Expert Review of Clinical Immunology

For Peer Review Only

Natural history and therapeutic options for COVID-19

Journal: Expert Review of Clinical Immunology

Manuscript ID ERM-2020--0073.R1

Manuscript Type: Review (Invited)

SARS-CoV-2, COVID-19, Physiopathology, Treatment, Chloroquine, Keywords: Azithromycin

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1 2 3 1 Natural history of COVID-19 and therapeutic options 4 5 6 2 7 8 3 9 10 4 11 12 5 13 14 15 6 16 17 7 18 For Peer Review Only 19 8 20 21 22 9 23 24 10 25 26 11 27 28 29 12 30 31 13 32 33 14 34 35 15 36 37 38 16 39 40 17 41 42 18 43 44 45 19 46 47 20 48 49 21 50 51 52 22 53 54 23 55 56 24 57 58 25 59 60

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1 2 3 26 Abstract 4 5 6 27 Introduction 7 8 9 28 COVID-19 presents benign forms in young patients who frequently present with anosmia. 10 11 29 Infants are rarely infected, while severe forms occur in patients over 65 years of age with 12 13 30 comorbidities, including hypertension and diabetes. Lymphopenia, eosinopenia, 14 15 16 31 thrombopenia, increased lactate dehydrogenase, troponin, C-reactive protein, D-dimers, and 17 18 32 low zinc levels areFor associated Peer with severity. Review Only 19 20 33 Areas covered 21 22 34 The authors review the literature and provide an overview of the current state of knowledge 23 24 25 35 regarding the natural history of and therapeutic options for COVID-19. 26 27 36 Expert opinion 28 29 37 Diagnosis should rely on PCR and not on clinical presumption. Because of discrepancies 30 31 32 38 between clinical symptoms, oxygen saturation or radiological signs on CT scans, pulse 33 34 39 oximetry and radiological investigation should be systematic. The disease evolves in 35 36 40 successive phases: an acute virological phase, and, in some patients, a cytokine storm phase; 37 38 39 41 an uncontrolled coagulopathy; and an acute respiratory distress syndrome. Therapeutic 40 41 42 options include antivirals, oxygen therapy, immunomodulators, anticoagulants and prolonged 42 43 43 mechanical treatment. Early diagnosis, care, and implementation of an antiviral treatment; the 44 45 44 use of immunomodulators at a later stage; and the quality of intensive care are critical 46 47 48 45 regarding mortality rates. The higher mortality observed in Western countries remains 49 50 46 unexplained. Pulmonary fibrosis may occur in some patients. Its future is unpredictable. 51 52 47 53 54 55 48 Keywords: SARS-CoV-2, COVID, Humans, Pathophysiology, Treatment, 56 57 49 Hydroxychloroquine, Azithromycin, Tocilizumab, Remdesivir, Care 58 59 60

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1 2 3 50 Highlights 4 5 6 51  Diagnosis of COVID-19 should rely on PCR and not on clinical presumption. 7 8 52  Because of “happy hypoxia”, pulse oximetry and radiological investigation should be 9 10 53 systematic. 11 12 13 54  The disease evolves in successive phases: an acute virological phase, and, in some 14 15 55 patients, a cytokine storm phase; an uncontrolled coagulopathy; and an acute 16 17 56 respiratory distress syndrome. 18 For Peer Review Only 19 20 57  Massive screening allowing early diagnosis, care (oxygen, anticoagulants), and 21 22 58 implementation of an antiviral treatment; the use of immunomodulators at a later 23 24 59 stage; and the quality of intensive care are critical regarding mortality rates. 25 26 27 60  Intravenous catheterization should be avoided, and early oral treatment in an 28 29 61 outpatient basis should be preferred. 30 31 62  Treatment with an oral combination of hydroxychloroquine, azithromycin and zinc 32 33 34 63 may represent the best current therapeutic option in relation to its antiviral and 35 36 64 immunomodulatory effects. 37 38 65  Preventive anticoagulants should be prescribed in patients with coagulopathy (positive 39 40 D-dimers). 41 66 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 67 1. Introduction 4 5 6 68 COVID-19 has a pleomorphic clinical presentation including asymptomatic individuals and 7 8 69 patients with mild to severe involvement with several evolutionary stages [1-3]. Age and 9 10 70 comorbidities including, notably, hypertension, diabetes and coronary heart disease are the 11 12 71 main risk factors for evolving toward severe infections [1-3]. Schematically, after the 13 14 15 72 incubation period, two main clinical presentations can occur: upper respiratory tract infections 16 17 73 (URTIs) with severe headaches, anosmia, ageusia (or dysgeusia) and rhinitis, which are 18 For Peer Review Only 19 74 mainly observed in young patients who then have a good clinical outcome; and lower 20 21 22 75 respiratory tract infections (LRTIs) with pneumonia symptoms that are observed more 23 24 76 frequently in patients with comorbidities and can be severe to fatal in older patients [1-3]. At 25 26 77 admission, prognosis can be assessed through the National Early Warning Score (NEWS-2), a 27 28 simple aggregate scoring system including respiration rate, oxygen saturation, systolic blood 29 78 30 31 79 pressure, pulse rate, level of consciousness or new confusion, and temperature. Age has been 32 33 80 added in a modified version of this score [4]. 34 35 81 During the onset of the COVID-19 outbreak, olfactory and gustative disorders, 36 37 38 82 including anosmia and ageusia were described in infected patients [5]. In Marseille, 3,497 39 40 83 adults who underwent PCR between 24 March and 25 April 2020 were asked the following 41 42 84 question prior to being tested for SARS-CoV-2: “Have you lost your sense of smell or taste in 43 44 45 85 the past two months?” The prevalence of the loss of smell and/or taste in COVID-19 patients 46 47 86 was 356/673 (53%), and the positive predictive value (PPV) for the diagnosis of COVID-19 48 49 87 by PCR was 67% when smell and taste disorders were reported (submitted). Asking patients 50 51 and healthcare workers (HCWs) about loss of smell and taste could be useful in areas where 52 88 53 54 89 testing for SARS-CoV-2 is politically or technically limited or impossible. Interestingly, 55 56 90 “happy hypoxemia”, a hypoxia observed in patients who are SARS-CoV-2 positive yet 57 58 91 comfortable and without dyspnea emphasizes the need to perform a low-dose CT-scan on 59 60

