Synthetic 6B di-, tri-, and tetrasaccharide-protein conjugates contain pneumococcal type 6A and 6B common and 6B-specific epitopes that elicit protective antibodies in mice

Abstract

The immunogenicity and protective capacity of Streptococcus pneumoniae 6B capsular polysaccharide (PS)-derived synthetic phosphate-containing disaccharide (Rha-ribitol-P-), trisaccharide (ribitol-P-Gal-Glc-), and tetrasaccharide (Rha-ribitol-P-Gal-Glc-)-protein conjugates in rabbits and mice were studied. In rabbits, all saccharides conjugated to keyhole limpet hemocyanin (KLH) evoked high levels of pneumococcal (Pn) type 6B antibodies that facilitated type-specific phagocytosis. Unlike the disaccharide rabbit antisera, tri- and tetrasaccharide rabbit antisera also reacted with 6A PS in an enzyme-linked immunosorbent assay (ELISA) and promoted phagocytosis of 6A pneumococci. All these rabbit antisera passively protected mice against a Pn 6B challenge. The disaccharide conjugate-induced antiserum, however, failed to protect mice against a 6A challenge. In mice, phagocytic and protective anti-Pn 6B antibodies were only induced by the tetrasaccharide conjugate and not by PS 6B or PS 6B-protein conjugates. These antibodies did not cross-react with 6A PS in ELISA and were unable to phagocytize 6A pneumococci. In conclusion, the disaccharide and tetrasaccharide conjugates already contain epitopes capable of inducing 6B-specific, fully protective antibodies in rabbits and mice, respectively.

FIG. 1

Schematic representation of synthetic di-, tri-, and tetrasaccharide conjugates. Di- and trisaccharides are overlapping fragments of the tetrasaccharide, the latter representing one repeating unit of 6B PS. Saccharides were coupled to KLH via a spacer at their reducing end.

FIG. 2

Rabbit antisera elicited by disaccharide OS conjugate were specific for serotype 6B. Di-, tri-, and tetrasaccharide conjugate antisera (n = 2) at 1 week after the second booster injection were analyzed by ELISA, inhibition ELISA, and a phagocytosis assay. Results are expressed as mean titers ± SD (n = 2). (A) Sera were analyzed for antibody titers against PS 6B and 6A by ELISA using respectively, 6B PS and 6A PS as the coating antigen. ∗, antibody titer not significantly above preimmunization antibody titer. (B) Inhibition of binding of sera to 6B PS. As inhibitors, 6B PS or 6A PS were used. (C) Promotion of phagocytosis of serotype 6B and 6A pneumococci, measured in a serotype-specific phagocytosis assay based on flow cytometry.

FIG. 3

Tetrasaccharide conjugate induced highest anti-PS 6B responses in mice. Mean antibody titers ± SD (n = 10) against 6B PS were determined by ELISA using antisera obtained at week 25 after primary immunization with 6B PS, 6B PS-KLH conjugate, or 6B di-, tri-, and tetrasaccharide-KLH conjugates. The tetrasaccharide conjugate induced significantly higher anti-PS 6B antibody titers than the disaccharide (P < 0.01), trisaccharide (P < 0.001), 6B PS (P < 0.001), 6B PS-KLH conjugate (P < 0.05), and saline (P < 0.0001).

FIG. 4

Tetrasaccharide conjugate-induced 6B-specific, phagocytic antibodies in mice. Di-, tri-, and tetrasaccharide conjugate antisera (n = 10) were analyzed at week 25 after primary immunization for sustained antibody levels by ELISA (A), inhibition ELISA (B), and phagocytosis assay (C). Results are expressed as mean titers ±SD (n = 10). Sera were analyzed for inhibition of binding of sera to 6B PS (coating antigen), with 6B PS or 6A PS as inhibitors in an inhibition ELISA (B) and for promotion of phagocytosis of serotype 6B and 6A pneumococci, measured in a serotype-specific phagocytosis assay based on flow cytometry (C). None of the control animals were able to produce anti-6B or -6A PS antibodies (data not shown). N.D., not done.

FIG. 5

OS conjugates contained epitopes for anti-6B PS antibodies induced by vaccination of either humans with a heptavalent conjugate vaccine or rabbits with 6B pneumococci. Binding to OS conjugates of rabbit anti-Pn 6B antiserum (A) or pooled sera from 10 infant 6B conjugate vaccinees (B) was inhibited by 6A PS and 6B PS. Results are expressed as means of two experiments ±SD. N.D., not done.

FIG. 6

Protective capacities of OS conjugates and OS conjugate-derived rabbit antisera in mice. Shown are percentages of survival for mice after serotype 6B and 6A challenge. (A) Passive protection of mice against serotype 6B and 6A challenge 2 h after intravenous injection with rabbit anticonjugate antisera or normal rabbit serum (control). (B) Active protection of mice against a 6B challenge after immunization with di-, tri-, or tetrasaccharide conjugates (second immunization experiment). Challenge was performed 25 weeks after primary immunization.