependent killing of these encapsulated organisms was investigated. Killing of encapsulated strains was not induced by these antibodies in conjunction with either human, mouse, rabbit or guinea-pig complement. Acapsular mutants were effectively killed by complement in the presence of antibodies against protein a . Killing was dependent on the presence of the 47-kDa protein a and was not influenced by the outer membrane protein subtype or lipopolysaccharide serotype of the strain. The killing-promoting activity could be absorbed from the sera with cells of strains with the same protein a , purified protein a , but not by purified lipopolysaccharide and capsular polysaccharide. Binding experiments showed that the encapsulated strain and its acapsular mutant bound antibodies against protein a with the same rate and to the same extent, indicating that the capsule probably interferes with complement activation or insertion of the membrane attack complex into the bacterial cell.     

Influence of the aminopeptide concentration on the growth of Haemophilus influenzae, type b, and the synthesis of its capsular polysaccharide

The influence of the aminopeptide concentration on the growth of H. influenzae b culture and the synthesis of H. influenzae b capsular polysaccharide was determined. The maximum amount of capsular polysaccharide was accumulated at the concentration of aminopeptide in the culture fluid reaching 50 ml/l. An increase in the aminopeptide concentration led to a decreased amount of synthesized polysaccharide and an increased amount of biomass. The decrease of the aminopeptide concentration to 10 ml/l resulted in decreased amounts of both biomass and synthesized polysaccharide.     

Determination of the structure of the Escherichia coli K100 capsular polysaccharide, cross-reactive with the capsule from type b Haemophilus influenzae

The structure of the Escherichia coli K100 capsular polysaccharide, cross-reactive with that from type b Haemophilus influenzae, was determined by using a combination of chemical and spectroscopic techniques. The structure of the K100 repeating unit was found to be----3)-beta-D-Ribf-(1----2)-D-ribitol-5-(PO4----. The K100 polysaccharide is thus identical in composition to, but different in linkage from, the H. influenzae type b capsular polysaccharide, which has beta-D-Ribf-(1----1)-D-ribitol linkages.     

Lipoproteins of Haemophilus influenzae type b.

Haemophilus influenzae type b Minn A produced 12 lipoproteins with apparent molecular weights of between 14,000 and 67,000. The lipoproteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of delipidated extracts of cells grown in [3H]palmitate. When the delipidated cell extracts were subjected to acid methanolysis, tritium was quantitatively recovered as palmitate and methyl palmitate, indicating that the [3H]palmitate had not been degraded and reincorporated into nonlipid material during cell growth. One of the lipoproteins comigrated with outer membrane protein (OMP) P6. OMP P6 was purified from [3H]palmitate-labeled cells. The purified protein preparation contained both amide- and ester-linked fatty acids. We conclude that (i) H. influenzae type b produces several lipoproteins, and (ii) one of these lipoproteins is OMP P6, a protein under consideration as a vaccine component.     

Haemophilus influenzae type b polysaccharide-protein conjugate vaccine

An Haemophilus influenzae type b capsular polysaccharide-protein conjugate has been prepared. The polysaccharide was coupled to the serotype II protein of group B meningococcus through the spacer 6-aminocaproic acid using cyanogen bromide and water soluble carbodiimide. The conjugate can be shown to be reproducible and is stable and highly immunogenic in mice and African green monkeys. Clinical evaluation of this conjugate in children 3 months to 4 years of age showed that it elicited an antibody titer to the polysaccharide moiety greater than 1000 ng/ml in children 8 months of age or older.     

Isotype concentrations of human antibodies to Haemophilus influenzae type b polysaccharide (Hib) in young adults immunized with the polysaccharide as such or conjugated to a protein (diphtheria toxoid)

Antibody responses of young adults to Haemophilus influenzae type b polysaccharide (Hib) or its protein conjugate were studied with special attention to the isotype composition of the antibodies. Three conclusions of interest can be made: 1) Immunoglobulin G (IgG) antibodies in polysaccharide-immunized volunteers displayed the subclass pattern previously found in antibodies to meningococcal type A polysaccharide. IgG1 was the predominant subclass in IgG antibodies of some individuals, IgG2 in others. Still others had the two subclasses in varying but more even proportions. 2) The conjugate vaccine induced a geometric mean response 2 to 3 times higher and an IgG response 4 times higher to Hib than the polysaccharide vaccine. 3) Anti-Hib antibodies induced by the conjugate vaccine still had essentially the same IgG subclass composition as anti-Hib antibodies induced by the polysaccharide. This composition was strikingly different from the composition of the anti-diphtheria toxoid response induced by the same conjugate vaccine.