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1 2 3 92 most patients to detect pneumonia at an early stage [6]. Most COVID patients are definitively 4 5 6 93 cured, but extreme caution is needed in patients with comorbidities and/or biological 7 8 94 parameter abnormalities such as lymphopenia, eosinopenia, increased D-dimers, troponin, 9 10 95 lactate dehydrogenase (LDH) or C-reactive protein (CRP) [1, 3]. Venous thromboembolism is 11 12 96 relatively common [7], is mainly characterized by pulmonary embolism, and is found in up to 13 14 15 97 one-third of critical cases [8]. Acute respiratory distress syndrome (ARDS), pulmonary 16 17 98 embolism and bacterial superinfection may result in a fatal evolution [9, 10]. Finally, delayed 18 For Peer Review Only 19 99 pulmonary fibrosis may occur in an as yet unknown proportion of patients [11]. 20 21 22 100 At the beginning of the health crisis, the use of chest X-rays was restricted to patients in 23 24 101 intensive care units due to its low value in detecting ground-glass opacities. However, low- 25 26 102 dose chest computed tomography (LDCT) appears to be a useful tool in the management of 27 28 patients with regards to diagnosing, assessing and quantifying disease severity and for 29 103 30 31 104 differential diagnosis. LDCT might be of interest in predicting lung fibrosis during healing 32 33 105 [12-15]. The main findings of COVID-19 pneumonia on chest CT include ground-glass 34 35 106 opacities, consolidation, and a crazy-paving pattern. These features are not specific, but the 36 37 38 107 distribution of lesions during COVID-19 pneumonia is more likely to be peripheral, 39 40 108 asymmetric and located in the lower lobes [16]. CT features revealed a good sensitivity and 41 42 109 specificity for COVID-19 diseases in centers where CT was used as a diagnostic tool [17]. 43 44 45 110 Furthermore, Li et al. developed a deep learning algorithm able to discriminate COVID-19 46 47 111 pneumonia from community-acquired pneumonia, with good results (Figure 1) [18]. COVID- 48 49 112 19 pneumonia is also characterized by the high prevalence of lung involvement in 50 51 paucisymptomatic patients. In our center, we decided to perform LDCT on all patients with a 52 113 53 54 114 positive PCR for COVID-19. Of the 2,065 LDCTs that were performed on COVID-19 55 56 115 patients, more than 70% revealed pneumonia. Of the 1,043 patients with a NEWS-2 score=0 57 58 116 who underwent LDCT, 628 (60.2%) had radiological abnormalities, including 494 (47.4%) 59 60

