Sodium caseinate

Trans-resveratrol (Res, 98% purity) and sodium caseinate (87% purity) were obtained from Sigma-Aldrich Chemical Co., Ltd. (St. Louis, MO, USA). Microbial transglutaminase (100 U/g) was supported by Jiangsu Yiming Biological Co., Ltd. (Taizhou, Jiangsu, China). Zein (97% protein content) was bought from Showa Sangyo (Tokyo, Japan).

Sodium caseinate, polydatin (HPLC grade, >95%) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). WPI was purchased from Davisco International, Inc. (Russel, MN, USA). Tween 20 (Biotechnology grade, ≥99%), resveratrol (trans-isomer, ≥98%), fish oil and peppermint oil were respectively obtained from Macklin Co. (Shanghai, China), Sango Biotech Co. (Shanghai, China), Chaopu Co. (Shanghai, China), Ltd. and Zixin Biotechnology Co., Ltd. (Shanghai, China). Sunflower oil (Duoli brand) was purchased from a local market (Wuxi, China). MCT (C8:C10 = 60:40) was purchased from Yong sheng Industry and Trade Co., Ltd. (Guangzhou, China). Other materials of analytical grade were purchased from Sino-Pharm CNCM Ltd. (Shanghai, China).

Artemether was supplied by Biotain Pharma Co., Ltd. (Xiamen, China). Zein was a kind gift from Flo Chemical Corporation (Ashburnham, MA, USA). Sodium caseinate was purchased from Sigma-Aldrich (St. Louis, MO, USA). Absolute ethanol was obtained from VWR Singapore Ltd. (Singapore). Ultrapure water was prepared by a Elix^® Essential 5 UV Water Purification System (Molsheim, France) or a Millipore Direct-Q^® Ultra-Pure Water System (Billerica, MA, USA) and used in all experiments. High performance liquid chromatography (HPLC) grade acetonitrile was supplied by Tedia (Fairfield, OH, USA). All other chemicals are of reagent grade and were obtained from either Sigma-Aldrich or Tokyo Chemical Industry (Tokyo, Japan).

Chemically defined diets used in Experiment 2 were prepared following the protocol described in Lee (2015) and Jang and Lee (2018). These diets differed in P:C ratio (1:8, 1:2, 2:1, and 8:1), but contained the same concentration of protein plus carbohydrate (P + C = 120 g/L). Sodium caseinate (Sigma C8654) and sucrose (Sigma S9378) were used as the source of protein and carbohydrate, respectively. All diets used in this experiment comprised the same concentrations of dietary lipids (0.3 g/L cholesterol, 4 g/L lecithin), salt mixtures (0.71 g/L KH₂PO₄, 3.73 g/L K₂HPO₄, 0.62 g/L MgSO₄, 1 g/L NaHCO₃), nucleic acids (0.57 g/L uridine, 0.64 g/L inosine), vitamin mixtures (0.002 g/L thiamine, 0.01 g/L riboflavin, 0.012 g/L nicotinic acid, 0.0167 g/L calcium pantothenate, 0.0025 g/L pyridoxine, 0.0002 g/L biotin, 0.003 g/L folic acid), preservatives (1 g/L Nipagin, 0.3% propionic acid), and solidifying agent (20 g/L agar).

Biotinylated peptide library consisting of 18-mer overlapping peptides (Mimotopes) were generated from sequence alignments of different CHIKV amino acid sequences as previously described [17] (link), [29] (link). Peptides were dissolved in dimethyl sulphoxide (DMSO) to obtain a stock concentration of approximately 15 µg/mL. All the peptides were screened in triplicates. Briefly, streptavidin-coated plates (Pierce) were first blocked with 1% sodium caseinate (Sigma-Aldrich) diluted in 0.1% PBST (0.1% Tween-20 in PBS), before coating with peptides diluted at 1∶1,000 in 0.1% PBST and incubated at room temperature for 1 hour on a rotating platform. Plates were then rinsed with 0.1% PBST before incubation with CHIKV-infected macaque serum samples (1∶2,000) diluted with 0.1% PBST for 1 hour. Plates were rinsed and then followed by incubation with the respective anti-monkey IgG antibodies conjugated to HRP diluted in 0.1% blocking buffer for 1 hour at room temperature to detect for peptide bound antibodies. Reaction was detected with TMB substrate solution (Sigma-Aldrich) and terminated with sulphuric acid (Sigma-Aldrich). Absorbance was read at 450 nm in a microplate autoreader (Tecan). Peptides are considered positive if absorbance values are higher than the mean ±3 standard deviation (SD) values of non-infected macaque serum controls. Data are presented as mean ± SEM.

The standard substrate was sodium caseinate (Sigma-Aldrich, St. Louis, MO, USA). Its solution was made up of 8 mL of 1 M NaOH, 36 g of urea, 10 mL of pre-prepared 22% sodium caseinate solution, 72 mL of water, and kept in a water bath at 25 °C for 30–60 min. Next, 10 mL of 1 M KH₂PO₄ and 4 g of urea were added. This was the substrate solution.
One milliliter of the enzyme solution was added to 5 mL of the substrate solution, the mixture was stirred and kept in a water bath at 25 °C for 10 min. Then 10 mL of 0.3 M trichloracetic acid was added, the mixture was stirred and filtered through Whatman No. 3 filter paper. Ten milliliters of 0.5 M NaOH was added to 5 mL of the filtrate, and 3 mL of phenol reagent [32 (link)] was rapidly added with stirring.
The standard solution was prepared by adding 0.145 mg of tyrosine (Sigma-Aldrich, St. Louis, MO, USA) to 5 mL of 0.2 M HCl. Ten milliliters of 0.5 M NaOH were added to 5 mL of the mixture, and 3 mL of phenol reagent [32 (link)] was rapidly added with stirring.
After 2–10 min, the tyrosine concentration was measured in a colorimeter with a red filter against the standard solution using a preliminarily made calibration [33 (link)]. The proteolytic activity of the EP was calculated as the amount of tyrosine per the EP portion weight used for the enzyme solution preparation.

Streptavidin-coated polystyrene 96-well microtiter plates (Pierce, Thermo Scientific) were blocked with 0.1% PBST supplemented with 1% w/v sodium caseinate (Sigma-Aldrich) for 1 h at RT before being coated with biotinylated 18-mer overlapping peptides synthesized based on consensus E2 glycoprotein sequence (Kam et al, 2012b (link)) for 1 h at RT. Mouse serum diluted 1:500 (mouse) or human plasma diluted 1:2,000 in 0.1% PBST and 0.1% w/v sodium caseinate were added and incubated for 1 h at RT followed by the addition of relevant HRP-conjugated goat secondary IgG (Santa Cruz, cat# sc-2005) to detect bound antibodies. Reactions were developed using 3,3′,5,5′-tetramethylbenzidine substrate (Sigma-Aldrich) and terminated by Stop reagent (Sigma-Aldrich). Absorbance at 450 nm was measured using TECAN Infinite® M200 microplate reader and analyzed using Magellan™ software.