Fungizone

The HeLa and A431 cell lines were maintained in Eagle's Minimum Essential Medium supplemented with 10% FBS and 1% antibiotics (100 U/mL penicillin, 100 µg/mL streptomycin) and 250 µg/mL fungizone. The cells were grown at 37°C in a humidified incubator set at 5% CO₂. Cells were subcultured by treating them with trypsin-EDTA (0.25% trypsin containing 0.01% EDTA) for 10 minutes.
Cytotoxicity was measured using the XTT cell proliferation Kit II and MPBA. The method described by Zheng et al. [8 (link)] was used to perform the assay. Both cell lines were seeded in a 96-well microtitre plate at a concentration of 1 × 10⁵ cells/mL. Cells were allowed to attach for 24 hrs at 37°C and 5% CO₂. The cells were exposed to the positive drug control Actinomycin D (Sigma-Aldrich, South Africa) with concentrations ranging between 0.5 µg/mL and 0.002 µg/mL. The microtitre plate was incubated for further 72 hrs and thereafter 50 µL XTT was added to a final concentration of 0.3 mg/mL to one set of plates and 20 µL PrestoBlue was added to another set of plates. The plates were incubated for further 2 hrs where after the absorbance of the colour complex was read at 490 nm with a reference wavelength set at 690 nm for XTT and at 570 nm with a reference wavelength set at 600 nm for PrestoBlue, using a BIO-TEK Power-Wave XS multiwell plate reader.

Viral strains used were provided by WHO Collaborating Center for arboviruses and viral hemorrhagic fever (CRORA) at the Institut Pasteur de Dakar. ZIKV and other flaviviruses strains isolated from mosquitoes and non-human vertebrates used in this study are described in Tables 1 and 2. Viral stocks were prepared by inoculating viral strains into AP 61 monolayer continuous cell lines in Leibovitz 15 (L-15) growth medium (GibcoBRL, Grand Island, NY, USA) supplemented with 5% foetal bovine serum (FBS) (GibcoBRL, Grand Island, NY, USA), 10% tryptose phosphate, penicillin-streptomycin and fungizone (Sigma, Gmbh, Germany). After 7 days of propagation, viral infection was tested by an indirect immunofluorescence assay (IFA) using specific hyperimmune mouse ascitic fluids as previously described [23 (link)] and supernatants from infected cells were collected as stocks for virus RNA isolation. ZIKV stocks were used for sequencing and evaluation of the sensitivity of the rRT-PCR assay. Other flaviviruses were used to evaluate the specificity of the assay.

Ae. albopictus mosquitoes (kindly provided by Illia Rochlin, Suffolk County Health Department, Yaphank, NY, USA) were originally collected in Suffolk County in 2014 and subsequently colonized in the NYSDOH Arbovirus Laboratory. F5–F7 female mosquitoes from New York were used for experimental feedings. Ae. aegypti mosquitoes used for preliminary experiments were collected by C. Mangudo in Salta, Argentina, in 2014 and initially colonized by V. Micieli and L.D. Kramer at the Centro de Estudios de Parasitología y Vectores (La Plata, Argentina) before being shipped to the NYSDOH Arbovirus Laboratory for maintenance. F4–F5 females from Argentina were used for experimental feedings. Ae. aegypti mosquitoes (kindly provided by G.D. Ebel, Colorado State University, Fort Collins, CO, USA) were originally collected in Poza Rica, Mexico. F7–F8 females from Mexico were used for experimental feedings. For preliminary blood feeding experiments, Ae. aegypti mosquitoes from Argentina were fed Zika virus PR stock virus diluted 1:1, 1:5, or 1:20 in defibrinated sheep blood (Colorado Serum Co., Denver, CO, USA) with 2.5% sucrose. For feedings with freshly propagated virus, supernatant from infected C6/36 cultures was harvested at 96 h after infection (multiplicity of infection ≈1.0) and diluted 1:1 with blood-sucrose mixture without freezing. Female mosquitoes, 4–7 days of age, were deprived of sucrose for 18–24 h and offered blood meal mixtures by use of a Hemotek membrane feeding system (Discovery Workshops, Acrington, UK) with a porcine sausage casing membrane. For all subsequent experiments assessing dose-dependent vector competence, similarly prepared fresh C6/36 cultures of Zika virus HND and Zika virus CAM were used to feed Ae. aegypti mosquitoes from Mexico and Ae.albopictus mosquitoes from New York. In addition to undiluted supernatant, 1:20, 1:400, and 1:8,000 dilutions were made in C6/36 maintenance media before being mixed with blood.
For all blood feeding experiments, mosquitoes were sedated with CO₂ after 1 h of feeding, and fully engorged mosquitoes were transferred to 0.6-L cartons and maintained at 27°C for experimental testing. Infection, dissemination, and transmission rates were determined as previously described (24 (link)) on day 14 or 21 after feeding. After the mosquitoes were sedated, the legs were removed from 12–30 mosquitoes and placed in 1 mL mosquito diluent (20% heat-inactivated fetal bovine serum in Dulbecco phosphate-buffered saline plus 50 μg/mL penicillin/streptomycin, 50 μg/mL gentamicin, and 2 μg/mL Fungizone [Sigma Aldrich, St. Louis, MO, USA]). For 30 minutes, mosquitoes were allowed to expectorate into capillary tubes containing ≈20 μL fetal bovine serum plus 50% sucrose (1:1), at which time the mixture was ejected into 250 μL mosquito diluent. Mosquito bodies were then placed in individual tubes with mosquito diluent. All samples were held at −80°C until tested. To test for infection, dissemination, and transmission, we processed and screened bodies, legs, and salivary secretions, respectively, by Zika virus–specific quantitative reverse transcription PCR (25 (link)). Zika virus body titers were calculated from standard curves based on infectious particle standards created from matched virus stocks. Data were analyzed by using GraphPad Prism version 4.0. Rates were compared by using Fisher exact tests, and dose dependence was evaluated and compared by using linear regression analyses.

