Therapeutic Immunomodulation for COVID-19 Treatment

Chloroquine: Modes of Action and Immunological Impact
Chloroquine (CQ) and its derivative hydroxychloroquine (HCQ) have gained traction as possible therapeutics for COVID-19. Both drugs are used as antimalarial agents and as immunomodulatory therapies for rheumatologic diseases. However, the application of CQ and HCQ to COVID-19 stems from their past use as antivirals (Savarino et al., 2003), including for SARS-CoV-1 (Keyaerts et al., 2004, Vincent et al., 2005). CQ and HCQ interfere with lysosomal activity and have been reported to have immunomodulatory effects. CQ augments antigen processing for MHC class I and II presentation, directly inhibits endosomal TLR7 and TLR9, and enhances the activity of regulatory T cells (Garulli et al., 2008, Lo et al., 2015, Schrezenmeier and Dörner, 2020, Thomé et al., 2013a, Thomé et al., 2013b). Early studies involving in vitro infection of host cells with SARS-CoV-2 demonstrated that both CQ and HCQ significantly impact endosomal maturation, resulting in increased sequestration of virion particles within endolysosomes. However, there has been conflicting evidence whether CQ is more potent than HCQ in reducing viral load (Liu et al., 2020d, Wang et al., 2020b, Yao et al., 2020a). Notably, one group reported that treatment of infected cells with HCQ before and during infection significantly reduced viral load, suggesting that combined prophylactic and therapeutic HCQ use yields maximum efficacy (Clementi et al., 2020). To better understand host immune responses to treatment, one group compared bulk transcriptomic changes in primary PBMCs treated with HCQ for 24 h to PBMCs from confirmed SARS-CoV-2 positive patients and controls, followed by a comparison of HCQ-treated primary macrophages to BAL and postmortem lung biopsies from COVID-19 patients (Corley et al., 2020). Across all comparisons, there was minimal overlap between host differential gene expression and genes altered by in vitro HCQ treatment. Thus, the potential mechanistic action of HCQ in the context of SARS-CoV-2 remains poorly defined.

Evaluation of HCQ Efficacy in Clinical Trials
Despite the apparent widespread use of HCQ and CQ to treat COVID-19 (Figure 6B), few controlled clinical trials have been performed so far and thus the potential benefits of these drugs for COVID-19 remains controversial. One of the earliest trials (2020-000890-25) was a single-arm, open-label trial of 600 mg daily HCQ in 20 COVID-19 patients. They reported that HCQ alone, or in combination with the antibiotic azithromycin (AZ), reduced viral load by day 6 (Gautret et al., 2020a). A follow-up trial in 80 patients treated with HCQ and AZ reported that 93% of patients had a negative PCR result on day 8 of treatment, and 81.3% were discharged within 10 days of treatment. However, it is important to note that both trials had no control arms (Gautret et al., 2020b). Rigorous statistical analyses by others that accounted for the patients excluded from the original analysis found limited evidence for HCQ monotherapy (Hulme et al., 2020, Lover, 2020). A double-blind RCT assessed HCQ monotherapy in the treatment of mild COVID-19 (ChiCTR2000029559) (Chen et al., 2020i). A total of 62 patients were enrolled; the treatment arm received 400 mg HCQ daily over 5 days. By day 6, patients who received HCQ had clinical resolution on average 1 day earlier than controls; no patients progressed to severe disease compared to four patients in the control arm. In a smaller RCT that treated 30 patients with mild COVID-19 (NCT04261517) with 400 mg HCQ for 7 days, there were no significant differences in the number of patients with negative PCR results on day 7 (all but one positive), median duration of hospitalization, time to fever resolution, or progression of disease on chest computed tomography (CT) (Chen et al., 2020d). The largest RCT to date enrolled 150 patients with mild COVID-19 across 16 centers in an open-label trial of HCQ and standard of care (ChiCTR2000029868). There were no significant differences between groups in conversion to negative SARS-CoV-2 RT-PCR result on day 28 or rate of symptom resolution; there were significantly more adverse events in the HCQ arm, though largely non-serious; they reported some evidence for faster normalization of C-reactive protein in the patients who received HCQ plus standard of care, but this finding was not significant (Tang et al., 2020b). A meta-analysis including most of the studies described here found no clinical benefits to patients receiving standard of care plus an HCQ regimen (Shamshirian et al., 2020).
Two studies have assessed HCQ efficacy in severe COVID-19. In a prospective study of 11 patients who had received 600 mg HCQ over 10 days with AZ on days 1–5, there were several patients with worsening clinical status and one death; 8 of 10 patients had a positive PCR result on day 10 (Molina et al., 2020). An ongoing double-blind RCT of patients with severe COVID-19 (NCT04323527) randomized 81 patients into high-dose HCQ (600 mg 2× per day for 10 days) or low-dose (450 mg/day for 5 days) treatment groups (Borba et al., 2020). Recruitment into the high-dose arm was halted prematurely due to poor safety outcomes. There was no significant difference in negative PCR results on day 4 or need for mechanical ventilation on day 6. Taken together, the clinical trials performed thus far to evaluate the efficacy of HCQ ± AZ for COVID-19 have not demonstrated clear evidence of clinical benefit in patients with severe disease. A search of ClinicalTrials.gov on April 27, 2020 found 140 clinical trials investigating HCQ. This number is rapidly growing, indicating the heightened interest in this therapeutic and pressing need for evidence-based recommendations.

