Research ArticleVACCINATION

A semisynthetic Streptococcus pneumoniae serotype 8 glycoconjugate vaccine

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Science Translational Medicine  08 Mar 2017:
Vol. 9, Issue 380, eaaf5347
DOI: 10.1126/scitranslmed.aaf5347
  • Fig. 1. AGA of ST8 CPS frameshift oligosaccharides.

    (A) ST8 CPS structure and nomenclature of tetrasaccharide frameshifts A to D. The cellobiuronic acid (CA) moiety is highlighted. d-Gal, d-Galactose. (B) Automated assembly of synthetic frameshifts (FSs) A(1), B(2), C(3), and D(4) using resin 5 and building blocks 6 to 11. (C) Overlay of 1H nuclear magnetic resonance (NMR) (600 MHz) spectra with commercially available ST8 CPS in D2O at 25°C. Red arrowheads indicate peaks from pneumococcal C-polysaccharide as reported (18). ppm, parts per million.

  • Fig. 2. Differential immune recognition of synthetic ST8 CPS frameshift oligosaccharides 1 to 4.

    (A) Glycan microarray analysis of pooled sera from Pneumovax 23–vaccinated humans (human reference serum mixture 007sp), rabbit ST8 typing serum (Typ. s.), and a protective murine mAb 28H11 at different concentrations (conc.). Corresponding data are means + SD of eight spots as technical replicates. (B) Inhibition of antibody binding by preadsorption with native ST8 CPS. Data are means + SD of eight spots as technical replicates. P values are determined by one-tailed, unpaired t test with Welch’s correction. Data are obtained from one representative of at least two independent experiments. MFI, mean fluorescence intensity; n.s., not significant.

  • Fig. 3. Evaluation of a synthetic ST8 CRM197–frameshift C(12) glycoconjugate in mice.

    (A) Conjugation of synthetic frameshift C tetrasaccharide 12 to CRM197. r.t., room temperature. (B) Immunization strategy. Mice (n = 3 per group) were subcutaneously immunized three times with CRM197–frameshift C(12) using different mouse strains and formulations. Serum was collected 1 week after the third immunization. (C) Structures of glycans 13 and 14. (D) Glycan-directed immune responses of mice, as assessed by glycan microarray, and cross-reactivity patterns toward ST8 CPS or tetrasaccharide 14. (E) Glycan binding specificities of sera reactive against ST8 frameshift C(3). DSAP, di-N-succinimidyl adipate; NaPi, sodium phosphate buffer; DMSO, dimethyl sulfoxide; BSA, bovine serum albumin; non-adj., nonadjuvanted.

  • Fig. 4. Binding of ST8 CPS–derived glycans and ST8 bacteria by mAb 1H8.

    (A) Glycan microarray analysis. Data are means + SD of eight spots as technical replicates from one representative of three independent experiments. a.u., arbitrary units. (B) Surface plasmon resonance of immobilized mAb 1H8 using ST8 CPS as analyte in the indicated concentrations. (C) Immunofluorescence of inactivated, fluorescein isothiocyanate (FITC)–labeled ST8 or ST1 pneumococci by 1H8 or IgG1 control mAb. Scale bars, 5 μm. bact., bacteria. (D) Flow cytometry of fluorescently labeled, inactivated pneumococci. Data are a representative result with gating strategy for binding after incubation with mAb 1H8 or IgG1 control (ctrl.) mAb and fluorescently labeled detection antibody and cumulated results of three labeling experiments. Data are means + SD of positive binding from n = 3 independent experiments. P values are determined by one-tailed, paired t test. DIC, differential interference contrast; RU, response units.

  • Fig. 5. Evaluation of mAb 1H8 to promote antibacterial activity in vitro and in vivo.

    (A) Opsonophagocytic killing assay (OPKA) using differentiated HL-60 cells as a phagocyte source. Data are colony-forming unit (CFU) reduction relative (Rel.) to control wells lacking an antibody source. Commercial ST8 antisera (1:18 dilution) are positive controls. Data are means + SD of n = 3 incubation wells of one representative of four independent experiments. P value is determined by one-tailed, unpaired t test with Welch’s correction. Ref., reference. (B to D) Passive immunization and lethal challenge with S. pneumoniae ST8. (B) Passive immunization strategy. Mice were treated intraperitoneally (i.p.) with 10 or 100 μg of mAb 1H8 or control IgG1 2 hours before lethal transnasal (t.n.) challenge with ST8 bacteria. Blood was withdrawn at regular intervals, and survival was monitored for 96 hours. (C) Survival of antibody-treated mice after ST8 infection. P values are determined by Mantel-Cox log-rank test of n = 5 to 6 mice between groups of equal antibody doses. (D) Bacterial burden in the blood of mice 30 hours after infection. Data are values from n = 5 to 6 individual mice and median. P values are determined by Mann-Whitney U test between groups of equal antibody doses.