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1 2 3 117 with minimal lung lesions, 118 (11.8%) with intermediate lesions and 11 (1%) with severe 4 5 6 118 lesions. Moreover, of the 1,370 LDCTs performed on patients without perceived dyspnea, 937 7 8 119 (68%) had pneumonia [3]. 9 10 120 Symptoms at the time of diagnosis of COVID-19 pneumonia do not appear to be related 11 12 121 to prognosis [1]. A meta-analysis of 1,558 patients found that significant risk factors for 13 14 15 122 mortality in COVID-19 were hypertension, diabetes, chronic obstructive pulmonary disease, 16 17 123 cardiovascular disease, and cerebrovascular disease [19, 20]. A study on 1,591 patients in the 18 For Peer Review Only 19 124 intensive care unit showed that the mortality rate was higher in patients over the age of 64 20 21 22 125 than in younger patients [20]. Furthermore, we showed that the percentage of lung 23 24 126 involvement quantified using the deep learning algorithm is an independent prognostic 25 26 127 marker, and the addition of lesion quantification significantly enhances the prediction model 27 28 based on comorbidities and NEWS-2 score (unpublished). 29 128 30 31 129 Reports of an increased incidence of acute pulmonary embolisms or intravascular 32 33 130 coagulopathy associated with COVID-19 have emerged in the literature [21]. The prevalence 34 35 131 of pulmonary embolism in COVID-19 has been reported to be approximately 20% in patients 36 37 38 132 with severe disease. Leonard-Lorant et al. found that a D-dimer threshold higher than 2,660 39 40 133 µg/L could detect all patients with a pulmonary embolus on chest CT after contrast injection 41 42 134 [8]. 43 44 45 135 COVID-19 might lead to sequelae such as lung fibrosis during the healing phase [22]. 46 47 136 The real prevalence and clinical impact of COVID-19 sequelae on the lungs, as well as on the 48 49 137 myocardium, requires further study. 50 51 52 138 Literature search methodology 53 54 139 A literature search was performed using the following keywords: SARS-CoV-2, COVID, 55 56 140 coronavirus, pathophysiology, natural history, treatment, and humans without restriction of 57 58 59 60

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1 2 3 141 the date or language. Medline, Google, and Google Scholar were used alongside 4 5 6 142 crossreferencing. 7 8 143 2. SARS-CoV-2 epidemiology 9 10 144 The first COVID-19 cases were identified in late December 2019 in Wuhan, China, and the 11 12 145 disease turned into a pandemic within a few weeks. As of 21 July 2020, more than fourteen 13 14 15 146 million cases have been reported globally, with more than 600,000 deaths. 16 17 147 (https://gisanddata.maps.arcgis.com/apps/opsdashboard/index.html#/bda7594740fd40299423 18 For Peer Review Only 19 148 467b48e9ecf6). The SARS-CoV-2 epidemic exhibits a bell-shaped incidence curve [23, 24] 20 21 22 149 (https://coronavirus.jhu.edu/data/new-cases; https://www.mediterranee-infection.com/covid- 23 24 150 19/; Figure 2), which is a typical epidemic curve. In Western countries in the Northern 25 26 151 hemisphere, the outbreak decreased dramatically during the spring, as is the case for 27 28 epidemics arising from other respiratory viruses. Moreover, it affected people differently 29 152 30 31 153 according to their age. Very few cases and less severe outcomes were observed in children 32 33 154 [25-31], while SARS-CoV-2 infections have been more frequent and severe in the elderly. For 34 35 155 instance, a large study conducted in Iceland on the general population found that children 36 37 38 156 under the age of 10 years were half as likely to be diagnosed with SARS-CoV-2 than children 39 40 157 over the age of 10 years or adults [31]. In addition, targeted screening did not diagnose 41 42 158 infections in children under the age of 10 years. This predominance of cases among adults 43 44 45 159 differs from the age distributions observed with other respiratory viruses, including endemic 46 47 160 coronaviruses [32]. Interestingly, several studies have detected immune responses to SARS- 48 49 161 CoV-2 in unexposed individuals. Thus, Grifoni et al. detected circulating SARS-CoV-2- 50 51 specific CD4+ and CD8+ T cells in ≈20-60% of SARS-CoV-2-unexposed individuals sampled 52 162 53 54 163 between 2015 and 2018 [33], and 10% of uninfected pregnant women exhibited IgG to 55 56 164 SARS-CoV-2 [34]. Conversely, increased IgG reactivity to endemic coronaviruses was 57 58 165 reported in SARS-CoV-2 infections, further suggesting crossimmunity [35]. As the four 59 60

