Pneumococcal type 22f polysaccharide absorption improves the specificity of a pneumococcal-polysaccharide enzyme-linked immunosorbent assay

Abstract

The specificity of the immune response to the 23-valent pneumococcal-polysaccharide (PS) vaccine in healthy adults and to a pneumococcal conjugate vaccine in infants was examined by measuring immunoglobulin G (IgG) antibody titers by enzyme-linked immunosorbent assay (ELISA) and the opsonophagocytosis assay. ELISA measures total antipneumococcal IgG titers including the titers of functional and nonfunctional antibodies, while the opsonophagocytosis assay measures only functional-antibody titers. Twenty-four pairs of pre- and post-pneumococcal vaccination sera from adults were evaluated (ELISA) for levels of IgG antibodies against serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F. Twelve of the pairs were also examined (opsonophagocytosis assay) for their functional activities. The correlation coefficients between assay results for most types ranged from 0.75 to 0.90, but the correlation coefficient was only about 0.6 for serotypes 4 and 19F. The specificities of these antibodies were further examined by the use of competitive ELISA inhibition. A number of heterologous polysaccharides (types 11A, 12F, 15B, 22F, and 33A) were used as inhibitors. Most of the sera tested showed cross-reacting antibodies, in addition to those removed by pneumococcal C PS absorption. Our data suggest the presence of a common epitope that is found on most pneumococcal PS but that is not absorbed by purified C PS. Use of a heterologous pneumococcal PS (22F) to adsorb the antibodies to the common epitope increased the correlation between the IgG ELISA results and the opsonophagocytosis assay results. The correlation coefficient improve from 0.66 to 0.92 for type 4 and from 0.63 to 0.80 for type 19F. These common-epitope antibodies were largely absent in infants at 7 months of age, suggesting the carbohydrate nature of the epitope.

FIG. 1

Competitive ELISA inhibition of pneumococcal PS type 9V antibody with homologous (type 9V) and heterologous (type 11A, 12F, 15B, 22F, and 33F) pneumococcal PSs. The inhibitors were used at 10 μg/ml.

FIG. 2

Specificities of antibodies reactive with pneumococcal type 19F PS following C PS absorption. (A) Results for a serum sample from an adult that had 6.6 μg of type 19F IgG per ml and that was functional by opsonophagocytosis assay. Heterologous pneumococcal PSs of types 1, 4, and 6B were used as inhibitors. The results for the homologous pneumococcal PS, the type 19F PS, is indicated by the broken line. (B) Results for a serum sample from an adult that had 7.7 μg of IgG to type 19F per ml but that was not opsonic for type 19F. OD, optical density; Pn, pneumococcal PS type.

FIG. 3

Determination of optimal type 22F PS concentration for use as an inhibitor. Competitive inhibition with type 22F PS on antibody binding in a serum sample from a healthy adult was done for the seven pneumococcal PS types present in the seven-valent conjugate vaccine. Pn, pneumococcal PS type.

FIG. 4

Comparative effects of C PS absorption and combined C PS and type 22F PS absorption on a serum sample from an unimmunized adult for 11 pneumococcal types. The C PS and type 22F PS were used at 1 and 2 μg/ml, respectively.

FIG. 5

Percent reduction of IgG antibody binding in pre- and postvaccination sera following type 22F PS absorption. The mean percent reduction for 10 preimmunization serum samples and 18 postimmunization serum samples are given for 11 pneumococcal types. Percent reduction was calculated as ([IgG] with C PS absorption only − [IgG] with C PS and 22F absorption)/ [IgG] with C PS absorption only × 100.

FIG. 6

Percent reduction in antibody binding to the type 6B PS by using type 22F PS absorption in postimmunization sera from seven 7-month-old infants. Percent reduction was calculated as described in the legend to Fig. 5.

FIG. 7

Correlation of pneumococcal type 4 ELISA and opsonophagocytosis assay titers. (A) After C PS absorption only. (B) After C PS plus type 22F PS absorption. The quadrant lines were drawn at the 1.0-μg/ml cutoff for ELISA and the log₂ (1:4) cutoff for the opsonophagocytosis assay titer. The numbers shown are the number of serum samples in each quadrant. The r values were 0.66 before type 22F PS absorption and 0.92 after type 22F PS absorption.

FIG. 8

Correlation of pneumococcal type 19F ELISA and opsonophagocytosis assay titers. (A) After C PS absorption. (B) After C PS plus type 22F PS absorption. The quadrant lines were drawn at the 1.0 μg/ml cutoff for the ELISA titer and the log₂ (1:4) cutoff for the opsonophagocytosis assay titer. The r value was 0.63 before type 22F PS absorption and 0.80 after type 22F PS absorption.