Mar1 5a3

Mice were pretreated with 2 mg of either MAR1-5A3 or mouse IgG₁ isotype control antibody (Leinco Technologies, Inc.) 1 day prior to infection with 1000 CFUs of Salmonella Typhimurium.

In some experiments, mice were treated with 2 mg of an Ifnar1-blocking antibody (MAR1-5A3, Leinco Technologies) by intraperitoneal injection one-day before virus infection. Mice were inoculated with ZIKV by a subcutaneous (via footpad) route with 10⁴ to 10⁶ FFU PFU of ZIKV in a volume of 50 μl PBS or by an intracranial route with 10⁴ FFU of ZIKV in a volume of 10 μl PBS. For survival studies, mice were monitored for 21–45 days. For enhanced safety, all mouse infection experiments with ZIKV were performed under A-BSL3 conditions.

DCs were cultured in 1.5 × 10⁵ cells/well in 200 μl, and where indicated pre-treated for 24h with CpG-A (5 μg/mL; InvivoGen), IFN-α (100U/ml, PBL Assay Science), Imiquimod (6 μM, Calbiochem), Poly I:C (25 μg/ml, Sigma), etomoxir (200 μM, Tocris Biochemicals), 5-(tetradecyloxy)-2-furoic acid (TOFA; 20 μM, Sigma Aldrich), UK-5099 (50 μM, Sigma Aldrich), MAR1-5A3 (5 μg/ml, Leinco Technologies) or GW6471 (3 μM; Tocris Biochemicals). pDCs were incubated overnight with 100 μM Gemfibrozil or 100 μM Muraglitazar (Santa Cruz). IFNα was detected using a mouse IFNα elisa kit (PBL assay science). TNF-α and IL-6 were measured using BD™ Cytometric Bead Array (CBA).

Wild-type male C57BL/6J mice (4 weeks of age) were purchased from Jackson Laboratory and housed in groups of up to 5 mice/cage at 18-24°C ambient temperatures and 40-60% humidity. Mice were fed a 20% protein diet (PicoLab 5053, Purina) and maintained on a 12 hrs light/dark cycle (6 am to 6 pm). For studies involving ZIKV challenge, mice were treated with 2 mg of an Ifnar1-blocking antibody (MAR1-5A3, Leinco Technologies) by i.p. injection one-day before virus inoculation. For prophylaxis experiments, mice were treated i.p. one day before virus challenge with purified individual mAbs and monitored for 21 days for survival. For therapeutic protection experiments, mice were treated i.p. one day after virus challenge with varying defined doses of purified individual Abs and monitored for 21 days for survival. For antibody-encoding RNA delivery studies, mice were inoculated with 40 μg of individual RNA by an i.m. route (into each quadricep and hamstring muscle groups) one day before virus challenge, with four injections of 50 μL of RNA/NLC complex per injection. For ZIKV infections, mice were inoculated by a subcutaneous (via footpad) route with 10³ FFU of ZIKV Dakar MA in a volume of 30 μL of PBS. Antibody ZIKV-117 was used as a positive control, and mAb FLU-5J8 or PBS were used as a negative control.

Ifnar1^−/− mice (Hwang et al., 1995 (link)) were backcrossed onto a C57BL/6 background. Mice were bred in a specific-pathogen-free facility at Washington University, or purchased (WT animals) from Jackson Laboratories. Mice were set up for timed-matings and at embryonic days E6.5 or E7.5 were inoculated with ZIKV by subcutaneous (footpad) route with 10³ FFU of ZIKV in 50 μl of PBS. Mice were sacrificed at E13.5, E15.5, or E16.5 depending on the experimental design. Placentas and fetuses were harvested from the infected mice. Fetus size was measured as the crown-rump length × occipito-frontal diameter of the head. In some experiments, WT mice were treated with indicated doses of an Ifnar blocking mouse MAb (MAR1-5A3) or isotype control mouse MAb (GIR-208) (produced by Leinco Technologies) (Sheehan et al., 2006 ; Sheehan et al., 2015 (link)) by intraperitoneal injection prior to and after ZIKV or DENV-3 infection.