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1 2 3 166 coronaviruses 229E, NL63, OC43 and HKU1 circulate endemically worldwide and massively 4 5 6 167 infect humans during the first years of life [32], exposure to these viruses may have conferred 7 8 168 crossimmunity to SARS-CoV-2 in a preferential way to children. 9 10 169 3. Natural history of the disease (Figure 3) 11 12 170 3.1 Transmission route 13 14 15 171 The main transmission route for SARS-CoV-2 is human-to-human transmission via 16 17 172 respiratory droplets and skin contact, with incubation times of 2–14 days (mean incubation 18 For Peer Review Only 19 173 time of approximately 6 days) [36, 37]. In addition to its presence in nasopharyngeal swabs, 20 21 22 174 bronchoalveolar lavage, and sputum, SARS-CoV-2 has been detected using molecular tools in 23 24 175 saliva, stools, urine, blood, tears, and conjunctival secretions [36, 38-40]. Live viruses have 25 26 176 been detected in feces, including nondiarrheal feces, implying that SARS-CoV-2 could be 27 28 transmitted through feces [36]. Its molecular detection around the toilet (doorknob, surface of 29 177 30 31 178 the toilet bowl, internal sink bowl) in the room of a patient who did not have diarrhea but who 32 33 179 did have positive stool samples for SARS-CoV-2, supports the hypothesis that fecal viral 34 35 180 shedding may be involved in transmission [41]. Live viruses have also been observed in saliva 36 37 38 181 [38]. Since saliva can be released through coughing and the influenza virus can be present in 39 40 182 respiratory droplets even during normal breathing, SARS-CoV-2 could be transmitted by 41 42 183 saliva directly or indirectly, even in patients without a cough or other respiratory symptoms 43 44 45 184 [38]. It has already been reported that asymptomatic carriers can spread SARS-CoV-2 [42]. 46 47 185 Although SARS-CoV-2 has been detected in the tears and conjunctival secretions of COVID- 48 49 186 19 patients with conjunctivitis, its transmission through the conjunctival route is debated [39]. 50 51 SARS-CoV-2 can survive outside the body for long periods of time; it can remain viable 52 187 53 54 188 in aerosols for up to three hours [43]. Viable SARS-CoV-2 was detected up to 72 hours after 55 56 189 experimental application to plastic and stainless steel. On copper and cardboard, no viable 57 58 190 SARS-CoV-2 was measured after four hours and 24 hours, respectively. Thus, transmission of 59 60

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1 2 3 191 SARS-CoV-2 by aerosols and fomites is likely. Investigations of a COVID-19 cluster in a 4 5 6 192 shopping mall support the hypothesis that the rapid spread of SARS-CoV-2 could result from 7 8 193 spread via fomites (elevator buttons, bathroom taps) or virus aerosolization in a confined 9 10 194 public spaces (bathrooms, elevators) [44]. 11 12 195 Nosocomial transmission including outbreaks of SARS-CoV-2 have occurred. Although 13 14 15 196 direct transmission is the most common route of transmission, contaminated surfaces that are 16 17 197 frequently contacted in healthcare facilities are a potential source. An extensive 18 For Peer Review Only 19 198 environmental study was performed in an intensive care unit (ICU) and general ward (GW) at 20 21 22 199 Wuhan Huoshenshan Hospital [45]. Nucleic acids from SARS-CoV-2 were observed on 23 24 200 surfaces that were frequently touched, such as computer mice, rubbish bins, and handrails on 25 26 201 patients’ beds, but were also found on floors and were sporadically observed on doorknobs 27 28 and on the sleeve cuffs and gloves of medical staff. Nucleic acids were also detected in the air 29 202 30 31 203 from a patient’s room, with an estimate that the transmission distance of SARS-CoV-2 could 32 33 204 be around four meters in a GW. However, since the detection of nucleic acids does not 34 35 205 indicate the amount of viable virus and the minimum infectious dose is unknown, the distance 36 37 38 206 of aerosol transmission cannot be strictly determined. These data imply a risk of infection for 39 40 207 medical personnel and other close contacts. However, appropriate precautions and adherence 41 42 208 to hand and environmental hygiene can effectively prevent infection since no member of the 43 44 45 209 hospital staff was infected with SARS-CoV-2 as of 30 March 2020 [45]. 46 47 210 3.2 Cellular life cycle of SARS-CoV-2 (Figure 4) 48 49 211 The SARS-CoV-2 is an enveloped RNA surrounded by spike glycoproteins [46-49]. 50 51 The virus enters cells through membrane fusion. The first step of the SARS-CoV-2 replicative 52 212 53 54 213 cycle is the attachment of the virus to the angiotensin-converting enzyme 2 (ACE2) 55 56 214 glycoprotein. The receptor-binding domain (RBD) amino acid sequences present in the S1 57 58 215 spike protein interact with the N-terminal region 30-41 and 82-93 of ACE2 that contains 59 60

