1. Introduction
Chikungunya fever (CHIKF) is caused by chikungunya virus (CHIKV), which is an arthropod-borne virus that was first identified in Tanzania in 1952–1953 [
1]. Acute infection is usually characterized by severe arthralgia and myalgia, which may persist for months or years after the initial febrile episode (for review, [
2]). The introduction of CHIKV into the Americas in December 2013 through the Caribbean islands has led to a resulting 2.3 million suspected cases, with more than 541 associated deaths as of December 2017 (PAHO;
http://www.paho.org).
Despite extensive studies, no effective antiviral drug is available for CHIKV prevention or treatment (for review, [
3]). Numerous compounds demonstrated anti-CHIKV properties in cell cultures, however failure to evaluate these compounds in suitable animal models has limited their potential use in humans [
4]. Current treatments have been mainly palliative using antipyretic, analgesic, and anti-inflammatory drugs [
5]. Chloroquine, which is a typical anti-malaria drug, has displayed antiviral properties in vitro [
6,
7,
8,
9,
10,
11]. CHIKV infection in Vero E6 cells was strongly inhibited following chloroquine treatment [
12,
13]. It was thus shown that chloroquine inhibit the early step of the viral infection in pre-treatment assay by modifying the endosomal pH, but also at some extant budding stage when being used as co-treatment [
10,
13]. The effectiveness of chloroquine in protecting against CHIKF was assessed in a double-blinded, placebo-controlled randomized trial in Reunion Island “CuraChik” conducted during the Reunion Island outbreak in 2006 [
12,
14]. Despite its antiviral potential, oral chloroquine treatment for five days in patients with acute chikungunya did not protect against severe disease. Moreover, the first analysis has shown that chronic arthralgia on day 300 post illness onset, was more frequent in patients receiving chloroquine [
12,
15]. This suggests that chloroquine may have exacerbated the disease and/or suppressed the antiviral immunity, leading to a chronic disease [
12].
To understand the reasons behind the failure of chloroquine treatment in CHIKV-infected patients, we performed two complementary studies: (i) a prophylactic chloroquine treatment in a preclinical non-human primate (NHP) model of CHIKV infection [
16], and (ii) a retrospective study of immunological parameters in patients that were recruited to the “CuraChik” clinical trial that was conducted during the Reunion Island outbreak in 2006.
4. Discussion
African populations have made use of the anti-inflammatory properties of chloroquine for decades to cure febrile illnesses that were presumed to be malaria (personal observation). Chloroquine is also widely used against auto-immune diseases, like lupus or rheumatoid arthritis [
29]. During the CHIKV outbreak in the Indian Ocean in 2005–2006, an increase in chloroquine use by the population of Reunion Island (an area free of malaria) was observed [
30]. Experimental results in vitro suggested that chloroquine significantly inhibited CHIKV replication (consistent with previous data obtained with other alphaviruses) [
12,
31,
32]. This raised important public health issues and led to a double-blind placebo-controlled randomized trial, that was conducted on Reunion Island and included adult patients with acute febrile arthralgia during the 2006 CHIKV epidemic. These patients received 600 mg of chloroquine per day for three days and then 300 mg for two days [
12]. Here, we conducted a preventive trial carried out in NHPs, a well-established animal model for studying CHIKV pathogenesis [
16], which was designed to assess whether the use of this drug could prevent CHIKV epidemic extension. The dose and administration route were determined on the basis of chloroquine pharmacokinetics in humans, to ensure that the data presented would be comparable and reproducible [
23]. Neither trial reported a significant therapeutic effect. Moreover, the chosen regimen expected to be more efficient, according to the in vitro data (15 days of treatment initiated five days before infection) exacerbated acute chikungunya disease in the macaques.
