Patricia Holder

Specific, sensitive, and quantitative enzyme-linked immunosorbent assay for human immunoglobulin G antibodies to anthrax toxin protective antigen.

The bioterrorism-associated human anthrax epidemic in the fall of 2001 highlighted the need for a sensitive, reproducible, and specific laboratory test for the confirmatory diagnosis of human anthrax. The Centers for Disease Control and Prevention developed, optimized, and rapidly qualified an enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) antibodies to Bacillus anthracis protective antigen (PA) in human serum. The qualified ELISA had a minimum detection limit of 0.06 µg/mL, a reliable lower limit of detection of 0.09 µg/mL, and a lower limit of quantification in undiluted serum specimens of 3.0 µg/mL anti-PA IgG. The diagnostic sensitivity of the assay was 97.8%, and the diagnostic specificity was 94.2%. A competitive inhibition anti-PA IgG ELISA was also developed to enhance diagnostic specificity to 100%. The anti-PA ELISAs proved valuable for the confirmation of cases of cutaneous and inhalational anthrax and evaluation of patients in whom the diagnosis of anthrax was being considered.

Randomized trial of the quantitative and functional antibody responses to a 7-valent pneumococcal conjugate vaccine and/or 23-valent polysaccharide vaccine among HIV-infected adults.

In a double-blinded, randomized trial, human immunodeficiency virus (HIV)-infected adults with > or = 200 CD4 cells/microl received placebo (PL), 7-valent conjugate, or 23-valent pneumococcal polysaccharide (PS) vaccine in one of the following two-dose combinations given 8 weeks apart: conjugate-conjugate, conjugate-polysaccharide, placebo-polysaccharide, placebo-placebo. A total of 67 persons completed the study. Neither significant increases in HIV viral load nor severe adverse reactions occurred in any group. After controlling for confounders, when compared with persons receiving placebo-polysaccharide, persons receiving conjugate-conjugate and conjugate-polysaccharide had higher antibody concentrations (serotypes 4, 6B, 9V and serotype 23F, respectively) and opsonophagocytic titers (functional antibody assay, serotypes 9V, 23F and serotypes 4, 6B, 9V, respectively) after the second dose (P<0.05). The second dose with either conjugate or polysaccharide following the first conjugate dose, however, produced no further increase in immune responses.

Specificity of the Antibody Response to the Pneumococcal Polysaccharide and Conjugate Vaccines in Human Immunodeficiency Virus-Infected Adults

ABSTRACT Nonspecific antibodies, which are thought to be nonprotective, have been shown to contribute a substantial proportion of the measured concentration in the standardized immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) for pneumococcal polysaccharide capsular antibodies. The presence of such antibodies in human immunodeficiency virus (HIV)-infected persons has not been evaluated. The amount of nonspecific antibodies is proportional to the reduction in IgG antibody concentration that occurs with serum absorption with the heterologous polysaccharide 22F. We measured the amount of nonspecific antibodies before and after vaccination with the pneumococcal conjugate vaccine (PCV; n = 33) or the pneumococcal polysaccharide vaccine (PPV; n = 34) in HIV-infected adults with CD4 counts of ≥200 cells/mm3. Blood was drawn before and 2 months after vaccination. For prevaccination sera, we found a substantial amount of nonspecific antibodies for serotypes 4, 6B, 9V, and 23F (23 to 47% of measured IgG concentration), but not for serotype 14. There tended to be proportionately less nonspecific antibodies in postvaccine sera than prevaccine sera for PCV, but not for PPV. Subjects with a low HIV viral load (≤400 copies/ml) had proportionately more nonspecific antibodies than those with higher viral load before and after both vaccines. After 22F absorption, the geometric mean concentrations of antibodies were significantly higher post-PCV than post-PPV for the high viral load group for all five serotypes, but for no serotypes in the low viral load group. These findings confirm that absorption with a heterologous pneumococcal polysaccharide (e.g., 22F) is necessary to remove nonspecific antibodies in a standardized IgG ELISA for pneumococcal capsular antibodies in HIV-infected adults.

