A comprehensive synthetic library of poly-N-acetyl glucosamines enabled vaccine against lethal challenges of Staphylococcus aureus

Abstract

Poly-β-(1-6)-N-acetylglucosamine (PNAG) is an important vaccine target, expressed on many pathogens. A critical hurdle in developing PNAG based vaccine is that the impacts of the number and the position of free amine vs N-acetylation on its antigenicity are not well understood. In this work, a divergent strategy is developed to synthesize a comprehensive library of 32 PNAG pentasaccharides. This library enables the identification of PNAG sequences with specific patterns of free amines as epitopes for vaccines against Staphylococcus aureus (S. aureus), an important human pathogen. Active vaccination with the conjugate of discovered PNAG epitope with mutant bacteriophage Qβ as a vaccine carrier as well as passive vaccination with diluted rabbit antisera provides mice with near complete protection against infections by S. aureus including methicillin-resistant S. aureus (MRSA). Thus, the comprehensive PNAG pentasaccharide library is an exciting tool to empower the design of next generation vaccines.

Fig. 1. Structures of the comprehensive library of PNAG pentasaccharides.

The five-digit number in the bracket for each compound codes for free amine (0) or N-acetamide (1) at residues ABCDE from the non-reducing end to the reducing end of the pentasaccharide, respectively. The five-digit number was then viewed as a binary number and converted to the decimal system as the compound number. For example, 01010 in binary number is equivalent to 10 in the decimal system. Thus, the PNAG pentasaccharide bearing N-acetylation at units B and D only is named as PNAG10.

Fig. 2. Structures and syntheses of key intermediates.

A Structures of two key linchpin pentasaccharide intermediates (1 and 2). B Synthesis of the reducing end glucosamine building block 8. C Syntheses of compound 13; D Syntheses of compound 1. Ac acetyl, Alloc allyloxycarbonyl, Bz benzoyl, TBDPS tert-butyldiphenylsilyl, Boc tert-butyloxycarbonyl, DIPEA diisopropylethylamine, Fmoc fluorenylmethoxycarbonyl, HATU hexafluorophosphate azabenzotriazole tetramethyl uronium, and Troc 2,2,2-trichloroethoxycarbonyl.

Fig. 3. Synthesis of the 32 membered comprehensive PNAG pentasaccharide library.

A Orthogonal deprotection of pentasaccharide 2. B Divergent syntheses of 16 PNAG pentasaccharides from the strategically protected pentasaccharide 1. C Divergent syntheses of 16 PNAG pentasaccharides from the strategically protected pentasaccharide 2. Ac acetyl, Alloc allyloxycarbonyl, Bz benzoyl, TBDPS tert-butyldiphenylsilyl, Boc tert-butyloxycarbonyl, DIPEA diisopropylethylamine, DMF dimethylformamide, Fmoc fluorenylmethoxycarbonyl, HATU hexafluorophosphate azabenzotriazole tetramethyl uronium, Troc 2,2,2-trichloroethoxycarbonyl, and TFA trifluoroacetic acid.

Fig. 4. Syntheses of conjugates.

Syntheses of A mQβ–PNAG, B TTHc–PNAG, and C BSA–PNAG conjugates. SBAP succinimidyl 3-(bromoacetamido)propionate, TCEP tris(2-carboxyethyl)phosphine, TThc tetanus toxoid heavy chain.

Fig. 5. Immunization of mice with mQβ–PNAG led to high levels and long lasting anti-PNAG IgG antibodies.

A C57Bl6 mouse (n = 5 per group) antibody responses at day 35 after immunization. The EC50 value (the fold of serum dilution that gives half-maximal binding) of the IgG titers to the immunizing oligosaccharide was plotted with each symbol representing one animal and the horizontal line is the geometric mean value of the titers within the group. The ELISA titers were determined using the BSA–PNAG conjugate containing the same PNAG structure as the immunizing Qβ–PNAG construct. One-way ANOVA allowed for rejection of the null hypothesis that all groups have the same mean IgG titers (P < 0.0001). Statistical significance was performed by Dunnett’s multiple comparisons post-hoc test. ****P < 0.0001; B Anti-PNAG0 IgG antibody responses of mice (n = 5) immunized with mQβ–PNAG0 monitored over time with mean titers plotted. Data are presented as mean values ± standard deviation of the titer numbers from five mice. The arrows indicate days of vaccination (days 0, 14, 28, 360, and 655). The antibody responses could be boosted more than 650 days after prime vaccination. Source data are provided as a Source Data file.

Fig. 6. Determination of the epitope profile of F598 mAb with the glycan microarray.

A Relative fluorescence unit (RFU) of F598 mAb binding with the library of 32 PNAG pentasaccharides. The glycans are grouped according to the number of NHAc units in the molecule. Each PNAG sequence is printed five times on the glycan microarray. The error bars represent the standard deviations of five individual spots. Data are presented as mean values ± standard deviation. F598 generally prefers highly acetylated PNAG sequences. Both the location and the number of NHAc units are important determinants of F598 binding. B Quantification of the preference of F598 for acetylation at each site of the PNAG pentasaccharide. The mean values are calculated from the values of the binding intensities of all 32 PNAG sequences to F598. Each PNAG sequence is printed five times on the glycan microarray. Data are presented as mean values ± standard deviation. Source data are provided as a Source Data file.