Corticosteroids for COVID-19 Therapy
Because of their anti-inflammatory activity, corticosteroids (CSs) are an adjuvant therapy for ARDS and cytokine storm. However, the broad immunosuppression mediated by CS does raise the possibility that treatment could interfere with the development of a proper immune response against the virus. A meta-analysis of 5,270 patients with MERS-CoV, SARS-CoV-1, or SARS-CoV-2 infection found that CS treatment was associated with higher mortality rate (Yang et al., 2020c). A more recent meta-analysis of only SARS-CoV-2 infection assessed 2,636 patients and found no mortality difference associated with CS treatment, including in a subset of patients with ARDS (Gangopadhyay et al., 2020). Other studies have reported associations with delayed viral clearance and increased complications in SARS and MERS patients (Sanders et al., 2020). In fact, the interim guidelines updated by the WHO on March 13, 2020 advise against giving systemic corticosteroids for COVID-19 (World Health Organization, 2020a). Yet, new data from COVID-19 are conflicting.
One group reported no significant difference in time to viral clearance between patients who received methylprednisolone orally (mild disease) or intravenously (i.v.) (severe) and those who did not (Fang et al., 2020). Retrospective studies from groups in China report that patients who were transferred to the ICU were less likely to have received CSs (Wang et al., 2020b) and that patients with ARDS who received methylprednisolone had reduced mortality risk (Wu et al., 2020a). In contrast, another retrospective analysis found that patients who received CSs were more likely to have either been admitted to the ICU or perished, although the CS-treated group also had significantly more comorbidities (Wang et al., 2020c). A smaller observational study of 31 patients found no association between corticosteroid treatment and time to viral clearance, length of hospital stay, or symptom duration (Zha et al., 2020). A larger study of adjuvant CSs in 244 patients with critical COVID-19 found no association with 28-day mortality; subgroup analysis of patients with ARDS found no association between treatment with CSs and clinical outcomes (Lu et al., 2020b). They also found that increased dosage was significantly associated with increased mortality risk. A retrospective review of 46 patients, of whom 26 received i.v. methylprednisolone, found that early, low-dose administration significantly improved SpO2 and chest CT, time to fever resolution, and time on supplemental oxygen therapy (Wang et al., 2020h). Others have published perspectives in support of early (Lee et al., 2020) and short-term, low-dose administration (Shang et al., 2020) based on anecdotal evidence but not clinical trials. Most of the current data on CS use in COVID-19 are from observational studies and support either modest clinical benefit or no meaningful effects. Larger RCTs are necessary to understand the risks and benefits of CSs for these patients; there are 22 trials evaluating various corticosteroids registered on ClinicalTrials.gov as of April 27, 2020.

Cytokine-Directed Therapy in COVID-19

Recombinant IFN as an Antiviral Treatment
One of the first defenses of the human body against RNA viruses like SARS-CoV-2 is the release of types I and III IFNs. It is important to note that type I IFN (IFNα/β) receptors are ubiquitously expressed, so IFNα/β signaling can result in not only antiviral effects, but also the activation of immune cells that potentially exacerbate pathogenesis. In contrast, type III IFN (also known as IFNλ) signals mainly in epithelial cells, as well as in a restricted pool of immune cells. Because type III IFNs have immunomodulatory functions, subsequent signaling could induce a potent antiviral effect without enhancing pathogenic inflammation (Andreakos et al., 2017, Prokunina-Olsson et al., 2020).
Recently, there has been a growing interest in the potential therapeutic impact of modulating the IFN response to disable COVID-19 pathogenesis. Before the current pandemic, groups have studied the role of IFNs in other betacoronavirus infections. One study of 40 patients with SARS-CoV-1 infection described unresolved elevated type I IFNs and ISGs in those with poor outcomes (Cameron et al., 2007). Others report that exogenous type I IFN does not improve outcomes when given with ribavirin in patients with MERS-CoV infection (Arabi et al., 2020), suggesting that the role of IFN as a therapeutic or prophylactic option may be strain or species specific (Sheahan et al., 2020). Interestingly, a recent study by Mount Sinai virology groups revealed that type I IFN signaling is impaired in the early response to SARS-CoV-2; in vitro, SARS-CoV-2 may be more susceptible to type I IFN than SARS-CoV-1 is (Blanco-Melo et al., 2020). Based on additional evidence that IFN responses to betacoronaviruses are altered as compared to other respiratory viruses (Blanco-Melo et al., 2020, Channappanavar et al., 2016, Okabayashi et al., 2006), trials of IFN-I/III administration have been initiated (NCT04343976, NCT04331899).