  • Fig. 6. Mapping protective and nonprotective glycotopes of ST8 frameshift C.

    (A) Glycan microarray analysis comparing ST8 CPS–recognizing mAb 1H8 and antisera of mice that recognize a nonprotective glycotope of frameshift C (see Fig. 3) by using synthetic ST8 and ST3 CPS–derived oligosaccharides. (B) Surface plasmon resonance of mAb 1H8 and serum of mouse 741 with immobilized glycan frameshift C(12) and disaccharide 18. (C) Glycan microarray of mAb 1H8 using oligosaccharides 20 to 24 for glycotope mapping. Boxes mark protective (blue) and nonprotective (red) glycotopes. Data are means + SD of fluorescence intensities of at least six spots as technical replicates from one representative of at least three independent experiments.

  • Fig. 7. Evaluation of semisynthetic glycoconjugates as vaccine candidates against ST8 in rabbits.

    (A) Immunization strategy. Rabbits (n = 3 per group) were subcutaneously immunized three times with CRM197 glycoconjugates or CRM197. Serum was collected at regular intervals. (B) Immune responses of one representative rabbit per group at day 35, as assessed by glycan microarray. (C to F) Immune response against ST8 CPS as assessed by polysaccharide ELISA. (C) Time course of ST8 CPS binding by rabbit sera (1:1600 dilutions). Data are means ± SD of n = 3 rabbits per group. (D) Evaluation of ST8 CPS binding by rabbit sera at day 35 (1:1600 dilution). Data are medians ± range of n = 3 rabbits per group. P values are determined by one-tailed Mann-Whitney U test. (E) ST8 CPS binding of rabbit sera at different dilutions. Data are means ± SD of n = 3 rabbits per group. (F) End point titers of rabbit sera (n = 3 per group) against ST8 CPS.

  • Fig. 8. Coformulation of semisynthetic ST8 glycoconjugates and Prevnar 13.

    (A) Adsorption of ST8 glycoconjugates to Prevnar 13 as assessed by flow cytometry. ST8 glycoconjugates were adsorbed to Prevnar 13 particles using 25, 100, or 400% of a full-dose equivalent and detected by flow cytometry using mAb 1H8 and an Alexa Fluor 635–labeled detection antibody (Ab). Controls are nontreated and CRM197-treated Prevnar 13 particles. Data are means + SD of the median fluorescence (Med. Fl) intensities of n = 3 independent experiments. (B) Immunization strategy. Rabbits (n = 3 per group) were subcutaneously immunized three times with Prevnar 13 with or without coadsorbed CRM197 glycoconjugates. Serum was collected at day 35. (C and D) Immune response to ST8 CPS as assessed by polysaccharide ELISA. (C) Evaluation of ST8 CPS binding by rabbit sera at day 35 (1:200 dilution). Data are medians ± range from n = 3 individual rabbits per group. P values are determined by one-tailed Mann-Whitney U test. (D) Comparison of ST8 CPS binding of rabbit sera from different groups. Data are means ± SD of n = 3 rabbits per group. (E) Comparison of opsonophagocytic killing of ST8 pneumococci by pooled rabbit sera. Data are means + SD of CFU reduction relative to control wells of n = 3 incubation wells as biological replicates at a 1:32 serum dilution. P values are determined by one-tailed, unpaired t test with Welch’s correction. (F) Comparison of opsonophagocytic killing by pooled rabbit sera and human reference serum 007sp at different serum dilutions. Data are means ± SD of CFU reduction relative to control wells of n = 3 incubation wells. Data in (E) and (F) are obtained from one representative of three independent experiments.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/380/eaaf5347/DC1

    Methods

    Fig. S1. AGA of tetrasaccharides 1 to 4.

    Fig. S2. Differential immune recognition of synthetic ST8 CPS frameshifts.

    Fig. S3. Solution-phase syntheses of ST8 CPS frameshift C(12) and disaccharide 13.

    Fig. S4. Characterization of the CRM197–frameshift C(12) glycoconjugate.

    Fig. S5. Time course of antiglycan immune responses after immunization with CRM197–frameshift C(12).

    Fig. S6. Characterization of mAbs raised against ST8 frameshift C(12).

    Fig. S7. Binding of ST8-related glycans by mAbs 1H8, 28H11, and 1F1.

    Fig. S8. Qualitative comparison of binding kinetics of mAb 1H8 toward ST8 CPS–derived glycans of different chain lengths.

    Fig. S9. Correlation between mAb 1H8 levels and bacterial burden in blood after passive immunization and lethal pneumococcal challenge.