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1 2 3 216 several sites for N-glycosylation. A cell surface protease, TMPRSS2, is responsible for spike 4 5 6 217 cleavage allowing the appropriate conformation for the S2 spike to expose the hidden fusion 7 8 218 peptide for insertion into the cellular membrane lipid bilayers. The viral nucleocapsid is thus 9 10 219 delivered into the cytoplasm through the endocytic vesicle. After acidification of the late 11 12 220 endosome, the action of cathepsin enables the uncoating of the genomic RNA and the 13 14 15 221 enzymes necessary for its replication. The genomic RNA is used as a template by the 16 17 222 replicase to synthesize the negative sense genomic RNAs (anti-genome), which are used as 18 For Peer Review Only 19 223 templates to synthesize the progeny positive sense genomes and subgenomic RNAs. Similarly 20 21 22 224 to SARS-CoV, the 5'-proximal two-thirds of the SARS-CoV-2 viral genome are translated 23 24 225 into polyproteins that give rise to several nonstructural proteins (Nsps) following 25 26 226 autoproteolytic processing. Among the Nsps, Nsp12 is an RNA-dependent RNA polymerase, 27 28 Nsp3 and Nsp5 are proteinases, Nsp13 is a helicase, Nsp14 and Nsp15 are ribonucleases, and 29 227 30 31 228 Nsp14 is a methyltransferase (involved in RNA cap formation). The 3'-proximal third 32 33 229 sequence serves as template for several subgenomic mRNAs that encode the viral structural 34 35 230 (the spike/S, the envelope/E, the membrane/M, and the nucleocapsid/N) and accessory 36 37 38 231 proteins. The S, E, and M proteins are synthesized and anchored on the endoplasmic 39 40 232 reticulum (ER) with the N protein translated in the cytosol. Posttranslational modifications of 41 42 233 viral proteins occur within the endoplasmic reticulum and trans-Golgi network vesicles. After 43 44 45 234 assembly in the ER-Golgi intermediate compartment (ERGIC), where the E protein plays an 46 47 235 essential role in virus assembly and the mature M protein shapes the virus, mature virions are 48 49 236 released from smooth-walled vesicles by exocytosis. 50 51 3.3 Immune response 52 237 53 54 238 Immune responses shape the clinical course of COVID-19. The hallmark of the disease 55 56 239 is the occurrence, in 10–20% of patients, of a sudden deterioration 7–10 days after the onset 57 58 240 of symptoms, increasing the risk of acute respiratory failure, organ support and ultimately a 59 60

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1 2 3 241 fatal outcome [27]. Early publications described lymphopenia mostly affecting T and B cells, 4 5 6 242 neutrophilia, and decreased eosinophil and monocyte counts [10, 27]. The degree of 7 8 243 eosinopenia, lymphopenia and neutrophilia, the latter sometimes expressed as the neutrophil- 9 10 244 to-lymphocyte ratio, was associated with and was predictive of clinical severity. In severe 11 12 245 cases, deficient induction of type I interferons at the initial stage, increased levels of some but 13 14 15 246 not all pro-inflammatory cytokines, most notably interleukin (IL)-6, increased levels of the 16 17 247 regulatory cytokine IL-10, an autoimmune signature, and an inconsistent specific antibody 18 For Peer Review Only 19 248 response to SARS-CoV-2 were reported [10, 27, 50-52]. Tissue investigations showed that 20 21 22 249 SARS-CoV-2 pulmonary tropism targeted alveolar cells. Severe pulmonary lesions were 23 24 250 associated with interstitial mononuclear infiltrates dominated by lymphocytes, CD4+ and 25 26 251 CD8+ T-cells, pulmonary edema, hyaline formation and pneumocyte desquamation without 27 28 histopathological evidence of sequestrated eosinophils [53]. Resident or recruited 29 252 30 31 253 immunosuppressive and inflammatory monocytes and macrophages (CD14+HLA-DRlo/neg) 32 33 254 tend to replace protective resident alveolar macrophages. Severe respiratory failure may arise 34 35 255 through two distinct pathways: either an atypical macrophage activation syndrome, or an 36 37 38 256 immune dysregulation status characterized by impaired monocyte activation and antigen 39 40 257 presentation [54]. The systemic passage of high levels of cytokines produced in the lungs may 41 42 258 contribute to a multivisceral failure syndrome [10]. Neutrophilia is associated with the 43 44 45 259 increased formation of extracellular traps (NETosis), contributing to inflammation, cytokine 46 47 260 dysregulation and autoimmune and thrombotic manifestations [55, 55]. SARS-CoV-2 was 48 49 261 reported to infect T lymphocytes and macrophages, resulting in impaired antigen presentation, 50 51 increased IL-6 production, and possibly in lymphocyte apoptosis in lymphoid organs [54, 56]. 52 262 53 54 263 Th1 polarization is involved in the efficient control of SARS-CoV-1 but not SARS-CoV-2 55 56 264 infection [57]. Crossreactive T-cell recognition between circulating seasonal human 57 58 265 coronavirus (HCoVs), the SARS-CoV and SARS-CoV-2 and crossreactive antibodies 59 60

