Radioantigen Binding Research Articles

Effectiveness Analyses May Underestimate Protection of Infants after Group C Meningococcal Immunization

Group C meningococcal conjugate-vaccine effectiveness in the United Kingdom declines from ~90% in the first year to 0% between 1 and 4 years after immunization in infants immunized at 2, 3, and 4 months of age and to 61% in toddlers given a single dose. Confidence intervals are wide, and the extent of protection is uncertain. Serum samples were obtained from children 3-5 years of age who were participants in a preschool booster-vaccine trial. Serum bactericidal activity was measured with human complement. Group C anticapsular antibody concentrations were measured by a radioantigen binding assay. Passive protection was analyzed in an infant rat bacteremia model. Serum samples from UK children who had been immunized 2-3 years earlier as infants or toddlers had higher levels of radioantigen binding, bactericidal activity, and passive protection than did historical control serum samples from unimmunized children (P<.05). A higher proportion of children immunized as infants had serum bactericidal activity titers > or =1 : 4 (considered to be protective) than those immunized as toddlers (61% vs. 24%; P<.01), but there were no significant differences in the proportion of serum samples conferring passive protection (50% and 41%, respectively; P=.4). We found no evidence of lower immunity in children immunized as infants than as toddlers. On the basis of serum bactericidal activity and/or passive protection, 40%-50% of both age groups are protected at 2-3 years after immunization, which was significantly greater than in unimmunized historical controls (<5%).

IGH V3-23*01 and its allele V3-23*03 differ in their capacity to form the canonical human antibody combining site specific for the capsular polysaccharide of Haemophilus influenzae type b.

The IGH V3-23*01 gene is used in the formation of the canonical combining site which dominates the human antibody repertoire to the Haemophilus influenzae type b (Hib) polysaccharide (PS). An allele of the human IGH V3-23*01 gene, known as V3-23*03, differs from V3-23*01 in nine bases, eight of which are located in the second complementarity determining region. These eight differences encode five amino acid substitutions. In this study we investigated whether the V3-23*03 sequence polymorphism affected Hib PS binding. We constructed two Fab fragments that had the canonical Hib PS combining site VH-VL configuration but that had either V3-23*01 or V3-23*03. Radioantigen binding assay showed that on a concentration basis the V3-23*03 Fab was 20-fold more effective in binding Hib PS than the V3-23*01 Fab. The V3-23*03 Fab was 4-fold more effective than the V3-23*01 Fab in mediating facilitated bactericidal activity against Hib organisms. These findings identify a functional consequence of V3-23 allelism, and suggest that utilization of the V3-23*03 gene in the human Hib PS repertoire would generate canonical antibodies with higher affinity and protective efficacy than canonical antibodies utilizing V3-23*01. Thus, IGH V gene allelic variation has the potential to impact the generation of protective immunity to Hib.

A high degree of natural immunologic priming to the capsular polysaccharide may not prevent Haemophilus influenzae type b meningitis.

A current debate is whether the immunologic priming of infants with Haemophilus influenzae type b (Hib) conjugate vaccines would be protective in the absence of circulating antibody to the capsular polysaccharide (PS). Data from the prevaccine era on the PS antibody responses of older children to Hib meningitis may be informative on this issue. PS antibody was assayed by radioantigen binding in sera taken in the first month postadmission in 47 children ages 2 to 136 months with culture-proved Hib meningitis. Sera obtained on admission had very low antibody concentrations, and the subsequent response during convalescence was age-dependent. The major finding is that some patients, including 10 of 11 children older than 2 years, had substantial antibody elevations within a few days of admission, increases resembling the response to PS vaccine in infants primed with PS-protein conjugate vaccines. In this group of patients with Hib meningitis, natural priming did not prevent infection. Hib may have the ability to invade despite the capacity for a vigorous antibody response.

Characterization of immune response as an indicator of Haemophilus influenzae type b vaccine efficacy.