HeLa cell lines (ATCC CCL2) used were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS; 10270106; Thermo Fisher Scientific), 1% Penicillin/Streptomycin (15140122; Thermo Fisher Scientific), and 0.1% Amphotericin B (Fungizone; 11510496; Thermo Fisher Scientific). Cell lines were cultured as a monolayer at 37°C and 5% CO₂. HeLa H2B-GFP, mCherry-Tubulin cell line was generated by transfecting mCherry-Tubulin expressing eukaryotic plasmid vector into HeLa H2B-GFP cells (Draviam et al., 2006 (link)). The HeLa mKate2-EB3 cell line was generated by transfecting a pmKate2-EB3 plasmid vector (#FP316; Evrogen) into HeLa cells. Plasmid transfection was carried out using DharmaFECT duo (T-2010; Dharmacon). The HeLa H2B-GFP, SiR-Tubulin cell line was generated by adding SiR-Tubulin dye, a paclitaxel-based fluorescent compound (Lukinavičius et al., 2014 (link); Spirochrome SC002; 100 nM) just 1 h before imaging. The HeLa FRT/TO cell line expressing siRNA-resistant MARK2-YFP-WT or KD mutant was generated by transfecting a Tet-inducible expression vector encoding siRNA-resistant MARK2-YFP-WT or KD, followed by colony picking (Zulkipli et al., 2018 (link)). Vectors bearing point mutants of MARK2 were generated by polymerase chain reaction–based point mutagenesis and confirmed by DNA sequencing (Hart et al., 2019 (link)). Fluorescent cells were enriched using a BD FACSAria III Cell Sorter for fluorescence-activated cell sorting (FACS).

Stock solutions (10 mM) of the peptides were prepared in 5% DMSO in bidistilled water and kept at −20°C Serial dilutions were carried out in HBSS/HEPES buffer (20 mM, containing 0.02% bovine serum albumin fraction V). Calcium mobilization assay was performed using the same method as previously described (Camarda and Calo, 2013 (link)). CHO cells with stable co-expression of human mu or kappa receptors and the C-terminally modified Gα_qi5 and CHO cells with co-expression of the delta-opioid receptor and the Gα_qG66Di5 protein were used. Dulbecco’s MEM/HAMS F12 (1:1) medium supplemented with 10% fetal bovine serum, penicillin (100 IU/mL), streptomycin (100 μg/mL), L-glutammine (2 mM), fungizone (1 μg/mL), geneticin (G418; 200 μg/mL) and hygromycin B (100 μg/mL) was used for cell culture. Cells were seeded at a density of 50,000 cells/well into 96-well black, clear-bottom plates and kept in the incubator at 37°C in 5% CO₂/humidified air. After 24 h the cell growth medium was aspired and loading medium, supplemented with probenecid (2.5 mM), calcium sensitive fluorescent dye Fluo-4 AM (3 µM), pluronic acid (0.01%) and HEPES (20 mM), was added. Then the plates were placed in the incubator again. After 30 min the loading solution was aspirated and 100 µL/well of assay buffer (HBSS supplemented with 20 mM HEPES, 2.5 mM probenecid, and 500 µM Brilliant Black) was added. Next, both plates (cell culture and compound plate) were placed in the FlexStation II reader (Molecular Device, Union City, CA 94587, United States), the on-line additions were carried out in a volume of 50 µL/well and the fluorescence changes were measured. Ligand efficacies, expressed as the intrinsic activity (α), were calculated as the E_max ratio of the tested compound and the standard agonist. At least three independent experiments for each assay were carried out in duplicate.
Curve fittings were performed using Graph Pad PRISM 5.0 (GraphPad Software Inc., San Diego, United States). Data have been statistically analyzed with one way ANOVA followed by the Dunnett’s test for multiple comparisons; p values < 0.05 were considered significant.

To assess the presence of PD-L1 and ARG1-specific lymphocytes, we performed a delayed-type hypersensitivity (DTH) test. DTH tests were assessed at baseline and at EOT. Briefly, we administered intradermal injections of the two peptides, without the adjuvant, at the lower back. The peptides were dissolved in sterile water and DMSO. We also administered a control injection, which included water and DMSO, but no peptide. At 48 h after the injection, skin reactions (induration) were measured; additionally, at the sites of ArgLong2- and PD-L1long1 injections, punch biopsies were acquired and cut into fragments. To identify fragments that contained skin-infiltrating lymphocytes (SKILs), fragments were cultured in 24-well plates in RPMI-1640 with 10% human serum and 100 U/mL interleukin-2 (IL-2), with penicillin, streptomycin, and fungizone, for 3–5 weeks to allow SKIL outgrowth. Every second or third day, half the medium was replaced with fresh medium containing IL-2, penicillin, streptomycin, and fungizone. After 3–5 weeks, SKILs were harvested, and a fraction was tested in ELISPOT assays. The remaining SKILs were cryopreserved.