Fig. 7. Immunization of rabbits with mQβ–PNAG conjugate induced significant anti-PNAG IgG antibodies.

A IgG antibody titers to the immunizing PNAG oligosaccharide in rabbit (n = 2 per group) sera on day 35 after prime vaccination. B IgG antibody titers in pooled rabbit sera from mQβ-conjugate or 5GlcNH₂–TT conjugate immunized animals (n = 2 per group) as well as titer of natural human IgG in pooled human serum against native PNAG polysaccharide purified from Acinetobacter baumannii. The numbers above symbols are the average titer numbers. Titers and 95% confidence intervals (CI) were determined by linear regression using log₁₀ values of the average of replicate serum dilutions to determine the X intercept and 95% CI when Y = 0.5 (OD₄₀₅ nm of ELISA plate reading). C Stacked bar graphs depicting the IgG signals at the serum dilution of 1:50,000 for each rabbit (n = 2) immunized with mQβ–PNAG0, mQβ–PNAG10, and mQβ–PNAG26 as well as pre-immune sera, respectively, on the array. The complete microarray results are provided in the Source Data file; D Normalized binding of the comprehensive library of PNAG pentasaccharides by IgG antibodies from post-immune sera of rabbits immunized with mQβ–PNAG0, mQβ–PNAG10, and mQβ–PNAG26, respectively, as well as pre-immune sera. PNAG sequences are grouped together according to the total number of acetamides in the molecules. The color scale bar is shown on the right with 100% indicating the strongest binding to a PNAG component and 0% indicating the weakest binder. For each antigen, the two rows represent sera from two rabbits per group immunized with the specific construct. Source data are provided as a Source Data file.

Fig. 8. Deposition of C1q onto purified PNAG polysaccharide and analysis of opsonic killing activities.

A Complement deposition tests were performed as described using pooled sera from rabbits (n = 2 per group) immunized with mQβ–PNAG conjugates, the 5GlcNH₂–TT conjugate, or from a sample of pooled normal human sera. Titers and 95% confidence intervals (CI) were determined by linear regression using log₁₀ values of the average of replicate serum dilutions to determine the X intercept and 95% CI when Y = 0.5 (OD₄₀₅ nm of ELISA plate reading). P values indicate the significance of the deviation of the slope of the titration curve from zero to identify sera with activity at P < 0.05. mQβ–PNAG10 and mQβ–PNAG26 conjugates were more potent than the mQβ–PNAG0 and 5GlcNH₂–TT conjugate in inducing C1q deposition onto purified PNAG. Normal human serum had no significant C1q depositing activity in spite of having a binding titer to PNAG (see Fig. 7B) consistent with prior reports that naturally acquired human antibody to PNAG is not functional due to the inability to activate the complement pathway^,. Titers were determined by simple linear regression. B Pooled sera from rabbits (n = 2 per group) immunized with mQβ–PNAG conjugate led to significantly higher levels of opsonic killing activities against S. aureus cells. Three aliquots were prepared from each pooled serum and the individual values of the three aliquots were presented. Source data are provided as a Source Data file.

Fig. 9. Protective effects of vaccination.

Immunization with mQβ–PNAG0 effectively A protected against S. aureus infection, and B reduced bacterial count in mouse kidney. mQβ–PNAG0 was significantly better than TTHc–PNAG0 in protecting mice and reducing disease burden (n = 20 for each group). Logrank tests were performed for statistical analysis. P values were presented in the graph. ****P < 0.0001. Source data are provided as a Source Data file.

Fig. 10. Protective effects of antisera from immunized rabbits.

Transfer of antisera from mQβ–PNAG immunized rabbits to mice A provided significant protection to mice (n = 10 per group) against the lethal challenges by S. aureus ATCC29213. Statistical analysis was performed with the logrank test. ****P < 0.0001; and B significantly reduced bacterial count in mouse kidneys. The combination of sera from mQβ–PNAG0 and mQβ–PNAG26 immunized rabbits provided complete protection to mice. Statistical analysis for survival was performed using the logrank test. Analysis of S. aureus cfu/gm was by Kruskal–Wallis non-parametric ANOVA (P < 0.0001 for overall effect of serum given). P values for pairwise comparisons are shown on graph by Dunn’s multiple comparisons test. Transfer of antisera from mQβ–PNAG immunized rabbits to mice C provided significant protection to mice against the lethal challenges by MRSA strain 1058 (n = 10 per group); statistical analysis for survival was performed using the logrank test; and D reduced bacterial count in mouse kidneys. Sera from mQβ–PNAG26 immunized rabbits provided the highest protection to mice. The horizontal line represents the median value of each group. Statistical analysis for survival was performed using the logrank test. Analysis of MRSA cfu/gm was by Kruskal–Wallis non-parametric ANOVA (P = 0.0967). P values for pairwise comparisons are shown on graph by Dunn’s multiple comparisons test. Source data are provided as a Source Data file.