Quantitation of antibodies to Haemophilus influenzae type b (Hib) polysaccharide has been an active area of investigation associated with the development of polysaccharide and subsequently polysaccharide-protein conjugate vaccines. These clinical studies indicate that there are several serologic parameters associated with Hib vaccine efficacy in infants. Efficacious vaccines elicit polysaccharide-specific antibodies in infants; they prime the immune system for an anamnestic response; the immune response is long-lived through the period of greatest risk for disease; and the elicited antibodies have functional activity as demonstrated in bactericidal and opsonophagocytic assays or protection in an infant rat challenge model. The immune response to different Hib vaccines varies both quantitatively and qualitatively. With the introduction of routine Hib vaccine immunization, vaccine performance can rely on these serologic parameters. Quantitative serologic assays, the radio-antigen binding and enzyme-linked immunosorbent assays, have been developed and standardized. The quality of the antigen as well as optimization of all assay steps and reagents are key to ensuring specific and reproducible antibody quantitation.

A simple and rapid microSepharose assay for GAD65 and ICA512 autoantibodies in diabetes

GAD65Ab and ICA512Ab are strongly associated with insulin-dependent (Type 1) diabetes mellitus. A novel, simple radio-antigen binding assay with microSepharose conjugated with monoclonal antibodies specific for human immunoglobulin light chains was developed to provide diagnostic sensitivity and specificity of GAD65Ab and ICA512Ab for Type 1 diabetes. The Receiver Operating Characteristic (ROC) curve was used to determine the upper level of Normal in 583 new onset Type 1 diabetic patients and in 829 matched controls. The sensitivity of GAD65Ab and ICA512Ab was 66% (384/583) and 41% (211/520), respectively, and the diagnostic specificity was 96% for both autoantibodies. Levels, but not frequency, of GAD65Ab were higher among female Type 1 diabetes patients, whereas ICA512Ab levels did not differ between males and females. Positivity for GAD65Ab, ICA512Ab or both showed a sensitivity of 74% and a specificity of 92% for Type 1 diabetes. This simple, one-step centrifugation, high-capacity radio-antigen binding assay has a high precision and reproducibility to accurately detect both GAD65Ab and ICA512Ab. This assay should also prove useful in other autoantibody assays against conformation-sensitive autoantigens.

Immunity to Haemophilus influenzae type b after reimmunization with oligosaccharide CRM197 conjugate vaccine in children with human immunodeficiency virus infection.

Immunity to Haemophilus influenzae type b after reimmunization with oligosaccharide CRM197 conjugate vaccine in children with human immunodeficiency virus infection.

An ELISA employing a Haemophilus influenzae type b oligosaccharide-human serum albumin conjugate correlates with the radioantigen binding assay

An ELISA measuring total Ig antibodies to the capsular polysaccharide of Haemophilus influenzae type b (HbPs) in human sera using an antigen composed of Haemophilus b oligosaccharides conjugated to human serum albumin (HbO-HA) was shown to have an excellent correlation to the radioantigen binding assay (RABA). When 214 sera with different anti-HbPs levels were assayed for total Ig by HbO-HA ELISA and by RABA the correlation coefficient was 0.917 and the paired t test p values was 0.575. Use of competitive ELISA employing soluble HbPs, HbO-HA and human albumin as competitors, showed that the HbO-HA ELISA was specific for antibodies to HbPs. The HbO-HA ELISA yielded reproducible results both within and between laboratories. The HbO-HA ELISA can also be used to determine the isotype of anti-HbPs antibodies by using isotype specific enzyme conjugates. The sum of the IgG, IgA and IgM HbO-HA ELISA results had excellent correlation to the RABA results (correlation coefficient = 0.976). Thus, the HbO-HA ELISA can be substituted for the classical RABA and also be utilized for quantitating the isotype of the anti-HbPs antibodies.

Effect of oligosaccharide chain length, exposed terminal group, and hapten loading on the antibody response of human adults and infants to vaccines consisting of Haemophilus influenzae type b capsular antigen unterminally coupled to the diphtheria protein CRM197.