Avidity determinations for Haemophilus influenzae type b anti-polyribosylribitol phosphate antibodies

ABSTRACT Determination of antibody avidity measurements can be difficult in human serum depending on the population evaluated. We evaluated three approaches for the determination of antibody avidity for immunoglobulin G (IgG). These approaches were (i) elution of bound antibody with increasing concentrations of a chaotropic agent using a single serum dilution, (ii) binding interference of multiple serum dilutions by a single concentration of a chaotrope, and (iii) elution of multiple serum dilutions by a single concentration of a chaotrope. Parameters that affect the determination of avidity measurements and their limitations were evaluated with pre- and post-Haemophilus influenzae type b conjugate vaccination sera (n = 89). We determined that elution of low-avidity antibodies present in multiple dilutions of the serum sample by a single concentration of a chaotrope (0.15 M sodium thiocyanate [NaSCN]) was optimal for the determination of avidity measurements throughout a wide range of IgG concentrations (0.94 to 304.6 μg/ml). The percent reduction in concentration as determined by the elution assay with 0.15 M NaSCN correlated highly (r = 0.84) with weighted averages obtained by an elution assay with multiple solutions of NaSCN. The correlation (r = 0.57) between elution and binding interference, when a single concentration of a chaotrope was used, was lower than the correlation between the two elution methods (r = 0.84). We found that the serum dilution, the heterogeneity of the antibody population, and the concentration of the chaotrope were the primary variables affecting avidity determinations. In this study, we present multiple analysis methods depending on the methodology used. We also present the factors that affect the analysis of avidity determinations given the polyclonal nature of human sera. This experimental approach should benefit the evaluation of similar antibodies induced by other bacterial polysaccharide vaccines.

Measurement of Serum Bactericidal Activity Specific for Haemophilus influenzae Type b by Using a Chromogenic and Fluorescent Metabolic Indicator

ABSTRACT We evaluated alamarBlue as a metabolic indicator in a standardized assay for the measurement of serum bactericidal activity (SBA) to Haemophilus influenzae type b (Hib) using sera containing natural and vaccine-induced anticapsular (polyribosylribitol phosphate) antibodies. SBA assays with a colorimetric and a fluorometric end point in the presence of alamarBlue were developed and compared to a standard SBA assay, where colony counts are performed to determine the titer (12). A colorimetric end point required a spectrophotometer, whereas a fluorometric end point required a fluorometer. Prevaccination sera (n = 27) and postvaccination sera (n = 13) were tested by all three methodologies, and the SBA titers obtained in the presence of alamarBlue were compared to those from the standard method. Both the colorimetric and the fluorometric SBA titers were significantly correlated (r = 0.87 and r = 0.95, respectively) with those of the standard assay (≥50% killing as the SBA titer end point), and titers were not significantly different when compared to those of the standard assay (P > 0.68). However, the fluorometric end point had superior performance and ease of titer determination compared to the colorimetric end point (95 versus 87% of SBA titers were within 2 dilutions of the standard titer). Hib SBA assays with alamarBlue are reproducible, faster (same-day assay), and easier to perform than the standardized assay, which requires manual or automated colony counts. These semiautomated methodologies result in increased sample throughput and collection of data in digital formats that can be exported to data analysis programs for determination of SBA titers.

Immunogenicity and reactogenicity to Haemophilus influenzae type B (Hib) conjugate vaccine among rural Alaska adults.

Background: Despite routine vaccination and declining disease rates, Haemophilusinfluenzae type b (Hib) invasive disease still occurs in rural Alaska. Colonization studiesindicate persistent transmission of Hib among village residents, including adults. As partof a project to eliminate Hib carriage in 3 rural villages, we evaluated a cohort of Alaskaadults for antibody response and reactogenicity to a single dose of Hib conjugate vaccine(HbOC).Methods: 75 previously unvaccinated, randomly-selected adults in one village received asingle dose of HbOC vaccine and completed a side-effects diary. Sera and oropharyngealspecimens were collected at baseline, 2 months and 1 year.Results: No participants were colonized with Hib or reported serious side-effects. Atbaseline, 97% of adults had IgG anti-PRP concentrations > 0.15 µg/mL, 69% >1 µg/mL,and 28% > 5 µg/mL. Two months post-vaccination, 100% of participants hadconcentrations > 0.15 µg/mL, 93% >1 µg/mL, and 86% >5 µg/mL. After 1 year, 98% hadIgG anti-PRP concentrations > 0.15 µg/mL, 86% > 1 µg/mL, and 67% >5 µg/mL. GMCswere 1.9, 33.3 and 8.4 µg/mL at baseline, 2 months and 1 year post-vaccine, respectively(p

