Comparison of naturally acquired and vaccine-induced antibodies to Haemophilus influenzae type b capsular polysaccharide.

The objective of this study was to assess qualitative differences in the types of Haemophilus influenzae type B (Hib) capsular polysaccharide (polyribosylribitol phosphate [PRP]) antibodies induced in children 15 to 27 months of age by (i) natural exposure, (ii) PRP vaccine, and by (iii) PRP-diphtheria toxoid conjugate vaccine, (iv) PRP-group B Neisseria meningitidis outer membrane vesicle conjugate vaccine, and (v) Haemophilus type B oligosaccharide conjugate vaccine (HbOC). The highest levels of total Hib-PRP antibody measured by radioimmunoassay and immunoglobulin G (IgG) measured by enzyme-linked immunosorbent assay were seen after HbOC immunization. IgG1 Hib-PRP antibodies predominated in all groups, and there were no differences between the groups in the proportion of IgG and IgA Hib-PRP antibodies. However, the proportions of IgM differed significantly by group. The highest proportions of IgM occurred in naturally acquired antibody and after PRP vaccine, and the lowest proportion occurred after HbOC vaccine. IgG light-chain V kappa type alpha PRP antibody was present in all groups, and the level correlated with the total IgG Hib-PRP antibody level. Therefore, HbOC induced the highest concentrations of V kappa II type alpha PRP antibody, and the naturally acquired antibody group had the lowest levels. IgG light-chain V kappa III antibody levels were also highest in the HbOC group, but there was no correlation between V kappa III antibody levels and total amount of IgG Hib-PRP antibody. These data demonstrate qualitative differences in the antibody repertoires induced by natural exposure, the Hib-PRP vaccine, and each of the different Hib conjugate vaccines. We doubt that there are major differences in the protection afforded by these different antibody repertoires, because these differences do not appear to correlate with differences in protective efficacy in older children.     

The repertoire of human antibody to the Haemophilus influenzae type b capsular polysaccharide.

Human antibody to the Haemophilus influenzae capsular polysaccharide (Hib CP) is restricted in diversity in the individual and the population with a limited number of variable region genes encoding antibody. Antibody to the Hib CP shows restricted isoelectric focusing gel patterns and light chain usage with frequent restriction to use of only kappa light chains. Shared cross-reactive idiotypes are expressed on antibody. The heavy chain of antibody to the Hib CP is predominantly encoded by two members of the VH3 family--LSG 6.1/M85-like and VH26/30P1-like. In VH the CDR1, based on complete identity in LSG 6.1/M85-like antibodies, CDR2, based on the suggestion of mutation in this region, and CDR3, based on conserved CDR3 usage in unrelated individuals, may be important for antigen binding. Six or more different VL gene families encode antibody. The predominant antibody of the majority of individuals uses the A2-V kappa II gene in germline or near germline configuration, which encodes an idiotype designated HibId-1. Antibody can also be encoded by V kappa I, non-A2 V kappa II, V kappa III, V kappa IV, V lambda II, and V lambda VII genes. Although different VL genes can be used, unrelated individuals appear to use the same V kappa III (A27), V lambda II (V lambda 2.1 and V lambda VII (4A) genes. The VL diversity accounts for differences in fine binding specificity, with A2-V kappa II genes not encoding E. coli K100 CP cross-reactive antibodies and V lambda VII genes and some of the non-A2 V kappa genes encoding cross-reactive antibodies. The arginine in CDR3 of both antibody kappa and lambda light chains and the asparagine in CDR2 of VL sequences and in CDR1 of LSG6.1-M85 VH sequences of antibody appear to be important residues for antigen binding. A relatively limited degree of somatic mutation has occurred in the non-A2 VL genes, V lambda VII, and the VH genes. Further studies comparing the polymorphism of germline V genes to antibody-encoding V genes are needed to clarify this issue. Research comparing this repertoire to repertoires directed to other bacterial CP and to self antigens and defining structure-antigen binding relationships is in progress.     

A simplification of the enzyme-linked immunospot technique. Increased sensitivity for cells secreting IgG antibodies to Haemophilus influenzae type b capsular polysaccharide.

