Serum samples and ethical considerations. This investigation was approved by the Ethical Committee of FIOCRUZ (CEP/IOC-CAAE:52892216.8.0000.5248 and 384/07) following the principles of the Declaration of Helsinki. MAYV serum samples used in this study were obtained from 18 patients with mayaro fever and diagnosed as positive when tested by a PCR test that was provided by the Leônidas and Maria Deane Institute of FIOCRUZ (Manaus, AM, Brazil) and Evandro Chagas Hospital (Manaus, AM, Brazil). The voluntary donor group was composed of residents of endemic areas that provided informed consent by a signed Free and Informed Consent Form (TCLE) for themselves or as the adult responsible for a minor without a definition of their health status, social group, age, sex, or race. Samples from healthy individuals were obtained from blood bank donors of Rio de Janeiro (HEMORIO). Biorepositories provided anonymous serum samples for dengue (Laboratory of Flavivirus, FIOCRUZ/IOC/RJ), chikungunya (LACEN/Ce), yellow fever (vaccinated individuals), and other laboratories of the Oswaldo Cruz Institute (Zika, C-hepatitis, Chagas disease, and leishmaniasis) that ensured the protection of patient privacy and negated the need for verbal or written consent.
Epitope mapping. The complete sequences of nonstructural (Q8QZ72) and structural (Q8QZ73) proteins of MAYV circulating in Brazil were obtained through access to the Uniprot database [http://www.uniprot.org/]. Microarrays of peptides were used to perform the mapping of linear B-cell epitopes, as described previously [45]. One hundred thirty-four linear B-cell IgM epitopes were identified using a pool (n=5) of patient's sera infected with MAYV (non-published data).
Synthetic gene, protein expression, and purification. Two copies of three epitopes and three copies of one epitope sequences were inserted into the green fluorescent protein (GFP, Aequorea victoria) core protein sequence [46] as described in Table 1. The final chimeric amino acid sequence was back transcribed into an optimized nucleic acid for expression in bacteria with end restriction sites for cloning into pET28a designed to include an amino terminus 6xHis tag. A synthetic gene fragment (GeneArt, ThermoFisher) was cloned in a single step into the expression vector and the resulting clone was confirmed by sequencing. For the expression of recombinant protein, the plasmid was transformed into Escherichia coli BL21 (DE3) by standard techniques. Production was induced for 3h at 37 °C with 0.8 mM of isopropyl β-D-1-thiogalactopyranoside. Cells were collected by centrifugation and disrupted by sonification in lysis buffer (300 mM NaCl, 50 mM Tris, pH 8.0). Inclusion bodies were collected by centrifugation, washed in lysis buffer with 0.5% Triton-X100, and resuspended in lysis buffer with 6M urea and 20 mM imidazole. Proteins were solubilized overnight at 4 °C, clarified by centrifugation, and Dx-MAYV-M was purified by affinity chromatography on a HisTrap™ column (GE Healthcare, Piscataway, NJ) connected to an Aktä prime chromatography system (GE Healthcare, Piscataway, NJ). Urea was removed by a gradient wash followed by a two-step elution with 250 and 500 mM imidazole. Fractions with Dx-MAYV-M, as confirmed by SDS-PAGE, were diluted and applied to the HiTrap™ Q HP column (GE Healthcare, Piscataway, NJ) to remove imidazole and to concentrate the chimeric protein. The final Dx-MAYV-M preparation was quantified spectrophotometrically and confirmed by SDS-PAGE.