Vaccines consisting of oligosaccharide (OS) derived from Haemophilus influenzae type b capsular polysaccharide and conjugated to carrier proteins had been shown capable of eliciting memory-type capsular polysaccharide of H. influenza type b antibody responses in human infants, but the structural variables governing immunogenicity were not defined. Here a series of conjugates were made with the diphtheria protein CRM197 and with uniterminally coupled OS haptens that varied in chain length, exposed terminal residue, or multiplicity of loading as defined by ribose/protein ratio. Adults were given a single injection, 1-yr-old infants were given a two-injection sequence, and capsular polysaccharide of H. influenzae type b antibody responses were assessed by radioantigen binding. Vaccines C-4r, C-6r, and C-12r, in which ribitol-ended OS of mean length 4, 6, or 12 repeat units were coupled at low hapten loading, were about equally immunogenic (geometric means 2 to 5 micrograms/ml in infants, 5 to 9 micrograms/ml in adults). Vaccine C7p was made with a higher loading of OS having mean length 7 repeat units and having mainly phosphate monoester at the exposed termini Vaccine C-7R was made from a portion of C-7p by enzymatic removal of most of the terminal phosphates. Compared to the C-4r, C-6r, and C-12r series, vaccines C-7p and C-7R induced geometric means about 10-fold higher in adults and 20-fold higher in infants. Thus OS chain length (in the range studied) and exposed terminus are less critical variables in this system than the extent of hapten loading.

Haemophilus influenzae b polysaccharide revaccination: a continued role for the unconjugated vaccine.

In a study of the issue of revaccination with Haemophilus influenzae b polysaccharide vaccine, 36 children who had been vaccinated with polysaccharide vaccine at age 18 to 20 months were stratified according to their 3-week postvaccination anticapsular antibody concentration determined by radioantigen binding assay, then sequentially assigned to one of three groups so as to have comparable distributions of peak antibody concentrations. These children were revaccinated with polysaccharide vaccine at 24 to 27, 30 to 33, or 36 to 39 months of age. The post-reimmunization geometric mean antibody concentrations in these three groups were 3.1, 3.0 and 7.8 micrograms/ml, respectively, and the percentages rising to greater than 1 microgram/ml were 75, 80 and 93%. The geometric means and antibody response rates were nearly identical to age-matched control groups not previously vaccinated. Thus there was no evidence of priming (memory induction) or of tolerance induction by the early (18 months old) primary vaccination.

Variable quantitation of Haemophilus influenzae type b anticapsular antibody by radioantigen binding assay

The measurement of antibody to Haemophilus influenzae type b capsular polysaccharide is important in the study of natural immunity and in the immunogenicity evaluation of H. influenzae type b vaccines. Several radioantigen binding assays (RABA) have been developed to measure H. influenzae type b anticapsular antibody, but recent immunogenicity data obtained with structurally similar vaccines suggest major differences in antibody quantitation in different laboratories. To evaluate interlaboratory variability in the measurement of anticapsular antibody levels, we blindly evaluated a sample of 40 pre- and postimmunization sera by eight RABAs in different laboratories. Evaluation of RABA methods revealed differences in polysaccharide antigens, radiolabeling methods, concentration and volume of antigen and antibody, and other assay methods. The reported results of assays varied significantly between laboratories (up to sixfold differences in geometric means), in part because of differences in assay sensitivity and different proportions of samples having undetectable levels of antibody (0 to 65% of specimens with undetectable levels). After standardizing the limit of sensitivity for all assays (0.125 microgram/ml), the results of all combinations of paired analyses of RABA assays correlated well (r = 0.88 to 0.99) but the geometric mean levels still varied as much as twofold. For individual sera, the differences between paired assays often were substantial (P less than or equal to 0.0001, paired t test), with some results varying as much as 64-fold. Differences were greatest for lower levels of antibody. There was good comparability and interlaboratory reproducibility of some assays but not of others. Intrinsic or extrinsic labeling of the antigen was not a major determinant of comparability. In most instances, the current variation in the quantitation of antibody levels by these assays precludes interassay comparisons. A standardized measurement of antibody needs to be developed to adequately compare results between different H. influenzae type b immunogenicity studies.

MUCOSAL AND SERUM ANTIBODY (Ab) RESPONSES TO PARENTERAL IMMUNIZATION OF 1-YR-OLD INFANTS WITH H. INFLUENZAE B CAPSULAR (PRP) ANTIGEN CONJUGATED TO A DIPH IHERIA PROTEIN CARRIER