A simplified enzyme-linked immunospot (ELISPOT) technique is described for the detection of cells secreting antibodies to tetanus toxoid (TT), diphtheria toxoid (DT) or Haemophilus influenzae type b capsular polysaccharide (PRP). By combining the cell suspension with the enzyme-linked secondary antibodies in one incubation, the second incubation and washing procedure could be omitted from the original technique. The simplified assay had the same sensitivity for anti-TT and anti-DT spot-forming cells as the ordinary ELISPOT assay. The IgG anti-PRP spots were, however, improved both in quality and in quantity (median: 40% more spots), while the detection of IgM and IgA anti-PRP spot-forming cells was the same in the two techniques. This simplified technique can probably also be used to save time in other antigen systems and should be considered when designing ELISPOT assays for the detection of polysaccharide-specific antibody-secreting cells.     

Functional capacities of clonal antibodies to Haemophilus influenzae type b polysaccharide.

Haemophilus influenzae type b (Hib) is an important pathogen for young children, and children can be protected with antibodies (Abs) to Hib polysaccharide (PS) capsule, a linear polymer of ribosyl ribitol phosphate. The structure of anti-Hib-PS Abs has been well characterized at the molecular level; about two-thirds of anti-Hib-PS Abs use a V kappa gene named A2, and the remaining anti-Hib-PS Abs use one of many other VL genes. In order to understand the structural basis for the variability in the function of these Abs, we prepared 18 clonally pure Abs from adults and studied their affinity, avidity, bactericidal potency in vitro, and ability to reduce bacteremia in newborn rats. Affinities and avidities were determined as the inverse of the concentrations of short (3 repeating units) and long (20 repeating units) ligands which could bind 50% of anti-Hib-PS Ab in solution, respectively. No significant correlations between the protection of newborn rats and affinity (r = 0.02) or avidity (r = 0.16) were observed. The amount of Ab required to kill 50% of bacteria in vitro decreased with avidity (r = -0.32), as expected. However, Abs with high affinity were unexpectedly found to have less bactericidal activity (r = 0.38). This suggests that avidity may be a better predictor of Ab function than affinity. Affinity and avidity results were negatively correlated (r = 0.76, P = 0.0022), and Abs that had A2 V kappa gene products had higher avidity (P < 0.05) and lower affinity (P = 0.06) than Abs that had other VL genes. A possible explanation of these observations is that the epitope for Abs with the A2 gene is within the Hib-PS chain itself, whereas the epitope for Abs with a non-A2 gene is the terminus of Hib-PS.     

Isolation of the capsular polysaccharide from culture supernatant of Haemophilus influenzae type b.

The capsular polysaccharide (CP) of Haemophilus influenzae type b is known to be spontaneously released from the cells in culture. The CP is precipitable from culture supernatant by the cationic detergent hexadecyltrimethylammonium. Most of the nucleic acid and some of the protein, but almost none of the endotoxin, in the supernatant are co-precipitated. Extraction of the precipitate with progressively stronger NaCl solutions separates nucleic acid and protein from the CP and also effects a molecular size fractionation. Residual endotoxin and protein can be reduced by extraction with cold phenol and ultracentrifugation. The resulting preparation has ribose, ribitol, and phosphate as principal components and contains less than 1% other sugars, protein, or nucleic acid; it elutes on Sepharose 2B as a symmetrical peak with Kav 0.51.     

Characterization of antibodies to capsular polysaccharide antigens of Haemophilus influenzae type b and Streptococcus pneumoniae in human immune globulin intravenous preparations

The most common infections in primary immune deficiency disease (PIDD) patients involve encapsulated bacteria, mainly Haemophilus influenzae type b (Hib) and Streptococcus pneumoniae (pneumococcus). Thus, it is important to know the titers of Hib- and pneumococcus-specific antibodies that are present in immune globulin (Ig) intravenous (IGIV) preparations used to treat PIDD. In this study, seven IGIV preparations were tested by enzyme-linked immunosorbent assay and opsonophagocytic activity for antibody titers to the capsular polysaccharides of Hib and five pneumococcal serotypes. Differences in Hib- and pneumococcus-specific antibody titer were observed among various IGIV preparations, with some products having higher- or lower-than-average titers. Opsonic activity also varied among preparations. As expected, IgG2 was the most active subclass of both binding and opsonic activity except against pneumococcal serotype 6B where IgG3 was the most active. This study determines antibody titers against capsular polysaccharides of Hib and pneumococcus in seven IGIV products that have been shown to be effective in reducing infections in PIDD patients. As donor antibody levels and manufacturing methods continue to change, it may prove useful from a regulatory point of view to reassess IGIV products periodically, to ensure that products maintain antibody levels that are important for the health of IGIV recipients.     

The regulation of the immune response of mice to Haemophilus influenzae type b capsular polysaccharide.

