Initial test immunizations. Six-week-old female BALB/c mice were subcutaneously injected, and sera were collected and treated as described previously (37). Immunizations were carried out with 10 μg each of ADDomer, ADDomer-tevCHIK (uncut), or ADDomer-tevCHIK quantitively cleaved by TEV protease, respectively. Specific antibodies in sera were detected by enzyme-linked immunosorbent assay (ELISA) in duplicate using 96-well plates coated with CHIK peptide (0.1 μg/ml). Horseradish peroxidase (HRP)–conjugated goat anti-mouse IgG 1030-05 (SouthernBiotech) was used for detection [concentration of 0.1 μg/ml in PBS with 1% bovine serum albumin (BSA)] for 1 hour at 37°C, supplemented with 100 μl of tetramethylbenzidine (TMB) substrate (Becton Dickinson) per well. Reactions were stopped by adding H2SO4 (0.2 N), and OD was measured in a microplate reader (Thermo Fisher Scientific) at 450 nm (fig. S7).

ADDomer-tevCHIK immunization experiments. Ten B6D2F1 mice per group received twice (study days 1 and 15) subcutaneously 40 μg per mouse of ADDomer (group 1) or quantitatively cleaved ADDomer-tevCHIK (group 2). Control mice were immunized twice with the buffer diluent. Mice were observed cage-side for clinical symptoms. Mice immunized with ADDomer (group 1) and ADDomer-tevCHIK (group 2) developed high serum IgG titers (<12,800) after the first and second immunization. Mice immunized with diluent did not induce ADDomer-specific IgG. IgM and IgG titers were <100 in serum samples collected before immunization. All mice immunized with ADDomer-tevCHIK sample developed CHIK-specific IgG after the first immunization (GMT, 2425; titer range, 400 to 6400). After the second immunization, IgG titers substantially increased (GMT, 19,400; titer range, 6400 to 51,200). Mice immunized with ADDomer (empty) and diluent did not develop CHIK-specific IgG titers. In contrast, only 1 of 10 mice immunized with ADDomer-tevCHIK developed detectable CHIK epitope–specific IgM after the first (titer, 1:50) and second (titer, 1:100) immunization. No CHIK-specific IgM could be determined in mice immunized with ADDomer or diluent.

Blood was collected by the facial vein technique at days 1, 14, and 35. Briefly, the mouse was gently and securely restrained in the operator’s nondominant hand. The hairless freckle on the side of the jaw was located and pricked with a sharp lancet held by operator’s free hand (sharp end of the lancet points at the far side of the mouse’s face, at the base of the far ear, or at the base of the far side of the mouth). About four to seven drops of blood were collected in an Eppendorf tube, and the mouse was released into its cage. Blood samples were centrifuged twice in a bench centrifuge for 5 min at 10,000g at 4°C, and collected serum was stored below −18°C before analyses. An ELISA protocol was developed using streptavidin-coated plates and the biotinylated CHIK peptide (table S2) following established protocols (Thermo Fisher Scientific). Briefly, streptavidin-coated plates were washed three times with 200 μl of wash buffer [25 mM tris-HCl (pH 7.2), 150 mM NaCl, 0.1% BSA, and 0.05% Tween 20]. Next, 100 μl of biotinylated CHIK peptide dissolved in dimethyl sulfoxide and diluted in PBS to a final concentration of 2.5 μg/ml in PBS was added to each well. Plates were incubated for 2 hours on a plate shaker at room temperature. After extensive washing, twofold serially diluted serum samples were added (100 μl per well). Plates were incubated for 1 hour with occasional shaking at room temperature. After three additional washing steps, 100 μl of goat anti-mouse IgM or HRP-conjugated IgG antibody (diluted 1:2000; Sigma-Aldrich), respectively, was added to each well, and plates were incubated for 45 min at room temperature. Plates were again extensively washed before adding 100 μl of color substrate (TMB) per well, followed by shaking. The color reaction was stopped with H2SO4 (0.2 N), and OD was measured at 450 nm. To determine serum IgM or IgG titers, a cutoff value was defined as mean absorption value of negative blank samples plus three times the SD.

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