Seventeen healthy infants age 9-15 mos received a 2-injection sequence of a PRP oligosaccharide conjugate vaccine. Sera and samples of nasal mucus obtained using saline washes (NW) were taken before the 1° and 2° injections and 1 mo and 6 mos after the 2°. A rise in total serum anti-PRP Ab [determined by radio-antigen binding (RAB)] occurred in 15 of 17. NW anti-PRP Ab developed in 6 of 17 as detected by RAB and in 8 of 17 as detected by an Ig class-specific immunosorbent assay (ELISA); these ELIS.A responses occurred as IgG (7 of 17), IgM (5 of 17), and IgA (4 of 17). Ab responses in NW were related to the magnitude of the serum Ab response: in 8 infants with post-2° serum Ab of <2 μg/ml, NW responses were detected in 0 of 8 by RAB and 1 of 8 by ELISA; in 9 infants with serum Ab >2 μg/ml, NW responses were detected in 6 of 9 by RAB and 7 of 9 by ELISA. At 6 mos after 2° injection, anti-PRP Ab was still detectable in NW in 6 of 16 subjects. Thus, a mucosal Ab component may contribute to the protective potential of conjugate vaccines.

EFFECT OF OLIGOSACCHARIDE (OS) CHAIN LENGTH, TERMINAL GROUP, AND HAPTEN LOADING ON THE SERUM ANTIBODY (Ab) RESPONSE OF 1-YR-OLD INFANTS TO H. INFLUENZAE B CAPSULAR ANTIGEN (PRP) CONJUGATED TO THE DIPHTHERIA PROTEIN CRM197

Vaccines consisting of OS derived from PRP and conjugated by reductive amination to the nontoxic diphtheria mutant toxin CRM 197 (C) were previously shown to elicit memory-type serum anti-PRP responses in human infants. Here we seek to define structural variables that control the infant's response. In the coupled OS, the nonreducing (and hence exposed) terminal group can be ribitol (r), ribose (R), or phosphate (p). A series of r-terminal OS of mean length 4, 6, or 12 repeat units were coupled to C at ca. 10 ribose/protein, and OS of 12 repeat units terminating in R or p were coupled at ca. 30 ribose/protein. A 2-injection sequence was given to healthy infants aged 9-15 mo (groups of 8-10). Anti-PRP Ab was assayed by radioantigen binding; at 1 mo after the 2° the geometric means (GM) and 95% confidence intervals (CI) were:Series C4r, C6r, C12r (all at low hapten loading) induced similar titers regardless of OS chain length. C12R and C12p (both with high loadings) induced similar titers that were >10-fold the r series.

POLYRIBOSYLPHOSPHATE (PRP) REVACCINATION TO PREVENT INVASIVE HAEMOPHILUS INFLUENZAE B (HIB) IN CHILDREN FIRST IMMUNIZED AT 18–20 MOS OF AGE

PRP vaccination (vax) of 18 mo old children at high risk for contracting invasive Hib disease is recommended by the ACIP. The timing of revax of such children has not been studied. Thus far, 30 children have been stratified according to their postvax anti-PRP antibody (Ab) level (determined by radioantigen binding assay) following PRP vax at 18-20 mo then sequentially assigned to 3 groups with comparable distribution of Ab levels to receive revax at 24, 30 or 36 mo of age; each group had children who had failed to respond to primary vax with PRP (<30% rise in Ab titer). The geometric mean titer +/- the standard deviation, the % responding to revax (>30% rise in Ab titer), and the % with presumed protective postvax Ab level (defined as >1.0 μg/ml)for each vaccine group were as follows:GMT's rose in both the 24-27 mo and the 30-33 mo groups; proportional rises were, as expected, more frequent in the latter. Adequate responses to revax were seen in some children who initially appeared to be “intrinsically” unresponsive to PRP vax.

HAEMOPHILUS INFLUENZAE TYPE B (Hib) CAPSULAR ANTIGEN (PRP) CONJUGATE VACCINE INDUCES ANTI-PRP ANTIBODY (Ab) IN CHILDREN FOLLOWING Hib INFECTIONS