The regulation of age-related antibody response to Haemophilus influenzae type b polysaccharide (HITB-PS) was studied by measuring the splenic plaque forming cells (PFC) following immunization with this capsular polysaccharide. The magnitude of PFC response to HITB-PS was found to be dose-related, enhanced by Freund's complete adjuvant and influenced by the genetic strain of mice. Priming with a low dose of HITB-PS did not induce a state of immunological unresponsiveness. Treatment with antilymphocyte serum significantly increased the PFC response to HITB-PS. Athymic nude mice showed an enhanced ability to induce both IgG and IgA-PFC responses as well as a significant increase in the biosynthesis of protein and mitogenicity in spleen cells. These findings suggest that the immune response to HITB-PS is regulated by the suppressor T cell. The magnitude of the IgM-PFC response induced by HITB-PS in mice increased gradually from two weeks of age and reached a plateau at 8 weeks. Treatment with fetuin resulted in the inhibition of direct IgM and IgG-PFC responses to HITB-PS; the suppressive effect on the immune response was more profound and lasting in young than in adult mice.     

Measurement of anti-Haemophilus influenzae type b capsular polysaccharide antibodies by ELISA

Measurement of antibodies to the capsular polysaccharide polyribosylribitolphosphate (PRP) of Haemophilus influenzae type b by ELISA is made difficult by the poor binding of this antigen to the solid phase. Six coating conditions were compared using immune and non-immune human sera. Direct coating with PRP was inefficient. Precoating with protamine or poly-L-lysine (PLL) yielded irreproducible results and high background levels. Assays with PRP conjugated with PLL as coat were not sensitive enough. In addition, anti-PRP antibodies, especially those belonging to the IgM class, crossreacted with PLL. Coating with avidin or streptavidin followed by incubation with biotin-coupled PRP was not satisfactory either, due to binding of certain sera in the absence of PRP. Coating with PRP coupled to tyramine resulted in low backgrounds and acceptable specific binding levels. However, the finding that the binding of a few sera was only partially inhibited by soluble PRP led us to include an inhibition step in every experiment. Only optical densities inhibited by the antigen were taken into account. In view of the lack of parallelism of dilution curves from different sera, no attempt was made to express the results in weight units. They were expressed in arbitrary units calculated by comparison with internal standards. Under such conditions, the assay permitted a reproducible (interassay coefficients of variation around 10%) determination of PRP-Ab belonging to the various immunoglobulin classes and IgG subclasses and showed a good correlation with results obtained using the Farr assay.     

Heteroimmunization to the capsular polysaccharide of Haemophilus influenzae type b induced by enteric cross-reacting bacteria.

Cross-reacting Escherichia coli strains Easter and 89 and Bacillus pumilis fed to newborn rabbits and E. coli fed to adult rhesus monkeys did not exert untoward reactions. The E. coli regularly colonized the newborns' intestinal tract from 1 to 7 weeks. High doses of E. coli were necessary to colonize adult primates. Colonization occurred in fewer newborn rabbits and lasted only 1 to 3 weeks with B. pumilis. Colonized newborn rabbits and adult rhesus had an active Haemophilus influenzae type b (HITB) immune response. In the rabbit, colonization resulted in accelerated induction of immunoglobulin (Ig) M-. IgA-, and IgG-producing cells in the spleen, mesenteric lymph nodes, and Peyer's patches after HITB challenge. E. coli-fed and control newborn primates were naturally colonized with nasopharyngeal and enteric cross-reacting bacteria and both groups rapidly developed HITB antibodies in the absence of the homologous organisms. Human newborn stool cultures, taken at the time of discharge from the nursery, showed a 0.9% carriage rate for cross-reacting E. coli. These "carrier" infants acquired HITB antibodies more rapidly than their age-matched "noncarrier" controls.     

Quantitation of antibodies to Haemophilus influenzae type b in humans by enzyme-linked immunosorbent assay.

The enzyme-linked immunosorbent assay was adapted to detect serum immunoglobulin G, immunoglobulin M, immunoglobulin A, and secretory immunoglobulin A antibodies to Haemophilus influenzae type b capsular polysaccharide in humans. I studied serum samples from 92 healthy children of various ages, 50 healthy adults, 24 patients with various H. influenzae type b infections, and 16 patients with clinical signs of epiglottis and cellulitis suspected to be caused by H. influenzae type b. The mean antibody titers of the sera from healthy children increased with age and reached adult levels in children more than 6 years old. A significant antibody response to capsular polysaccharide was observed in serum samples from the majority of patients with infections due to H. influenzae type b and in 4 of 16 patients with clinical signs of epiglottis and cellulitis. In addition to the enzyme-linked immunosorbent assay, the antibody responses of patients were tested by a bactericidal assay. When the two methods were compared, there was no evident correlation (r, about 0.22). The enzyme-linked immunosorbent assay was further adapted to test secretory immunoglobulin A antibodies specific to capsular polysaccharide in nasopharynx secretions and in milk samples from lactating women. Antibodies were detected in 12 of 24 secretions and 9 of 11 milk samples.     