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1 2 3 266 between SARS-CoV-2 and SARS-CoV have been reported [58-60]. Interestingly, CD4+ T 4 5 6 267 cells from COVID-19 patients targeted the N and C terminal regions of the S protein equally, 7 8 268 whereas CD4+ T cells from noninfected patients only targeted the C terminal region, which is 9 10 269 highly homologous with the seasonal HCoVs S protein [59]. Recently, Grifoni et al., used 11 12 270 HLA class I and II and predicted peptide megapools detected circulating SARS-CoV-2- 13 14 + + 15 271 specific CD4 and CD8 T cells in 100% and ≈70% of patients with resolved COVID-19, 16 17 272 respectively [33]. Interestingly, such cells were also detected in ≈20–60% of SARS-CoV-2- 18 For Peer Review Only 19 273 unexposed individuals collected between 2015 and 2018. It should also be noted that all 20 20 21 22 274 SARS-CoV-2-unexposed individuals exhibited IgG to HCoV-OC43 and HCoV-NL63. 23 24 275 Another study showed the presence of anti-SARS-CoV-2 humoral responses elicited by 25 26 276 patients previously infected with endemic coronaviruses [34]. 27 28 It is thus hypothesized that past infection with HCoVs causing upper respiratory infection 29 277 30 31 278 may confer SARS-CoV-2 cellular immunity as the result of CD4+ T cellular crossreactivity. 32 33 279 Regarding the antibody response, between 47% and 100% of COVID-19 patients seroconvert 34 35 280 within fifteen days after the onset of symptoms [61, 62, 62]. Recovery after mild or moderate 36 37 38 281 forms is associated with neutralizing antibodies, whereas patients with severe forms of the 39 40 282 disease present early and high levels of non-neutralizing and possibly deleterious antibodies 41 42 283 [61, 63-65]. 43 44 45 284 Thrombotic phenomena and lung vessels obstructive thrombo-inflammatory syndrome 46 47 285 play a role in severe and critical COVID-19 presentations. Similar to SARS-CoV-1, an 48 49 286 autoimmune signature including antiphospholipid antibodies, e.g., anticardiolipin IgA, anti-β2 50 51 glycoprotein 1 IgA, IgG and IgM, is present in SARS-CoV-2 infected patients [65]. It is 52 287 53 54 288 assumed that autoimmune markers are associated with the occurrence of thromboembolic 55 56 289 phenomena, but direct evidence is lacking. Lupus anticoagulant has also been reported as a 57 58 59 60

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1 2 3 290 possibly frequent finding in up to 25 of 56 patients (45%), but again, the causative or 4 5 6 291 predictive relation to thrombotic events during COVID has not been assessed [50]. 7 8 292 To summarize, our current understanding of immune responses to SARS-CoV-2 is that of an 9 10 293 early choice between two distinct pathways. An efficient monocyte-macrophage, CD4+ and 11 12 294 CD8+ T cellular response accompanied by a controlled inflammatory response enables virus 13 14 15 295 control and swift recovery. Conversely, a status of SARS-CoV-2-induced immune 16 17 296 dysregulation associated with low levels of type I interferons, an IL-6-driven inflammatory 18 For Peer Review Only 19 297 status with immunosuppressive and inflammatory monocytes and macrophages, a defective 20 21 22 298 antigen presentation, an extensive organ immunopathogenesis and a prominent anti-SARS- 23 24 299 CoV-2 and autoimmune antibody response is associated with severe forms of the disease. 25 26 300 Investigation of the SARS-CoV-2 immune response has already given insights into multiple 27 28 immune modulating therapies, from repurposed molecules (antimalarials, chlorpromazine, 29 301 30 31 302 antibiotics) to antivirals to monoclonal antibodies directed at cytokines (anti-IL-6, anti-IL-1) 32 33 303 or innate pathways (C5/C5a). Whether the crossreactivity of CD4+ T-cell lymphocytes 34 35 304 epitopes and antibodies with seasonal HCoVs can protect against SARS-CoV-2 and SARS- 36 37 38 305 CoV needs to be further explored. 39 40 306 3.4 Immunopathogenic phase: the cytokine storm 41 42 307 On approximately the tenth day of infection, COVID-19-associated pneumonia may 43 44 45 308 evolve toward acute respiratory failure due to ARDS requiring ICU admission and high-flow 46 47 309 oxygen or mechanical ventilation, with a severe prognosis [2]. The underlying mechanisms of 48 49 310 these complications are immunological rather than due to the virus itself, which in most cases, 50 51 is no longer detectable at this stage. 52 311 53 54 312 Persistence of viral RNA detection in respiratory specimens has been reported even 55 56 313 three weeks after disease onset [66] and in 10-20% of patients at day 10 [3]. However, disease 57 58 314 in some patients became aggravated while the virus was no longer detectable using 59 60