Immunologic Response to Haemophilus influenzae Type b (Hib) Conjugate Vaccine and Risk Factors for Carriage among Hib Carriers and Noncarriers in Southwestern Alaska

ABSTRACT Continued Haemophilus influenzae type b (Hib) carriage in rural Alaska contributes to the ongoing risk of invasive disease. Community-wide Hib carriage surveys were conducted in three villages in southwestern Alaska. Sixteen carriers and 32 age- and village-matched controls were enrolled and were vaccinated with Hib oligosaccharide-CRM197 conjugate vaccine. Serum immunoglobulin G (IgG) concentration, antibody avidity, and serum bactericidal activity (SBA) were measured prior to Hib vaccination and 2 and 12 months after vaccination. We identified no demographic or behavioral factors associated with Hib colonization. Prior to vaccination, Hib carriers had a higher IgG geometric mean concentration than controls did (8.2 versus 1.6 μg/ml; P < 0.001) and a higher SBA geometric mean titer (7,132 versus 1,235; P = 0.006). Both groups responded to vaccination with increased IgG and SBA. These data illustrate the role of Hib colonization as an immunizing event and show that Hib carriers in communities with ongoing transmission have no evidence of reduced immune responsiveness that may have put them at risk for colonization.

Concomitant administration of recombinant PsaA and PCV7 reduces Streptococcus pneumoniae serotype 19A colonization in a murine model.

A murine colonization model was used to determine the effect of co-administering 7-valent polysaccharide-protein conjugate vaccine and pneumococcal surface adhesin A. Mice were challenged intranasally with either PCV7 serotypes, 4 or 14, or a non-PCV7 serotype, 19A. Post-challenge samples were evaluated for IgG antibody levels, opsonophagocytic activity, and nasopharyngeal colonization. No interference was observed between immune responses from the concomitant and individual immunizations. Concomitant immunizations reduced carriage for tested serotypes; largest reduction was observed for 19A. From these mouse studies, co-administering pneumococcal antigens appear to expand coverage and reduce colonization against a non-PCV7 serotype without inhibiting immunogenicity to other serotypes.

Pneumococcal antibodies in a child with type 14 pneumococcal conjugate vaccine failure

We measured the concentration, opsonic activity, and avidity of serotype-specific serum antibodies in a pneumococcal conjugate vaccine (PnCRM7) efficacy trial participant who contracted serotype 14 pneumococcal bacteremia following dose 3 of PnCRM7. Controls included 18 PnCRM7- and 10 MnCC-vaccinated children without invasive pneumococcal disease (IPD). The child with vaccine failure had 4.98mcg/mL of serotype 14 antibodies 10 days before disease onset; these antibodies had greater opsonic activity and lower avidity than those of control PnCRM7 recipients. The child had no booster response to a fourth dose of PnCRM7 for most vaccine serotypes. We conclude that antibody concentration, functional activity and avidity do not predict individual protection against IPD, and immunological correlates of protection are only useful at the population level.

Fluorescent Multivalent Opsonophagocytic Assay for Measurement of Functional Antibodies to Streptococcus pneumoniae

ABSTRACT We developed fluorescent mono- and multivalent opsonophagocytic assays (fOPA and fmOPA, respectively) specific for seven Streptococcus pneumoniae serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F). Bacterial survival was quantitated with alamar blue, a fluorescent metabolic indicator. Both fOPA and fmOPA allow for determination of viability endpoints for up to seven serotypes with high levels of agreement to the reference method. The fmOPA eliminates colony counting, reduces serum volume, and produces results in 1 day.