Interlaboratory study evaluating quantitation of antibodies to Haemophilus influenzae type b polysaccharide by enzyme-linked immunosorbent assay.

An interlaboratory study was conducted to determine whether an enzyme-linked immunosorbent assay (ELISA) with an antigen preparation composed of various-sized fragments of Haemophilus influenzae type b polysaccharide conjugated to human serum albumin could be standardized across laboratories and whether the ELISA-derived results from different laboratories are equivalent to those obtained by the standard radioactive antigen binding assay (RABA) for quantitation of anti-H, influenzae type b polysaccharide antibodies. Twenty coded human serum samples were quantitated by ELISA in 11 laboratories and by RABA in 5 laboratories. The mean RABA-derived values served as the basis for all comparisons. While the overall correspondence of antibody values between the two methods was good, significant differences were found among some of the 11 ELISA data sets and among the mean RABA values. Seven laboratories generated higher ELISA antibody values for low-titered sera. Four laboratories generated antibody concentrations that were not statistically different between the two assay methods. The results therefore indicate that the ELISA can tolerate substantial variations in protocol, such as the use of different plates and different antibody reagents, without affecting the quantitation of serum antibodies. However, attention should be focused on low-titered sera, as some assay conditions may yield spurious results. This ELISA is a serologic assay which can serve as an alternative to the RABA for quantitation of antibodies to H. influenzae type h polysaccharide.     

Relation between enzyme-linked immunosorbent assay and radioimmunoassay for detection of antibodies to the capsular polysaccharide of Haemophilus influenzae type b.

The measurement of antibodies to the capsular polysaccharide (PRP) of Haemophilus influenzae type b (Hib) is important because vaccines inducing such antibodies are now available. We developed and evaluated an enzyme-linked immunosorbent assay (ELISA) for detection of these antibodies based on direct coating of the plates with tyraminated PRP. The assay fulfilled the requirements for parallel line assays; it was sensitive, specific, and reproducible with a coefficient of variation between days of 19%. Results from the ELISA were compared with results from radioimmunoassay and a correlation coefficient of 0.93 was found. Results obtained by the two methods were proportional and the relation was independent of the antibody level. The relation between them was also unaffected by the contribution of different antibody isotypes, indicating that these were measured to the same extent by both methods. ELISA employing direct coating of the plates with tyraminated PRP represents a useful alternative for detection of antibodies when studying immunogenicity of Hib vaccines.     

The human antibody V region repertoire to the type B capsular polysaccharide of Haemophilus influenzae.

The V region repertoire of the human antibody response to the type b capsular polysaccharide of Haemophilus influenzae (Hib-PS) is being defined at the molecular level using antibodies purified from serum of immunized adults. The VH of this response is restricted to the VHIII subgroup while the VL can be divided into two categories. The most common VL, expressed in > 90% of adults and usually constituting the majority of a subjects anti-Hib-PS antibody response, is restricted to the product of a single V kappa II gene known as A2 that probably lacks somatic mutations. The product of the A2 gene is invariably joined to one of several J kappa products by an inserted arginine at the V kappa-J kappa junction. In contrast to the restricted nature of the dominant VL clonotype, the second category of VL constitutes a heterogeneous group of at least seven different VL gene products that often contain somatic mutations and generally exhibit crossreactivity with a related polysaccharide from E. coli. Elucidation of anti-Hib-PS V regions at the molecular level will permit examination of structure-function relationships among these clinically important antibodies and should make the V region repertoire to Hib-PS a useful model for studying human V gene responses.     

Variable region sequences of a protective human monoclonal antibody specific for the Haemophilus influenzae type b capsular polysaccharide.

A hybridoma secreting a human immunoglobulin G2 kappa monoclonal antibody (MAb) specific for the capsular polysaccharide of Haemophilus influenzae type b (Hib) was isolated. This MAb, designated CA4, was bactericidal to Hib in vitro and protected infant rats from Hib bacteremia. Nucleotide sequence analysis of CA4 variable (V) region cDNA showed that the heavy (H)-chain V region was of subgroup III and was 96% identical to the VH germ line gene segment DP77 (V3-21). The light (L)-chain V region was of the kappa subgroup III and was 94% identical to the A27 (Humkv325) germ line gene, which is commonly used by rheumatoid factors and other autoantibodies. MAb CA4 did not have rheumatoid factor activity and did not react with histones, DNA, or chromatin. These findings identify an additional VHIII gene segment which can contribute to the anti-Hib capsular polysaccharide repertoire and demonstrate that a VL gene commonly encoding autoantibodies can be utilized for protective immunity.     