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1 2 3 315 conventional detection methods. It cannot be ruled out that, although not detected, the virus 4 5 6 316 was still present in cellular reservoirs not accessible to detection and can continue to activate a 7 8 317 pro-inflammatory response. However, it is likely that the virus is at the origin of the triggering 9 10 318 of a pro-inflammatory reaction that then self-amplifies. 11 12 319 Elevated circulating inflammatory cytokine concentrations have been reported in 13 14 15 320 patients, notably interleukin (IL)-1β, IL-6, MCP-1, IP-10, MIP-1α, IL-2, IL-10, revealing a 16 17 321 so-called “cytokine storm”, described in other inflammatory diseases such as macrophage 18 For Peer Review Only 19 322 activation syndrome (MAS) or systemic inflammatory response syndrome [67, 68]. 20 21 22 323 Strikingly, and consistently with immunopathophysiology, IL-6 levels are a near perfect 23 24 324 predictor of subsequent acute respiratory failure [69]. 25 26 325 In vitro experiments and animal models of other SARS-CoV infections have shown that the 27 28 virus induces alveolar epithelial cell necrosis, the release of viral particles and of cytoplasmic 29 326 30 31 327 proinflammatory danger-associated molecular patterns (DAMPS), including ATP, nucleic 32 33 328 acids, or IL-1α [70]. In the meantime, SARS-CoV inhibits or delays IFNα/β production by 34 35 329 alveolar epithelial cells, which normally constitute the initial anti-viral defense [71]. 36 37 38 330 Alternatively, SARS-CoV induces a robust but delayed IFNα/β response by the plasmacytoid 39 40 331 dendritic cells and macrophages, which, together with DAMPS, trigger chemokine production 41 42 332 and the recruitment of inflammatory monocytes/macrophages into the lungs [71]. In 43 44 45 333 inflammatory monocytes/macrophages, the virus induces NF-κB activation and the 46 47 334 transcription of several pro-inflammatory cytokines, notably IL-1β and IL-18 precursors, and 48 49 335 the constituents of the NLRP3 (for NOD-like receptor family, pyrin domain containing 3) 50 51 inflammasome [72, 73]. NLRP3 assembles in the cytoplasm after a second danger cell signal, 52 336 53 54 337 consisting in K+ efflux, ATP, lysosome degradation or production of mitochondrial reactive 55 56 338 oxygen species (ROS) [74]. Once assembled, NLRP3 activates caspase 1 and the processing 57 58 339 of biologically inactive IL-1β and IL-18 precursors into biologically active cytokines [74]. 59 60

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1 2 3 340 Importantly, SARS-CoV behaves as a membrane K+ channel and thus directly activates 4 5 6 341 NLRP3 assembly [73, 75]. In SARS-CoV animal models, the excessive NLRP3 activation 7 8 342 and IL-1β or IL-18 production induce a cytokine storm, ARDS and death, whereas blocking 9 10 343 NLRP3, the IL-1 receptor type 1 or IL-18 are protective [72]. IL-1β is a major pro- 11 12 344 inflammatory cytokine known to induce fever via prostaglandin E2 secretion, neutrophilia via 13 14 15 345 GCSF production, liver acute-phase protein synthesis and a Th-17 immune response through 16 17 346 IL-6 secretion [76]. IL-18 is known to induce IFNγ production by Th-1 lymphocytes and NK 18 For Peer Review Only 19 347 cells [77]. By binding to its receptor on inflammatory monocytes/macrophages, IL-1β induces 20 21 22 348 a harmful cytokine loop consisting of excess NLRP3, IL-1β/IL-6, IL-18/IFNγ synthesis, 23 24 349 initiating the cytokine storm [57]. NLRP3 also induces pyroptosis, a programmed cell death 25 26 350 process leading to the uncontrolled release of IL-1β, IL-18 and SARS-CoV2 particles [74]. 27 28 At this stage of COVID-19, patients usually present with high-grade fever (>38.5°C), 29 351 30 31 352 moderate increased neutrophils, and elevated CRP concentrations (>150 mg/L), which are the 32 33 353 hallmarks of an IL-1/IL-6 signature [78]. Later, hyperferritinemia, diffuse coagulopathy and 34 35 354 cytopenia may appear, constituting the hallmarks of an IL-18/IFNγ signature [78]. Thus, the 36 37 38 355 absence of or delayed primary immune defenses against SARS-CoV2 encourages the 39 40 356 persistence of the virus, which in some individuals stimulates an uncontrolled self-stimulating 41 42 357 inflammatory loop and a cytokine storm that are initially located in the lungs but may diffuse 43 44 45 358 systemically and induce multi-organ failure. 46 47 359 3.5 Coagulopathy and thrombosis 48 49 360 Evidence of abnormal coagulation parameters associated with COVID-19 appeared in 50 51 early reports from China [1]. The most common hemostatic abnormalities observed are mild 52 361 53 54 362 thrombocytopenia and increased fibrinogen and D-dimers. Elevated D-dimers upon admission 55 56 363 are associated with increased mortality [79, 80]. 57 58 364 Although the SARS-CoV-2 virus does not appear to have intrinsic procoagulant effects, the 59 60

