2.1. Prophylactic Treatment in a Preclinical NHP Model

Three- to four-year-old male cynomolgus macaques (Macaca fascicularis), weighing 3–4 kg, were imported from Mauritius (negative for SIV, STLV, herpes B virus, filoviruses, SRV-1, SRV-2, measles, Dengue virus, and CHIKV) and were housed in a BSL3 facility (Permit Number A 92-032-02), in accordance with Office for Laboratory Animal Welfare (OLAW, Bethesda, MD, USA; #A5826-01) standards. Studies were approved by the regional animal care and use committee in accordance with European directive 63/2010/EU: “CREEA Ile de France Sud”, Fontenay aux Roses, decision #A08-012 dated 7 July 2008. Treatment and sampling procedures caused no suffering. At the end of the study, animals were sedated and euthanized by the intra-venous (i.v.) injection of a lethal dose of pentobarbital.

The CHIKV strain LR2006-OPY1 was used, as previously described [16]. The in vitro titer was 10⁸ CCID50/mL in BHK21 cells and 1.8 × 10¹⁰ ± 0.9 vRNA equivalents per mL. In vivo titer was obtained by infection of eight cynomologus macaques with serial dilutions of the virus stock. The 50% animal infectious dose (AID50) was estimated at 7.07 × 10³ ± 3.15 vRNA copies [17].

Animals were treated with chloroquine once daily either orally (dose of 7 or 14 mg/kg, 0.83 g/mL in saccharose syrup; 5 mg/mL chloroquine sulfate (Baby Nivaquine^®, Avantis-Pharma, Antony, France), or subcutaneously (14 mg/kg chloroquine diphosphate (Sigma, Saint Louis, MO, USA); 30 mg/mL in PBS. Subcutaneous treatments were thus 1.6 to 2 mL of a PBS solution. The days with treatment but not bleeding, animals received chloroquine after containment, but not sedation. Animals were sedated with ketamine (10 mg/kg, Rhône-Mérieux, Marcy l’Etoile, France) before handling. Clinical examinations, rectal temperature, and weight measurements were performed 10 min after sedation, before bleeding. Macaques were inoculated i.v. with 100 AID50 in 1 mL PBS, 1 h after the 6th treatment. Plasma chloroquine concentration was determined by high-performance liquid chromatography (HPLC), as described elsewhere [18]. Aspartate transaminase (AST), alanine transaminase (ALT), and Complement-Reactive Protein (CRP) levels were evaluated using Gamme DPC Kit AST/GOT or ALT/GPT (Thermo Electron Corporation/Thermo Fisher Scientific, Saint Herblain, France), following the manufacturer’s instructions. Complete blood count was obtained with a Micro Diff II apparatus (Beckman Coulter, Villepinte, France).

Human monocytes were isolated from PBMCs with CD14 magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) and were cultured for six days in 24-well plates containing DMEM (^©Glutamax, Gibco Lifesciences/Thermo Fisher Scientific, Saint Herblain, France), supplemented with 10% fetal calf serum (FCS, (PAA, Les Mureaux, France)), M-CSF and GM-CSF (10 and 1 ng/mL, R&D Systems, Abingdon, UK). Macaque fibroblasts were obtained from tendons after necropsy (macaque negative for CHIKV or Dengue virus infection as assessed by antibodies and PCR evaluation). Briefly, the tendons were minced and plated in 3 cm diameter Petri dishes that were saturated with FCS. Tendon pieces are immerged within DMEM, 20% FCS and explant are cultured at 37 °C, 9% CO2, water saturated atmosphere. After 7 to 10 days, fibroblasts might be seen migrating from the explants that should be discarded (Supplementary Figure S1A). Then, the cells were passaged every three days in DMEM, 10% FCS, and stable up to 13 passages. The resulting macrophages (Day 7 post-isolation, 10⁵ cells/well) or fibroblasts (passage 5, day 23 post-isolation, 4 × 10⁵ cells/well), respectively, were incubated with various concentrations of chloroquine for 24 h, before being infected by incubation with CHIKV (multiplicity of infection (MOI) at 1 or 3.4, respectively) for two hours at 37 °C. They were then thoroughly washed five times and 900 µL of culture medium supplemented by chloroquine at the same concentration was subsequently added.

Viral RNA was prepared from 100 µL of cell-free plasma collected in EDTA tubes, and quantified, as previously described [16]. The standard RNA template dilution gave a correlation coefficient of 98–99% over seven orders of magnitude, with a sensitivity of 10³ vRNA/mL or 20 copies per sample.

Inactivated purified CHIKV (kindly provided by Alere, Brisbane, Australia) was immobilized on 96-well Maxisorp plates (Nunc, Roskilde, Denmark). Wells were blocked by overnight incubation at 4 °C with 0.05% Tween-20 (v/v), 5% non-fat dried milk (w/v) in PBS (PBST-milk). Plasma samples were diluted 1:150 to 1:109,000 in PBST-milk and were incubated for one hour at 37 °C in the plates. Horseradish peroxidase (HRP)-conjugated anti-rhesus IgG and anti-human IgM (Southern Biotech, Birmingham, AL, USA) were used to detect macaque IgG and IgM, respectively. Reactions were developed with TMB substrate (3, 3′, 5, 5′–Tetramethylbenzidine, Sigma) and were terminated by adding stop reagent (Sigma). Samples from non-infected NHPs were used as controls. ELISA was performed in duplicate.

IFNα/β bioassay was performed as previously described [19]. Plasma levels of selected cytokines and growth factors were measured using the Miliplex Non-Human Primate Cytokine Magnetic Bead Panel for 23 soluble markers: GM-CSF, TGFα, G-CSF, IFNγ, IL-2, IL-10, IL-15, sCD40L, IL-17, IL-1Ra, IL-13, IL-1β, IL-4, IL-5, IL-6, IL-8, MIP-1α, MCP-1 (CCL2), TNFα,MIP-1β, IL-12–23(p40),IFNγ,IL-18; or, the Human Cytokine assay Magnetic Bead on 10 soluble factors: Eotaxin, GM-CSF, IFNα2, IL-12(p70), IL-1Ra, IL-6, IL-8, IP-10, MCP-1, TNFα, respectively (Millipore, Darmstadt, Germany), according to the manufacturer’s instruction. Data was acquired with a Bio-Plex Instrument 200 and analyzed with Bio-Plex Manager Software version 6.1 (Bio-Rad, Hercules, CA, USA). IFNγ ELISPOT was performed, as previously described [20], with heat-inactivated CHIKV (45 min, 56 °C twice; 10⁷ virions per well in 100 µL).