Type III group B streptococcal polysaccharide induces antibodies that cross-react with Streptococcus pneumoniae type 14

Abstract

Covalent linkage of a bacterial polysaccharide to a protein greatly enhances the carbohydrate's immunogenicity and its binding to solid surfaces in immunoassays. These findings have spurred the development of glycoconjugate vaccines to prevent serious bacterial infections as well as the use of glycoconjugates as coating antigens in bioassays. We evaluated sera from women immunized with unconjugated group B streptococcal (GBS) type III (GBS III) polysaccharide (IIIPS) or with IIIPS covalently linked to tetanus toxoid to assess specificity, sensitivity, and parallelism in dilution curves in two GBS III enzyme-linked immunosorbent assays (ELISAs). One assay used IIIPS mixed with methylated human serum albumin (IIIPS + mHSA) as the coating antigen, and the other used IIIPS covalently linked to HSA (III-HSA). Each coating antigen was associated with a highly specific GBS III bioassay. The sensitivity was higher in the III-HSA ELISA, in which conjugated IIIPS is bound to the plates. Parallelism in titration curves was observed in the III-HSA but not in the IIIPS + mHSA ELISA. The excellent correlation between the concentrations of GBS IIIPS-specific immunoglobulin G (IgG) and the opsonophagocytic activity of these antibodies indicated that the III-HSA assay can predict functionality of vaccine-induced IgG against GBS III disease. The structure of the repeating unit of the capsular polysaccharide of GBS III differs from that of Streptococcus pneumoniae type 14 (Pn14 PS) only by the presence on GBS III of a sialic acid residue at the end of the side chain. The majority of healthy adults responding to GBS III vaccines with a fourfold or greater increase in GBS III-specific IgG antibodies developed antibodies cross-reacting with Pn14 PS (i.e., desialylated GBS IIIPS). The proportion of GBS vaccine responders who developed IgG to the desialylated IIIPS did not depend on whether IIIPS was given in the unconjugated or conjugated form. When present, these vaccine-induced cross-reacting antibodies conferred in vitro antibody-mediated opsonophagocytosis and killing of both GBS III and Pn14, two pathogens that cause invasive disease in young infants.

FIG. 1.

Competitive-inhibition IgG ELISAs demonstrate antigenic specificity of a pooled standard human reference serum (SHRS III) in GBS III ELISA using either III-HSA (left panel) or IIIPS + mHSA (right panel) as the coating antigen. The percent inhibition of binding of the IIIPS antibodies in SHRS III was determined by comparison of the A_405/630 in the presence and absence of inhibitors as indicated. ATTC, American Type Culture Collection.

FIG. 2.

Standard curves from the GBS III ELISA show a relationship between the quantity of specific immunoglobulin and A_405/630. (Left panel) Reaction of reference serum 19 with plates coated with III-HSA (filled triangles) or IIIPS + mHSA (open triangles). (Right panel) Reaction of SHRS III with plates coated with III-HSA (filled triangles) or IIIPS + mHSA (open triangles). The arrows indicate the sensitivities of the III-HSA ELISA (filled arrows) and the IIIPS + mHSA ELISA (open arrows). The results represent means ± SDs for six experiments.

FIG. 3.

Spearman rank correlation coefficient (r_s) comparing IIIPS-specific antibodies in sera from healthy adults given GBS vaccines (III-TT, n = 30; IIIPS, n = 30) as measured by ELISAs with a suboptimal (by 2 orders of magnitude) III-HSA coating concentration (y axis) or with III + mHSA (x axis).

FIG. 4.

Comparison of naturally acquired (preimmunization) (left panels) and vaccine-induced (III-TT, n = 30; IIIPS, n = 30) (right panels) IIIPS-specific antibodies in human sera as quantitated by IgG ELISA (y axis) and by RABA (x axis). The coating antigens used in the ELISAs are shown in the upper left corner of each panel. CPS, capsular polysaccharide.

FIG. 5.

Comparison of IIIPS-specific antibodies in human sera elicited by unconjugated IIIPS vaccine (n = 30) (left panels) or by III-TT conjugate vaccine (n = 30) (right panels), as quantitated by ELISA (y axis) and RABA (x axis). The coating antigens used in the ELISAs are shown in the upper left corner of each panel. CPS, capsular polysaccharide.