Synthesis of MAPs containing bispecific multi-antigen-peptides. For the preparation of dendrimeric bispecific multi-antigen peptides (MAP), was used a standard solid-phase synthesis protocol and the octameric Fmoc8-Lys4-Lys2-Lys-В-Ala Wang resin as described previously [47]. preparation of the dendrimer multi antigen peptides (MAPs), was used the protocol of synthesis in solid phase and the tetrameric Fmoc4-Lys2-Lys-β-Ala Wang resin as described previously. Each peptide epitope [nsP1-20 (RIRLLLQGGNGVKQTVD) and E2-11 (YRTFGAERGGS RTLDSR)] was linked in tandem with the addition of two extra Gly residues to improve the presentation of antigens to antibodies. Briefly, the construct was prepared in an automated peptide synthesizer (PSS8-model, Shimadzu, Kyoto, Japan) and the side chains of octafunctional Fmoc-amino acids were protected with TFA-labile protecting groups as required. Residues corresponding to the monovalent (‘tail’) part of the construct, up to the first (bis-Fmoc) Lys residue initiating the dendrimer structure, were incorporated via single couplings. Once sequence assembly was completed, the Fmoc groups were removed and the peptide-resin was cleaved and fully deprotected with TFA/H2O/EDT/TIS (94/2.5/2.5/1.0 v/v, 90 min). The peptides were precipitated by the addition of chilled diethyl ether followed by centrifugation. The resulting pellet was taken up into aqueous AcOH (10% v/v), dried, and stored as a lyophilized powder. When necessary, the MAP was dissolved in water, centrifuged (10,000 g, 60min, 15 oC) and the supernatant filtered by a centricon filter. Synthetic peptides were purified by high-performance liquid chromatography (HPLC) using a reverse-phase column (Vydac C18) and characterized by mass spectrometry at the National Institute of Quality Control on Health (INCQS) of FIOCRUZ, Rio de Janeiro, Brazil.
In-house ELISA optimization and procedure. Optimal target quantities along with dilution of patient serum and secondary antibody were determined by a checkerboard titration [48]. After optimization, Dx-MAYV-M chimera was used at 300 ng/well in coating buffer (0.05 M carbonate-bicarbonate, pH 9.6) to sensitize high-binding 96-well microplates (Jet Biofil®, Guangzhou, China) overnight at 4 °C. Microplates were rinsed and blocked with TBSM (TBS with 1% fat-free dry milk) for 1h at 37 °C and washed with TBST before the addition of patient serum samples (100 μl, diluted 1:150 in TBS). Plates were incubated for 90 min at 37 °C, washed in TBST, and then incubated with alkaline phosphatase-conjugated goat anti-human IgM (1:20,000) for 90 min at 37 °C. Next, plates were washed five times before the addition of 100 μl of PNPP (p-nitrophenyl phosphate, 1mg/ml, Thermo Fisher, USA). After a 15 min incubation at room temperature in the dark, the absorbance at 405 nm was measured in a FlexStation® 3 Multi-Model microplate reader (Molecular Devices, USA).
Molecular modeling. As three-dimensional structure data does not exist for Dx-MAYV-M, a theoretical model from the primary sequence of the chimeric protein was generated on the I-TASSER server (http://zhanglab. ccmb.med.umich.edu/I-TASSER/), which combines the methods of threading, ab initio modeling, and structural refinement [49]. A model with a TM-score value greater than 0.70 and a reasonable quality c-score was chosen for visualization using Visual Molecular Dynamic (VMD) 1.9 [50].
Statistical analysis. Data were encoded and analyzed using scatter computer graphic software (GraphPad Prism version 6, San Diego-CA, USA). Descriptive statistics were presented as geometric mean ± SD. Reactivity performance of the enzyme-linked immunosorbent assay was evaluated by receiver operating characteristic curve (ROC) curve analysis. The ROC curve also produces a table that relates the specificity, sensitivity, and cutoff (cutoff). The cutoff selection criteria were 100% specificity and 100% sensitivity. To test the normality of datasets, the D'Agostino & Pearson test and Kologorov-Smirnv test followed by Student's t-test was used, and the variance homogeneity assumption was confirmed. The ANOVA (Brown-Forsythe and Welch) parametric test was chosen for analysis of variance with confidence intervals. This index is referred to as reactivity index (RI) and all results <1.00 were considered negative. However, samples were deemed inconclusive (or in the gray zone) if the RI values fell into the undetermined zone, which was hypothesized as RI values of 1.0 ± 10%.