A radioactive antigen-binding assay for the measurement of antibody to Haemophilus influenzae type b capsular polysaccharide

A new polyethylene glycol (PEG) radioimmunoprecipitation assay was developed for the detection of antibody to Haemophilus influenzae b capsular polysaccharide, polyribosylribitol phosphate (PRP). The radioactive antigen, [3H]PRP, with a high specific activity, was produced by growing the organism in the presence of [3H]ribose and was purified by hydroxylapatite and Sepharose 4B column chromatography. In the assay, PEG (12.5%) was used to separate antibody-bound [3H]PRP from free [3H]PRP. The assay covered the range of 0.5 and 20 ng antibody/assay at a maximum sensitivity of 0.5 approximately 1.0 ng antibody/assay. With various dilutions (1-20 ng antibody/assay) of S. Klein reference antiserum, the within-run coefficient of variation (CV) of 10 replicates ranged from 3.5 to 8.5%. Average CVs of 8.9% and 11.0% were obtained in the between-run and day-to-day reproducibility studies. The binding of [3H]PRP to S. Klein reference antiserum was severely inhibited by a minute amount of non-radioactive PRP; however, no significant interference was found in the presence of high concentrations of polysaccharides from Escherichia coli K100 and Streptococcus pneumoniae indicating that the RIA was highly specific for antibody to H. influenzae b PRP. The present RIA is a simple, specific, sensitive and reproducible procedure for the evaluation of antibody responses of young animals and infants to H. influenzae b vaccines and infections.     

Avidity of specific IgG antibodies elicited by immunisation against Haemophilus influenzae type b.

AIM--To investigate the avidity of specific IgG polyribosyl ribitol phosphate (PRP) antibodies induced by three Haemophilus influenzae type b (Hib) conjugate vaccines: PRP-meningococcal outer membrane protein complex (PRP-OMP), PRP-non-toxic mutant diphtheria toxin, CRM197 (HbOC) and PRP-tetanus toxoid (PRP-T). METHODS--One hundred and ten infants were immunised with one of the vaccines at two, three and four months of age. Blood samples were taken after each vaccination and serum stored at -20 degrees C. Serum samples collected after the third course (that is, at five months of age) were submitted to antibody avidity assessment, using a urea enzyme linked immunosorbent assay (ELISA). RESULTS--All three conjugate vaccines elicited IgG PRP antibodies of high median avidity. The resultant antibody populations were heterogeneous with regard to avidity, which in turn was independent of PRP antibody concentration. CONCLUSIONS--With the recent findings of a correlation between bactericidal killing and affinity, our data highlight the need for a protective level to be expressed qualitatively as well as quantitatively.     

Characterization and mitogenic activity of Haemophilus influenzae type B capsular polysaccharide.

Studies were conducted on the characterization of Haemophilus influenzae type b polysaccharide (HITB-PS) and its mitogenic activity upon peripheral lymphocytes. This capsular polysaccharide was found to contain hexosamines and hexoses in addition to the main components of ribose and ribitol phosphate. The molecular weight of HITB-PS was determined as 585,000. The affinity constant of HITB-PS to unfractionated lymphocytes was 3.13 X 10(3) M-1 with 1.11 X 10(4) binding sites per cell. HITB-PS was found to be mitogenic for both human T and B lymphocytes. At optimum doses, a three to five fold increase in 3H-thymidine incorporation into T and B cells was observed. Higher than optimum doses resulted in suppression of this mitogenicity. The effect of concanavalin A (Con A) mitogenicity was detected in T and B cells treated with effective as well as suppressive doses of HITB-PS; the mitogenic activities of Con A and HITB-PS were found to be independent of each other.     

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.     

Quantitation of rheumatoid factors by enzyme immunoassay using biotinylated human IgG

An enzyme-linked immunosorbent assay (ELISA) using biotinylated human IgG for the detection of rheumatoid factor (RF) in human sera has been developed. This assay avoids some of the problems encountered with earlier ELISA methods used for RF detection. Accurate RF titers of human sera were determined using a simple computer program developed for curve fitting of ELISA data.