Ptarget

Murine pre-B cells L1.2 were cultured in RPMI 1640 containing 1 mM L-glutamine, 1 mM sodium pyruvate, 10% FBS and 50 μM 2-mercaptoethanol. Transfections of L1.2 cells were performed using Amaxa nucleofector (Lonza) (program U-015), according to the manufacturer’s instructions. Cells were plated at 1 × 10⁶/mL and, after 24 h, 4 × 10⁶ cells were nucleofected with 3 mg endotoxin-free plasmids pTarget or pTarget-CXCR3A. The vector pTarget was from Promega and was used as negative control. In pTarget-CXCR3A construct (a kind gift from Alan Wells, Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA) CXCR3A (NM_001504.1) was cloned into the EcoRI and SalI sites of pTarget. After 4 h from transfection, in order to enhance CXCR3A cell surface expression, transient transfectants were incubated overnight at 37 °C in medium supplemented with 10 mM sodium butyrate (Sigma-Aldrich). The transfection efficiency was evaluated as GFP expression by flow cytometry using control vector pmaxGFP included in Amaxa nucleofector kit. Cell surface expression of CXCR3A was evaluated by flow cytometry using an anti-human CXCR3 PE-conjugated mAb (Thermo Fisher Scientific).

The ompL37 PCR product amplified from L. interrogans sv. Copenhageni strain Fiocruz L1-130 genome, as described above, was cloned into the E. coli expression vector pAE [27 (link)] for expression of an OmpL37 recombinant protein (rOMPL37) with an N-terminal 6×His tag. For construction of the DNA vaccine, ompL37 was amplified using the primers OmpL37-pTargeT-For (5ˈ-ACCATGGGAGATCAGATCAACTTAG) and OmpL37-Rev and cloned into the mammalian expression vector pTargeT (Promega, Madison, WI, USA). Both plasmid constructs were confirmed by PCR, restriction digestion and DNA sequencing.

The pTARGET (Promega Corporation, Madison, WI, USA) vector, containing the cDNA of wild type or mutated human pendrin [9 (link)], was used for functional tests.
The pEYFPN1 vector (Clontech, Mountain View, CA, USA), containing the cDNA of wild type or mutated human pendrin, was used for co-localization and determination of pendrin expression levels via imaging. Following transfection of this construct in cells, pendrin is produced with the enhanced yellow fluorescent protein (EYFP) fused to its C-terminus [27 (link)].
Sequence alterations in the cDNA of pendrin were obtained using the QuikChange^® site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) and the primers listed in the Table S6.
The sequence of all plasmid inserts was verified by Sanger sequencing (Microsynth AG).

For pseudovirus production, full-length rev-env cassettes were cloned into one of two mammalian expression vectors, pcDNA 3.1 Directional/V5-His-TOPO (Invitrogen, Carlsbad, CA) or pTarget (Promega, Madison, WI). The amplicon sequence selected for cloning was the one closest to a participant’s consensus that was generated from at least five sequences. The resulting clones were sequenced to ensure an exact match to the original amplicon sequence and where cloned inserts differed from the parental sequence, mutagenesis was performed to ensure a match with the parental sequence. Env-pseudotyped viruses were generated by co-transfecting envelope clones with a clade B backbone pSG3ΔEnv (NIH AIDS Research and Reference Reagent Program) in HEK293T cells as previously described [27 (link),28 (link)]. Pseudovirus functionality was determined by measuring luciferase expression after infecting TZM-bl cells (NIH AIDS Research and Reference Reagent Program, ARRRP). Relative luminescence units (RLUs) of ≥100,000 were considered ideal and 30,000 RLUs were accepted in cases where readings were 2.5 x the background; <2.5 times the background were considered negative.