Cytokine Blockade
Hyperinflammatory responses and elevated levels of inflammatory cytokines, including IL-6, -8, and -10, have been shown to correlate with COVID-19 severity (Chen et al., 2020h, Diao et al., 2020, Gong et al., 2020, Moore and June, 2020, Wan et al., 2020a, Xu et al., 2020b). The drivers of this cytokine storm remain to be established, but they are likely triggered initially by a combination of viral PAMPs and host danger signals. The heterogeneous response between patients suggests other factors are involved, possibly including the SARS-CoV-2 receptor, ACE2 (Hirano and Murakami, 2020).
Several studies have begun to report the cellular programs that may contribute to the cytokine storm detected in COVID-19 patients. One group reported that in the context of generalized lymphopenia, certain subsets of CD4 T cells that express GM-CSF and IL-6 are more abundant in severe COVID-19 patients than in COVID-19 patients who do not require intensive care (Zhou et al., 2020b). Reports that other major proinflammatory cytokines (TNF-α, IFN-ɣ, IL-2) and chemokines (CCL2, CCL3, CCL4) are elevated underscore a potentially pathogenic TH1/2 program in COVID-19 (Diao et al., 2020, Giamarellos-Bourboulis et al., 2020). Histological and single-cell analyses identified monocytes and macrophages as other potent sources of inflammatory cytokines in COVID-19 cytokine storm (Chen et al., 2020h, Giamarellos-Bourboulis et al., 2020, Law et al., 2005, Moore and June, 2020, Zhou et al., 2020b). Studies of other betacoronavirus infections, including SARS-CoV-1 and MERS-CoV, have also identified similar hyperactivation of monocytes, macrophages, and DCs as a driver of cytokine-mediated immunopathology in humans (Cheung et al., 2005, Chien et al., 2006, Huang et al., 2020c, Konig et al., 2020, Wang et al., 2005, Wong et al., 2004, Xu et al., 2020b, Zhou et al., 2020b).
Following preliminary reports of IL-6 as a critical cytokine in COVID-19-associated CRS, monoclonal antibodies that target the IL-6 signaling pathway have been proposed as therapeutic candidates (Moore and June, 2020) (Figure 6C). The commercial anti-IL-6R antibodies tocilizumab (Actemra) and sarilumab (Kevzara) and the anti-IL-6 antibody siltuximab (Sylvant) are now being tested for efficacy in managing COVID-19 CRS and pneumonia in 13 ongoing clinical trials (Table 2 ). To date, only one group has reported preliminary results from a cohort of 20 COVID-19 patients treated with a single administration of tocilizumab (400 mg, i.v.), along with lopinavir, methylprednisolone, and oxygen therapy (ChiCTR2000029765) (Xu et al., 2020b). The single observation study found recuperated lymphocyte counts in 10 of 19 patients and resolution of lung opacities in 19 of 20 patients on chest CT; 19 of 20 patients were discharged. All patients experienced an improvement in symptoms, and no subsequent pulmonary infections were reported. A second report described an association between use of tocilizumab and reduced likelihood of ICU admission and mechanical ventilation. Still, in 30 declining patients with severe COVID-19 pneumonia, this retrospective study did not report significant improvement in mortality on weighted analysis (Roumier et al., 2020). Nevertheless, these studies are encouraging, but like other treatment approaches, larger RCTs are needed.
Table 2 Clinical Trials Evaluating the Efficacy of IL-6/IL-6R Blockade Therapy
Clinical Trial Intervention
NCT04331795 (COVIDOSE)NCT04320615 (COVACTA)NCT04332913 (TOSCA)NCT04317092 (TOCOVID-19)NCT04335071 (CORON-ACT)NCT04315480ChiCTR2000029765 tocilizumab
NCT04315298 sarilumab
NCT04310228 tocilizumabfavipiravir
NCT04306705 (TACOS) tocilizumabcontinuous renal replacement therapystandard of care
NCT04332094 (TOCOVID) tocilizumabazithromycinhydroxychloroquine
NCT04341870 (CORIMUNO-VIRO) sarilumabazithromycinhydroxychloroquine
NCT0433z638 (COV-AID) tocilizumabsiltuximabanakinrastandard of care
In addition to the IL-6 signaling pathway, other cytokine- and chemokine-associated elements, including IL-1R, GM-CSF, and the chemokine receptor CCR5, have been proposed as potential targets for blockade to manage COVID-19 CRS (Figure 6C). Finally, complement activation was shown to be overactivated in lungs of COVID-19 patients. Although results from the randomized trial are not yet published, anti-C5a monoclonal antibody therapy showed benefits in two critically ill COVID-19 patients (Gao et al., 2020d).