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.

An analytical model applied to a multicenter pneumococcal enzyme-linked immunosorbent assay study.

Pneumococcal conjugate vaccines will eventually be licensed after favorable results from phase III efficacy trials. After licensure of a conjugate vaccine for invasive pneumococcal disease in infants, new conjugate vaccines will likely be licensed primarily on the basis of immunogenicity data rather than clinical efficacy. Analytical methods must therefore be developed, evaluated, and validated to compare immunogenicity results accurately within and between laboratories for different vaccines. At present no analytical technique is uniformly accepted and used in vaccine evaluation studies to determine the acceptable level of agreement between a laboratory result and the assigned value for a given serum sample. This multicenter study describes the magnitude of agreement among 12 laboratories quantifying an identical series of 48 pneumococcal serum specimens from 24 individuals (quality-control sera) by a consensus immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) developed for this study. After provisional or trial antibody concentrations were assigned to the quality-control serum samples for this study, four methods for comparison of a series of laboratory-determined values with the assigned concentrations were evaluated. The percent error between assigned values and laboratory-determined concentrations proved to be the most informative of the four methods. We present guidelines that a laboratory may follow to analyze a series of quality-control sera to determine if it can reproduce the assigned antibody concentrations within an acceptable level of tolerance. While this study focused on a pneumococcal IgG ELISA, the methods that we describe are easily generalizable to other immunological assays.

Primary human immune response to Neisseria meningitidis serogroup C in interleukin-12-treated severe combined immunodeficient mice engrafted with human peripheral blood lymphocytes.

Lack of primary immune response in severe combined immunodeficient (SCID) mice engrafted with human peripheral blood lymphocytes (hu-PBL) has limited the applicability of this model. Use of human cytokines, in particular interleukin (IL)-12, was studied in the hu-PBL-SCID model. SCID mice were treated with IL-12 and reconstituted with hu-PBL in T replacement factor. The hu-PBL-SCID mice were immunized with serogroup C meningococcal polysaccharide (MCPS). The MCPS-specific antibody response was determined by ELISA. Thirteen of the 15 immunized, IL-12-treated hu-PBL-SCID mice demonstrated a primary human antibody response to MCPS ranging from 0.25 to 3.3 microg/mL, while no MCPS-specific antibody response was detectable in the 18 controls. Expression of cross-reactive idiotypic markers found on human anti-MCPS antibodies in the immunized hu-PBL-SCID mice was similar to that observed in immunized volunteers.

Determination of parallelism and nonparallelism in bioassay dilution curves.

There is a lack of consensus among investigators who use a variety of immunoassay techniques (e.g., enzyme-linked immunosorbent assay [ELISA] and radioimmunoassay) regarding the protocols for describing and forming standard reference or calibration curves and interpolating serum antibody concentrations. This confounds the issue of detecting the presence or absence of parallelism between standard reference serum and serially diluted serum sample curves. These curves must be parallel to support the assumption that the antibody-binding characteristics are similar enough to allow the determination of antibody levels in the diluted serum sample. There is no universal and widely adopted strategy for assessing parallelism in bioassays, and without an assurance of parallelism, investigators are not able to calculate reliable estimates for antibody concentrations in serum samples. Furthermore, single-point (dilution) serum assays do not provide information related to parallelism and nonparallelism, and this, too, may lead to considerable error when calculating antibody concentrations. When assay methodology, technique, and precision improve to the extent that standard reference serum and serially diluted serum sample curves are fit with little error, standard analysis of variance techniques are overly sensitive to negligible departures from parallelism. We present a series of guidelines that compose a protocol for assessing parallelism between bioassay dilution curves that are applicable to data derived from ELISAs. These criteria should be applicable, with minor modifications, to most immunoassay experimental situations and, most importantly, are not dependent on the mathematical model used to form the standard reference curve. These guidelines have evolved in our laboratories over the past 4 years during the performance of thousands of ELISAs for antibodies to the capsular polysaccharides of Neisseria meningitidis groups A and C and Haemophilus influenzae type b.

Multicenter comparison of Neisseria meningitidis serogroup C anti-capsular polysaccharide antibody levels measured by a standardized enzyme-linked immunosorbent assay.

A standardized enzyme-linked immunosorbent assay (ELISA) was used by 11 laboratories to measure levels of total serum antibody to Neisseria meningitidis serogroup C capsular polysaccharide in 16 unpaired pre- and postvaccination serum samples. Twelve serum samples were from adults, and four were from children aged 2, 3, 5, and 9. The between-laboratory coefficient of variation for pre- and postvaccination sera ranged from 16 to 59% and 11 to 21%, respectively. The average percent difference (absolute value) from the between-laboratory means for all prevaccination sera measured by each laboratory was 24%, whereas the average percent difference was 13% for all postvaccination sera. A postvaccination quality control serum was diluted three times to give optical densities on the high, middle, and low portions of the standard reference curve. The three dilutions were assayed by the 11 laboratories a total of 241 times and yielded an overall coefficient of variation of 20%. Antibody-binding inhibition curves showed that the standardized ELISA was specific for N. meningitidis serogroup C capsular polysaccharide antibody. Fifty percent inhibition of seven serum samples was obtained after reaction with an average concentration of 0.9 micrograms of meningococcal serogroup C polysaccharide per ml; an average of 93% inhibition was obtained with 50 micrograms of polysaccharide per ml. The acceptance and use of this standardized ELISA will reduce between-laboratory assay variability and ensure a more accurate and reproducible assessment of immunogenicity for vaccines under development.