FIG. 6.

Competitive-inhibition RABA demonstrating the specificity of GBS IIIPS-specific vaccine-induced antibodies in a standard human reference serum pool (SHRS III). The percent inhibition of binding of SHRS III to IIIPS in solution was determined by comparison of counts per minute in the presence and absence of inhibitor.

FIG. 7.

Correlation of vaccine-induced (III-TT, n = 30; IIIPS, n = 30) IIIPS-specific antibodies in human sera, as measured by ELISA (y axis) and opsonophagocytic killing (x axis) of GBS III (A) or Pn14 (B). Opsonophagocytic killing is the log₁₀ reduction in CFU (A) or the titer resulting in 50% reduction in CFU (B). The coating antigens used in the ELISAs are shown in the upper left corner of each panel. CPS, capsular polysaccharide.

FIG. 8.

Dilution curves from GBS III ELISA in which III-HSA was used at an optimal coating concentration (filled triangles) or at a suboptimal coating concentration (2 orders of magnitude lower than optimal) (open triangles). The relationship between the avidity of the antibodies and their binding to ELISA plates is evident. (Left panel) Reaction of a GBS III vaccine-induced serum of low avidity (avidity index, 12%). (Middle panel) Reaction of a GBS III-TT conjugate vaccine-induced serum pool (SHRS III) of intermediate avidity (avidity index, 35%). (Right panel) Reaction of tetravalent GBS polysaccharide vaccine-induced reference serum 19 of high avidity (avidity index, 71%). Results are means ± SDs for four experiments.

FIG. 9.

Reverse cumulative distribution plot of the fold increase in IIIPS-specific IgG in sera 8 weeks after immunization with unconjugated IIIPS (n = 30) (upper panel) or III-TT conjugate vaccine (n = 30) (lower panel). The proportions of vaccine-induced IgG that recognized native IIIPS (solid lines) or Pn14 PS (i.e., desialylated IIIPS) (dotted lines) are indicated.

FIG. 10.

Competitive-inhibition ELISAs (upper panels) and the corresponding competitive inhibition of opsonophagocytic killing of GBS III (strain M781) by human PMNs in the presence of endogenous complement (lower panels) demonstrate the functional activity of IIIPS-specific antibodies that cross-react with Pn14 PS. The results shown are for sera from three volunteers immunized with unconjugated IIIPS that represent the observed categories of antibody specificity: left panels, all GBS IIIPS-specific antibodies cross-react with Pn14 PS (A21); middle panels, a subpopulation of GBS IIIPS-specific antibodies cross-react with Pn14 PS (B30); right panels, no GBS IIIPS-specific antibodies cross-react with Pn14 PS (A33). The upper panels show the percent inhibition of binding of IIIPS-induced IgG, as determined by comparison of A_405/630 in the presence and absence of inhibitor. The lower panels show the percent reduction in CFU in the presence of inhibitors. The following inhibitors were used: Pn14 PS (filled squares), IIIPS (open squares), and III-HSA (open circles).

FIG. 11.

Competitive-inhibition ELISAs (upper panels) and the corresponding competitive inhibition of opsonophagocytic killing of GBS III (strain M781) by human PMNs in the presence of endogenous complement (lower panels) demonstrate the functional activity of IIIPS-specific antibodies that cross-react with Pn14 PS. The results shown are for sera from three volunteers immunized with III-TT conjugate vaccine. These sera represent the observed categories of antibody specificity: left panels, all GBS IIIPS-specific antibodies cross-react with Pn14 PS (B2); middle panels, a subpopulation of GBS IIIPS-specific antibodies cross-react with Pn14 PS (B28); right panels, no GBS IIIPS-specific antibodies cross-react with Pn14 PS (B20). The upper panels show the percent inhibition of binding of III-TT-induced IgG, as determined by comparison of A_405/630 in the presence and absence of inhibitor. The lower panels show the percent reduction in CFU in the presence of inhibitors. The following inhibitors were used: Pn14 PS (filled squares), IIIPS (open squares), and III-HSA (open circles).