PRP vaccine is a poor immunogen when administered to some children following Hib infection or their sibs. To date, we have immunized 18 patients (mean age 19 mos., range 7-45 mos; 14 meningitis) with PRP-diphtheria toxoid conjugate vaccine (PRP-D) containing 20 μg PRP. Children were inoculated 1-8 mos. (15 ≤ 2 mos.) post illness with 1 or 2 doses of PRP-D. Serum was obtained for anti-PRP Ab (radioantigen binding assay at Connaught Laboratories) prior to and 1 month post vaccine. Serology is available on 13 patients, 6 of whom were Ab negative ( < 0.012 μg/ml) pre-vac (4 were ≤ 16 mos. when immunized). Anti-PRP rises developed in 11/13 patients and 5/6 pre-vac Ab negative patients. The pre- and post-geometric mean titers were 0.13 and 2.0 μg/ml, respectively (p < 0.001 for log transformed data). One patient (pre-vac Ab neg), a non-responder to routine PRP given at 24 mos., developed 0.28 ug/ml anti-PRP Ab following PRP-D at 26 mos. Anti-PRP Ab developed in 3/3 sibs, (8, 11, and 24 mos.), 2 of whom were Ab negative pre-vac. Our data indicate that young children who may not have developed anti-PRP within 1 or 2 mos. of Hib infection usually respond to 1 or more doses of PRP-D and suggest that children at increased risk for Hib disease will likely respond to PRP conjugate vaccines.

Serum opsonic activity after immunization of adults with Haemophilus influenzae type b-diphtheria toxoid conjugate vaccine

We measured the uptake of radiolabeled Haemophilus influenzae type b by human polymorphonuclear leukocytes. Haemophilus influenzae type b strains were preopsonized in individual sera from six adults immunized with type b polysaccharide vaccine (PRP) or six adults immunized with PRP covalently coupled to diphtheria toxoid (PRP-D vaccine). Serum was heat inactivated before use, and exogenous human complement was added. Of the 12 subjects, 3 had high levels of opsonic activity (greater than 40% of immune control) in their preimmunization serum. This activity did not correlate with the concentrations of anti-PRP antibody and was unaffected by absorption of anti-PRP antibody. At 1 month after vaccination, the serum of PRP-D subjects had higher opsonic activity than that from subjects who received PRP (5% serum, mean PRP-D = 86%, mean PRP = 53%, P = 0.001). After 12 months, both groups had higher serum opsonic activity than before immunization (P less than 0.02), but there was no difference between the two groups (mean PRP-D = 48%, mean PRP = 51%). In postimmunization serum, opsonic activity induced by PRP-D or PRP vaccines correlated directly with anti-PRP antibody concentrations as measured by a radioantigen binding assay. We conclude that both vaccines induce opsonic activity, opsonic activity induced by immunization of adults correlates well with the concentration of anti-PRP antibody achieved, and in preimmune sera with low concentrations of anti-PRP antibody, factors other than anti-PRP antibody contribute to opsonic activity.

1103 IgG1 ANTIBODY RESPONSES OF CHILDREN AND ADULTS IMMUNIZED WITH HAEMOPHILUS INFLUENZAE TYPE b (Hib) POLYSACCHARIDE VACCINE (PRP)

IgG was purified by gel filtration of sera from 3 adults immunized with PRP. The ratio of IgGl to IgG2 anti-PRP was determined in affinity purified anti-PRP. We also determined IgG1 anti-PRP by measuring anti-PRP radioantigen binding activity before and after column immunoabsorption of IgGl employing a highly specific monoclonal prepared locally (HG-11). 38–46% of the post-vaccine serum IgG anti-PRP was of the IgGl subclass. An ELISA was then used to measure IgG and IgGl anti-PRP antibody in sera from 16 normal adults; and from 97 children 12 to 83 mos of age and 8 adults immunized with PRP. Only 2 of 16 unimmunized adults had IgGl liters >1:49. The IgGl anti-PRP antibody 2 mo after immunization was age-dependent.12 of 35 children had high IgG antiPRP responses (post vaccine titers >1:400) but no detectable IgGl antibody (IgGl titer <1:49). Thus, IgGl is an important component of the IgG antiPRP response of some but not all immunized subjects. In addition, although serum IgGl reaches adult concentrations in the first year of life, most children of this age cannot respond to immunization with IgGl anti-PRP whereas many older children can.

