Repurposing of the anti-malaria drug chloroquine for Zika Virus treatment and prophylaxis

Abstract

One of the major challenges of the current Zika virus (ZIKV) epidemic is to prevent congenital foetal abnormalities, including microcephaly, following ZIKV infection of pregnant women. Given the urgent need for ZIKV prophylaxis and treatment, repurposing of approved drugs appears to be a viable and immediate solution. We demonstrate that the common anti-malaria drug chloroquine (CQ) extends the lifespan of ZIKV-infected interferon signalling-deficient AG129 mice. However, the severity of ZIKV infection in these mice precludes the study of foetal (vertical) viral transmission. Here, we show that interferon signalling-competent SJL mice support chronic ZIKV infection. Infected dams and sires are both able to transmit ZIKV to the offspring, making this an ideal model for in vivo validation of compounds shown to suppress ZIKV in cell culture. Administration of CQ to ZIKV-infected pregnant SJL mice during mid-late gestation significantly attenuated vertical transmission, reducing the ZIKV load in the foetal brain more than 20-fold. Given the limited side effects of CQ, its lack of contraindications in pregnant women, and its worldwide availability and low cost, we suggest that CQ could be considered for the treatment and prophylaxis of ZIKV.

Figure 1

CQ inhibits ZIKV infection and apoptosis in human neurospheres. (a–d) Human iPSC-derived neurospheres were infected with ZIKV^BR (multiplicity of infection = 1) and immediately treated with CQ at 5 µM, 20 µM, or 40 µM. MOCK = uninfected neurospheres. DMSO = DMSO treated neurospheres. Quantification of (a) neurosphere size at 96 h post-infection, (b) ZIKV-positive cells, (c) cleaved caspase 3 (CC3)-positive cells, and (d) CC3 and ZIKV double-positive cells. Data are the mean ± SEM of triplicates. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared with “ZIKV” one-way ANOVA with Dunnett’s multiple comparisons test.

Figure 2

CQ attenuates ZIKV infection in AG129 mice. (a) Survival curves for control and CQ- treated mice (n = 6 and 5, respectively). Note that 60% of the CQ-treated mice were euthanized on day 15. p = 0.0075 by log-rank Mantel–Cox test. (b) Weight loss following ZIKV infection in control and CQ-treated mice. Data are the mean ± SEM. *p < 0.05, **p < 0.01 compared with controls by unpaired t-test with Welch’s correction. (c,d) Visual appearance health scores in ZIKV-infected control (c) and CQ-treated (d) mice. Note that by day 13, all control mice had died but 60% of CQ-treated mice were alive.

Figure 3

Mouse model of chronic ZIKV infection for testing therapeutic interventions. (a) Female (n = 5) and (b) male (n = 8) SJL mice were infected with 10⁸ PFU ZIKV^BR retro-orbitally. Blood samples were taken every 3 days for 50 days and analysed for viral RNA by qRT-PCR. (c) qRT-PCR analysis of viral RNA in the testes of male mice 3 months after ZIKV^BR infection. Mean ± SEM of 8 mice.

Figure 4

Horizontal and vertical sexual transmission of ZIKV in SJL mice. (a) Efficient sexual transmission of ZIKV^BR was observed from infected males to uninfected females, but not from infected females to uninfected males. SJL male and female mice (n = 5) were infected with ZIKV^BR (10⁸ PFU retro-orbitally) and co-housed for 14 days with uninfected females and males, respectively. Viral RNA was measured by qRT-PCR of serum samples obtained from the sires and dams on day 14. Each point represents one animal. Data are the mean ± SD. n.d.: not detected (b) Vertical transmission of ZIKV^BR to the offspring of infected sires and dams. RNA from the newborn heads was prepared on postnatal day 1 for qRT-PCR analysis of viral RNA. Each point represents one day 1 pup. Data are the mean ± SEM of the offspring of 4 infected dams/uninfected sires or 4 infected sires/uninfected dams as indicated.

Figure 5

CQ represses ZIKV infection in SJL mice and reduces vertical transmission. (a) Schematic of the experimental design. SJL dams were infected with ZIKV (2 × 10⁵ PFU) on E12.5. On E13.5, they were randomly assigned to receive vehicle or CQ (30 mg/kg/day) in the drinking water. On E18.5, mice were euthanized for collection of blood and foetuses. (b) qRT-PCR of viral RNA. Each point represents one animal. Data are the mean ± SD of 3 (vehicle-treated) or 5 (CQ-treated) mice. *p < 0.05 by Student’s t-test. (c) qRT-PCR of viral RNA in foetal head extracts. Each point represents one foetus. Data are the mean ± SD of 8 foetuses pooled from 3 independent litters (control) or 5 foetuses pooled from 2 independent litters (CQ). *p < 0.05 by Student’s t-test. (d) Representative images of foetal brain sections from control, ZIKV-infected, and ZIKV-infected/CQ-treated mice on E18.5. Sections were stained with a primary antibody against Flavivirus Group Antigen (brown) and counterstained with Mayer’s hematoxylin (blue). Scale bar, 4 mm. (e) Quantification of ZIKV-infected cells in foetal brain sections from control, ZIKV-infected, and ZIKV-infected/CQ-treated mice. Data are the mean ± SEM of 6 sections per condition (3 embryos, 2 sections per embryo). *p < 0.05 compared with untreated ZIKV-infected mice by one-way ANOVA with Dunnett’s multiple comparisons test.