    Fig. S10. Divergent total syntheses of ST8 frameshift C–related glycans 21 to 24 from versatile precursor 32.

    Fig. S11. Total synthesis of ST8 tetrasaccharide 15.

    Fig. S12. Binding characterization of mAbs 1H8 and 1F1 to ST8 CPS–derived substructures by glycan microarray.

    Fig. S13. Determination of affinities of mAb 1H8 toward synthetic ST8 CPS–related oligosaccharides.

    Fig. S14. Conjugation of synthetic oligosaccharides to CRM197 using the bifunctional spacer bis(4-nitrophenyl)adipate (DNAP).

    Fig. S15. Evaluation of CRM197 glycoconjugates of trisaccharide 21 and tetrasaccharide 22 in mice.

    Fig. S16. Immune response against semisynthetic ST8 glycoconjugates in rabbits.

    Fig. S17. Adsorption of ST8 glycoconjugates to Prevnar 13, as assessed by flow cytometry.

    Fig. S18. Effect of coformulation of semisynthetic ST8 glycoconjugates with Prevnar 13 on the immune response against several pneumococcal CPSs.

    Table S1. Sequences of automated assembly of protected ST8 CPS–related tetrasaccharide frameshifts.

    Table S2. Antipolysaccharide IgG end point titers of rabbits immunized with Prevnar 13 alone or coformulated with semisynthetic ST8 glycoconjugates.

    Spectra

    References (3945)

  • Supplementary Material for:

    A semisynthetic Streptococcus pneumoniae serotype 8 glycoconjugate vaccine

    Benjamin Schumann, Heung Sik Hahm, Sharavathi G. Parameswarappa, Katrin Reppe, Annette Wahlbrink, Subramanian Govindan, Paulina Kaplonek, Liise-anne Pirofski, Martin Witzenrath, Chakkumkal Anish, Claney L. Pereira*, Peter H. Seeberger*

    *Corresponding author. Email: peter.seeberger{at}mpikg.mpg.de (P.H.S.); claneylebev.pereira{at}vaxxilon.com (C.L.P.)

    Published 8 March 2017, Sci. Transl. Med. 9, eaaf5347 (2017)
    DOI: 10.1126/scitranslmed.aaf5347

    This PDF file includes:

    • Methods
    • Fig. S1. AGA of tetrasaccharides 1 to 4.
    • Fig. S2. Differential immune recognition of synthetic ST8 CPS frameshifts.
    • Fig. S3. Solution-phase syntheses of ST8 CPS frameshift C(12) and disaccharide 13.
    • Fig. S4. Characterization of the CRM197–frameshift C(12) glycoconjugate.
    • Fig. S5. Time course of antiglycan immune responses after immunization with CRM197–frameshift C(12).
    • Fig. S6. Characterization of mAbs raised against ST8 frameshift C(12).
    • Fig. S7. Binding of ST8-related glycans by mAbs 1H8, 28H11, and 1F1.
    • Fig. S8. Qualitative comparison of binding kinetics of mAb 1H8 toward ST8 CPS–derived glycans of different chain lengths.
    • Fig. S9. Correlation between mAb 1H8 levels and bacterial burden in blood after passive immunization and lethal pneumococcal challenge.
    • Fig. S10. Divergent total syntheses of ST8 frameshift C–related glycans 21 to 24 from versatile precursor 32.
    • Fig. S11. Total synthesis of ST8 tetrasaccharide 15.
    • Fig. S12. Binding characterization of mAbs 1H8 and 1F1 to ST8 CPS–derived substructures by glycan microarray.
    • Fig. S13. Determination of affinities of mAb 1H8 toward synthetic ST8 CPS–related oligosaccharides.
    • Fig. S14. Conjugation of synthetic oligosaccharides to CRM197 using the bifunctional spacer bis(4-nitrophenyl)adipate (DNAP).
    • Fig. S15. Evaluation of CRM197 glycoconjugates of trisaccharide 21 and tetrasaccharide 22 in mice.
    • Fig. S16. Immune response against semisynthetic ST8 glycoconjugates in rabbits.
    • Fig. S17. Adsorption of ST8 glycoconjugates to Prevnar 13, as assessed by flow cytometry.
    • Fig. S18. Effect of coformulation of semisynthetic ST8 glycoconjugates with Prevnar 13 on the immune response against several pneumococcal CPSs.
    • Table S1. Sequences of automated assembly of protected ST8 CPS–related tetrasaccharide frameshifts.
    • Table S2. Antipolysaccharide IgG end point titers of rabbits immunized with Prevnar 13 alone or coformulated with semisynthetic ST8 glycoconjugates.
    • Spectra
    • References (3945)

    [Download PDF]

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