Site-Specific Conjugation for Fully Controlled Glycoconjugate Vaccine Preparation

Abstract

Glycoconjugate vaccines are formed by covalently link a carbohydrate antigen to a carrier protein whose role is to achieve a long lasting immune response directed against the carbohydrate antigen. The nature of the sugar antigen, its length, its ratio per carrier protein and the conjugation chemistry impact on both structure and the immune response of a glycoconjugate vaccine. In addition it has long been assumed that the sites at which the carbohydrate antigen is attached can also have an impact. These important issue can now be addressed owing to the development of novel chemoselective ligation reactions as well as techniques such as site-selective mutagenesis, glycoengineering, or extension of the genetic code. The preparation and characterization of homogeneous bivalent pneumococcal vaccines is reported. The preparation and characterization of homogeneous bivalent pneumococcal vaccines is reported. A synthetic tetrasaccharide representative of the serotype 14 capsular polysaccharide of Streptococcus pneumoniae has been linked using the thiol/maleimide coupling chemistry to four different Pneumococcal surface adhesin A (PsaA) mutants, each harboring a single cysteine mutation at a defined position. Humoral response of these 1 to 1 carbohydrate antigen/PsaA conjugates have been assessed in mice. Our results showed that the carbohydrate antigen-PsaA connectivity impacts the anti-carrier response and raise questions about the design of glycoconjugate vaccine whereby the protein plays the dual role of immunogen and carrier.

Keywords: chemoselective ligation; cysteine mutagenesis; glycoconjugate vaccine; pneumococcal vaccine; protein conjugation; thio/maleimide ligation.

Figure 1

(A) Structure of tetrasaccharide antigen 1 from S. pneumoniae serotype 14 capsule and its activation with a maleimide linker; reagents and conditions: (a) 3-maleimidopropionic acid N-hydrosysuccimide ester (1.45 equiv), DIEA (2 equiv), DMF, RT, overnight, 58%. (B) Ribbon diagram of PsaA (green). Regions containing known Th and B-epitopes are colored in red. Lysine side-chain are represented in green except the four lysines targeted for mutagenesis (in blue). Representation based on the 1PSZ PDB file, with a resolution of 2.0 Å (Lawrence et al., 1998).

Figure 2

mPsaA K309C characterization as a representative example. Analysis of histidine tag removal from mutant mPsaA K309C by SDS-PAGE (A) and by Western blot (B). Lane 1: unstained protein marker; Lane 2: tagged mPsaA K309C; Lane 3: purified mPsaA K309C after removal of poly-histidine tag.

Figure 3

Comparison of size exclusion chromatography profile of Pn14TS-mPsaA K247C (red trace) and Pn14TS-mPsaA K309C (Black trace) conjugates. Column HiLoad 15/600 Superdex™ 75 pg (GE Healthcare) column and PBS 0.1 M, pH 7.3 as eluent.

Figure 4

Analysis of bioconjugation efficiency by SDS PAGE and mass spectrometry. (A) Comparison of each mPsaA mutant before, mix of before and after, and after conjugation. Lanes 1–3: mPsaA K237C; Lanes 4–6: mPsaA K247C; Lanes 7–9: mPsaA K242C; Lanes 10–12: mPsaA K309C. Two micrograms protein sample/lane, 12% SDS-PAGE, 100 V, 2 h; (B) MALDI MS spectrum of mPsaA K309C (top spectrum) and Pn14TS-mPsaA K309C (bottom spectrum).

Figure 5

Titers of anti-mPsaA (coated on microtiter plates) IgG Abs of mice immunized with Pn14TS-mPsaA K237C, K242C, K247C, K309C, PBS 1 week after the 2nd (J21) (A) and the 3rd (J35) immunization (B). The serum samples data presented as geometric mean titer ± standard deviation of five mice per group. Statistical analysis was performed using one-way ANOVA with Tukey analysis for multiple comparisons. Statistical difference between the groups is ^*P < 0.05.