972 PLACENTAL TRANSFER OF ANTIBODY: CORRELATION WITH SUBCLASS COMPOSITION

Previous studies indicate that IgGl concentrations are higher in cord than in maternal sera whereas IgG2 is thought to cross the placenta less well. Transfer of specific subclasses has not been related to the concentrations of specific antibodies of differing subclass composition. Therefore, we measured the concentration of IgGl and IgG2 and of IgG-anti-tetanus toxoid (TT), IgG-anti-H.influenzae, type b capsular polysaccharide (PEP) and anti-group A streptococcal carbohydrate (GAG) in 16 paired maternal (M) and cord (I) sera. Measurement of IgGl and IgG2 was performed by a competetive radioantigen binding assay. Measurement of antl—TT, anti—PHP and anti—GAC was accomplished with an IgG-solM phase RIA, an IgG—ELISA, and a radioactive antigen binding assay, respectively. Previous studies have shown that anti-TT of adults is 90% IgGl, anti-PRP is 40% IgGl, 60% IgG2, and anti-GAC is 90% IgG2. By paired t-test, mean IgGl was higher in I than M sera(p<.001), mean I/M=1.78±.59. IgG2 was not different inland M sera, mean I/M=0.98. Anti-TT was higher in I than M(p=0.002), mean I/M=2.228±1.17 and anti-GAC was not different mean I/M=1.07±.63. Anti-PRP was lower in I than M sera (p=0.05), mean I/M=0.88. Thus, IgGl but not IgG2 is selectively transferred across the placenta. As a result, anti-TT (predominantly IgGl) is higher in infant than maternal sera, whereas anti-GAC (predominantly IgG2) is not. The reason for lower concentrations of anti-PRP in I than M sera is unclear.

1030 PASSIVE IMMUNIZATION OF INFANTS AGAINST HAEMOPHILUS INFLUENZAE TYPE B (HIB) BY MATERNAL VACCINATION

Prior to a planned conception, 24 women were immunized with purified HIB capsular polysaccharide to attempt to extend their offspring's period of passive antibody protection against systemic HIB disease. All women had an antibody rise. Geometric mean titer prior to immunization was 1.2 μg/ml and at 1, 7-10, and 19-27 months after immunization was 35 μg/ml, 23 μg/ml, and 16 μg/ml respectively as measured by radioantigen binding. Most post-immunization antibody was of the IgG class as determined by an enzyme-linked immunosorbent assay. However, there was a wide range of post-immunization titers (2.7-310 μg/ml at 7-10 months post-immunization). The low magnitude antibody responders also had low IgG antibody levels that were not boostable. The offspring of 6 women, born 11½ to 17½ months after immunization, had antibody titers markedly higher than those of age-matched infants of unvaccinated women. Their titers persisted above the estimated protective level through 6-12 months of age. Four infants, all born to low-responder women, had titers similar to natural antibody titers. These results suggest that the interval of passive antibody protection can be extended. This approach may be especially useful in populations highly susceptible to HIB and may be applicable to the prevention of other childhood and neonatal infections.

958 PRODUCTION OF MUCOSAL ANTIBODY FOLLOWING PARENTERAL VACCINATION WITH HAEMOPHILUS INFLUENZAE TYPE B CAPSULAR POLYSACCHARIDE (PRP)

Anti-PRP antibody (ab) in sera and mucosal secretions from 5 children (>18 mo of age) and 4 adults was quantified by radioantigen binding, before and 3 weeks after immunization with 15 μg of PRP. Total IgA in secretions was quantified by laser-nephelometry. 8 of 9 subjects had detectable serum ab prior to vaccination and all produced an increase (children 4.5-640X, adults 3.3-48X) in serum ab following immunization. Prior to vaccination 3 of 5 children had detectable ab in nasal secretions (519-1828 ng ab/mg IgA). 2 of the 3 children with preimmunization nasal ab had an increase of ab following vaccination (2.3 & 7.7X) and 1 had no response. Both children with nondetectable preimmunization nasal ab levels had detectable levels following vaccination (1026 & 2093 ng ab/mg IgA). All adults had ab in nasal mucus (120-1917 ng ab/mg IgA) and 3 of 4 adults had ab in parotid saliva (127-241 ng ab/mg IgA) prior to vaccination. 3 of 4 adults had an increase (1.6-6.2X) and 1 had a transient decrease of nasal ab and all adults had an increase in salivary ab (3-8.5X in those with detectable preimmunization levels and to 1419 ng ab/mg IgA in the other subject) following vaccination. The subjects who did not have an increase in mucosal ab had the highest preimmunization mucosal ab titers. We conclude that, unlike vaccination with many other non-replicating antigens, PRP immunization often increases mucosal as well as serum ab.