This discrepancy between the efficacy of chloroquine in in vitro experiments involving Vero-E6 cells [
12,
13] and the results of clinical testing could be accounted for by the unfavorable balance of the antiviral and immunomodulatory effects of chloroquine in vivo. CHIKV induces IFN-β in fibroblasts, allowing for the intrinsic control of infection [
33]. Conversely, chloroquine inhibits IFN-I responses in other paradigms [
34] and these deleterious effects might have been missed in Vero-E6 cells, which do not produce IFN-I [
35]. On the other hand, monocytes [
36,
37] and macrophages [
16,
22] are IFN-competent cells that are susceptible to CHIKV infection and are also critical to CHIKV pathogenesis. Given our observed antiviral effects of chloroquine in NHP-derived macrophages, the failure of chloroquine treatment in preventing and treating CHIKV infection in NHPs would reflect the undesired effects of chloroquine. Chloroquine exacerbates infection in other animal models by inducing a greater proinflammatory cytokine profile in Semliki forest virus (SFV) and encephalomyocarditis virus infected mice [
38,
39]. These two studies reported the effects on viral load similar to those describe here, however it was difficult to reconcile the observation with the known anti-inflammatory properties of chloroquine [
40]. Chloroquine has known immune-regulating properties and has been used to treat rheumatoid arthritis and lupus erythematosus by reducing the inflammatory mediators that are present during the acute-phase response [
40,
41,
42].
Whereas, the viremia in the placebo group was cleared by 5 dpi, CHIKV vRNA remained detectable in chloroquine-treated NHPs up to 12 dpi, two days after the end of treatment. IFN-I production was not impaired and was correlated with viral load, which is consistent with human data from Reunion Island [
33]. Previous studies [
33,
43] have indicated a probable role of IFN-I in controlling viral replication. Nonetheless, despite a decrease of four to five orders of magnitude in virus levels between 5 and 7 dpi, chloroquine treatment in NHPs did not result in the completed clearance of CHIKV (
Figure 2C). This indicates that the IFN-I-driven response is not capable of complete virus clearance in the treated macaques, a notion that was previously proposed by Werneke et al. [
43]. This highlights the potential underlying immune-deficiencies that may prolong viremia during chloroquine treatment.
In the curative trial in human patients, chloroquine treatment did not modify the clinical and biological status, or the virus levels between days 1 and 3. Interestingly, despite a randomization procedure at baseline on CHIKV viral load, the immunological assays of the first sample revealed that patients that were included in the chloroquine group had a more severe disease (higher levels of IFNα, IL-6, IL-8, and MCP-1). This concordance with the higher levels of CRP observed in the chloroquine group at inclusion. To assess the potential impact of chloroquine administration in CHIKV treatment, we compared the profile of the immunological markers overtime between the two groups using thre GEE approach We found that chloroquine treatment was associated with a faster decrease of the level of Eotaxin, IL-6 and MCP1 over time. Furthermore, 300 days after treatment, advanced age, female gender and chloroquine treatment were independently associated with persistent arthralgia at D300. This may reflect the negative impact of chloroquine on the immunological response favoring a possible delayed of the CHIKV clearance, as it was shown in the NHP model.
Thus, the clearance of residual CHIKV depends on other antiviral responses that may be impaired by chloroquine treatment. We found that both CHIKV-specific humoral and cellular responses were delayed at 15 dpi. Subsequently, these responses recovered and were of similar levels to those that were observed in the placebo group at 23 dpi (13 days after the end of chloroquine treatment) (see
Table 2 and
Figure 5). Antigen-specific responses depend on the quality of antigen processing and presentation, which may be affected by chloroquine treatment [
44]. Chloroquine inhibits TLR3 signalling, which is an important pathway in the response to viral infections [
45], and antigen presentation on MHC class II molecules [
44,
46,
47]. Possibly, this also impaired helper T cell activation throughout treatment, accounting for the hampered antigen-specific responses in chloroquine-treated macaques both from B cells (
Table 1) and cytotoxic T cells (CTL,
Figure 5). Conversely, chloroquine also promotes MHC class I presentation and CD8+ CTL responses [
46,
48], which would result in an immune boost situation rather than initial priming by CD4+ T cells. Thus, the initiation of continuous treatment for five days before CHIKV infection may have suppressed the anti-CHIKV response and concealed the anti-CHIKV activity of chloroquine. Viremia was detected one day later in chloroquine-treated macaques (2 versus 1 d.p.i. in the placebo group), suggesting that antiviral activity did occur in vivo, but it was likely to be outweighed by the immuno-suppressive effect of chloroquine. This highlights the importance of choosing the correct dose and administration schedule in achieving the best treatment outcome. For example, a single dose at peak fever, after antigen presentation to CD4+ helper lymphocytes has been initiated, might improve the recovery phase if it increases the CTL response [
48]. The macaque model of CHIKV will be useful for testing this and other hypotheses.