Chemically Enhanced Immunogenicity of Bacteria by Supramolecular Functionalization with an Adjuvant

Abstract Many pathogens blunt immune responses because they lack immunogenic structural features, which typically results in disease. Here, we show evidence suggesting that pathogen immunogenicity can be chemically enhanced. Using supramolecular host‐guest chemistry, we complexed onto the surface of a poorly immunogenic bacterium (Staphylococcus aureus) a TLR7 agonist‐based adjuvant. “Adjuvanted” bacteria were readily recognized by macrophages and induced a more pro‐inflammatory immunophenotype. Future applications of this concept could yield treatment modalities that bolster the immune system's response to pathogenic microbes.


Chemicals
Chemicals were obtained commercially from Merck (Darmstadt, Germany), TCI (Tokyo, Japan) or Cyclodextrin-Shop (Tilburg, The Netherlands) and used without further purification; these were deemed free of any significant endotoxin contamination by respective manufacturers. Amino acids were obtained from either Bachem (Bubendorf, Switzerland) or Iris Biotech (Marktredwitz, Germany). Solvents were obtained from Actu-All (Oss, The Netherlands), Biosolve (Valkenswaard, The Netherlands) or Merck (Darmstadt, Germany). Acetonitrile, N,N-Dimethylformamide and Dimethylsulfoxide were dried using 4Å molecular sieves Merck (Darmstadt, Germany) unless stated otherwise.
Reactions were carried out under normal atmosphere unless stated otherwise. Column chromatography was performed with 40-63 µm silica from Screening Devices (Amersfoort, The Netherlands). SPPS was carried out either by a Biotage Syro II (Uppsala, Sweden) or by hand using fritted tubes (6, 10 or 25 mL) from Screening Devices (Amersfoort, The Netherlands) and in-house N2 flow/vacuum.

Mass spectrometry
Mass spectrometry was performed using a Bruker Microflex Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometer (Billerica, MA, United States).

NMR
1 H NMR, COSY and 13 C NMR of the dyes were recorded on a Bruker AV-300 spectrometer (300 MHz) (Billerica, MA, United States) in methanol-d4. Quantification of the number of β-CD units per polymer with 1 H NMR and DOSY was done in D2O using a Bruker Avance III spectrometer (500 MHz), equipped with a 5 mm TXI probe.

Photometry
Absorbance spectra were recorded using an Ultrospec 2100 pro (Amersham Biosciences, Little Chalfont, United Kingdom).

Indole-COOH (2)
6-bromohexanoic acid (2.5 g, 12.6 mmol) was dissolved in dry acetonitrile (10.0 mL) followed by addition of 2,3,3trimethylindolenine (1.0 mL, 6.3 mmol). The brown solution was stirred at 60°C for 60 hours whereupon it turned into a dark red solution. Acetonitrile was removed by vacuum and after dissolving the remaining solid in MeOH the product was precipitated in ethyl acetate. Filtration over a sintered glass filter (P3) followed by washing with ethyl acetate and drying in vacuo yielded a light pink solid (447.0 mg, 25.8 % isolated yield) which was used without further purification.
The solid was dissolved in acetone and the product was precipitated in diethyl ether. The pink supernatant was decanted and the slightly orange solid was redissolved and reprecipitated in diethyl ether; this process was repeated until the supernatant was colorless. The orange precipitate was then filtrated using a sintered glass filter (P3) and dried in vacuo resulting in an orange solid (1.2 g, 89.6% isolated yield) which was used without further purification.

Phth-Cy5-COOH (4)
0.4 mmol) and pyridine (152.0 µL, 1.9 mmol) were added and the mixture was refluxed overnight. After cooling down, the solvents were evaporated and the residue was dissolved in acetone followed by precipitation in diethyl ether. The suspension was poured onto a sintered glass filter (P3) followed by washing with diethyl ether and drying in vacuo. The solid was then dissolved in 1:4 methanol/ethyl acetate and purified by silica column chromatography using a gradient of methanol:ethyl acetate 1:4→1:1. The fractions containing product were combined and the solvent was removed in vacuo, resulting in a blue solid (84.2 mg, 33.9% isolated yield).

NH2-Cy5-COOH (5)
The amine was deprotected using a protocol from literature [2] . 20.0 mL of 33 wt. % methylamine in ethanol was added to Phth-Cy5-COOH (4) (44.0 mg, 67.0 µmol), and the colorless solution was stirred for 2 hours at room temperature. The solvents were then removed by evaporation, resulting in a blue solid which was redissolved in 2.0 mL of acetonitrile containing 0.1% TFA, followed by addition of 2.0 mL of water containing 0.1% TFA. Purification by semi-preparative HPLC yielded a blue solid after lyophilization (23.2 mg, 65.8% isolated yield).

H NMR spectrum with integrated peaks and annotation of PIBMA[389]-CD[85] (6) confirming presence of β-CD, which was used for calculation of the average amount of β-CD per polymer unit.
The amount of β-CD per polymer was determined as previously described [3,5]  This was rounded to 85 for clarity of illustration and calculation.

Quantification of Cy5
The molar extinction coefficient of NH2-Cy5-COOH was determined as previously described; [2] briefly: a weighed amount of Cy5 was dissolved in water to create a 5 mM stock solution. From this stock a dilution range from 7.5 µm to 0.25 µm was made in triplicate, of which the absorption was measured at Absmax = 640 nm. Using linear regression, the molar extinction coefficient was then determined to be 62900 L -1 ·mol -1 ·cm -1 in water. This was used with the Lambert-Beer equation to determine the dye molarity in a 3.

Quantification of CL307 (the adjuvant) on polymer
The molar extinction coefficient of CL307 was estimated as previously described; [2] briefly: a weighed quantity of CL307 was dissolved in water to yield a 1 mM stock solution. From this stock a dilution range from 7.5 µm to 0.25 µm was made in triplicate, of which the absorption was measured at Absmax = 298 nm. Using linear regression, the molar extinction coefficient was determined to be 5200 L -1 ·mol -1 ·cm -1 in water. This was used with the Lambert-Beer equation to determine the CL307 molarity in a 0. 5 Figure