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1 2 3 365 significant increase in cytokines, also known as a cytokine storm, induces the activation of 4 5 6 366 coagulation and thrombin generation leading a state of major hypercoagulability. Initial 7 8 367 pulmonary findings during autopsy showed the presence of diffuse alveolar lesions with 9 10 368 infiltration of macrophages and CD4+ T lymphocytes around thrombosed small vessels with 11 12 369 significant hemorrhages. The formation of microthrombi in the pulmonary microvessels 13 14 15 370 appears to be involved in the pathogenesis of the respiratory picture observed in patients with 16 17 371 SARS-CoV-2 infection [9]. This form of thrombosis involving the immune cells is referred to 18 For Peer Review Only 19 372 as immunothrombosis [81]. In addition, hypoxia induced by respiratory impairment may 20 21 22 373 cause thrombosis not only by increasing blood viscosity but also by increasing hypoxia- 23 24 374 inducible transcription factors [82]. Moreover, the tropism of the virus for ACE2 receptors 25 26 375 could induce the activation of endothelial cells, disruption of their natural antithrombotic 27 28 properties, and apoptosis. Endothelialopathy may also contribute to the pathophysiology of 29 376 30 31 377 microcirculatory thrombosis [83]. Based on the literature currently available, the 32 33 378 coagulopathy and vasculopathy mechanisms are uncertain. Furthermore, patients with severe 34 35 379 COVID-19 present a hypercoagulability that predisposes them to thrombotic events. This 36 37 38 380 condition explains the presence of high levels of D-dimer. Despite coagulopathy, bleeding 39 40 381 manifestations have not been described. In later stages of COVID-19, sepsis-induced 41 42 382 coagulopathy and disseminated intravascular coagulation have been reported [83]. 43 44 45 383 Many reports describe a high risk of venous thromboembolism. Notably, a high frequency of 46 47 384 pulmonary embolism (20 from 27%) was reported in ICU patients while patients were 48 49 385 receiving a standard dose of venous thromboembolism (VTE) prophylaxis [84, 85]. However, 50 51 a discrepancy between pulmonary embolism and deep venous thrombosis has been observed. 52 386 53 54 387 In view of these data, the use of anticoagulants could reduce the vicious circle of 55 56 388 inflammation-coagulation observed in patients with a severe form of the infection [86]. 57 58 389 Moreover, a protective effect of heparin in patients with most severe COVID-19 infections 59 60

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1 2 3 390 and increased D-dimers has recently been reported [87]. 4 5 6 391 4. Therapeutic options (Tables 1 & 2) 7 8 392 Therapeutic options include antiviral and nonantiviral molecules. Potential antiviral drugs 9 10 393 according to in vitro results and chemical structure are detailed in Table 1. Three chemical 11 12 394 classes are particularly represented: 4-aminoquinolines, phenothiazines, which are chemically 13 14 15 395 related to methylene blue, and ribonucleic analogues. Zinc is of particular interest because it 16 17 396 is a nontoxic micronutrient with direct antiviral activity on RNA-dependent RNA polymerase 18 For Peer Review Only 19 397 of Nidovirales with demonstrated activity in vitro and in a clinical study with the synergistic 20 21 22 398 association combining hydroxychloroquine (HCQ) and azithromycin (AZ). Interferon has 23 24 399 been administered in several Chinese studies, but its efficacy remains unclear. Nonantiviral 25 26 400 molecules are mainly represented by immunomodulators with corticosteroids, anti-interleukin 27 28 6, and hydroxychloroquine since the latter molecule has both antiviral and anti-inflammatory 29 401 30 31 402 effects. Increasing evidence stresses the critical importance of early anticoagulants in patients 32 33 403 with coagulopathy (positive D-dimers), and this is further supported by the fact that lung 34 35 404 embolism may be the direct cause of death in up to 30% of cases at autopsy [9]. 36 37 38 405 4.1 Drugs active against SARS-CoV-2 39 40 406 Testing for molecules that are potentially active against SARS-CoV2 has been based on 41 42 407 different approaches. These included the selective testing of molecules previously shown to 43 44 45 408 be active against SARS-CoV and/or MERS-CoV or suspected to have a broad enough range 46 47 409 of activity to merit testing on SARS-CoV-2; high throughput testing of drug libraries; and in 48 49 410 silico prediction followed by confirmation of drug activity in vitro. Based on previous studies 50 51 on SARS-CoV, chloroquine (CQ) and hydroxychloroquine were among the earliest tested 52 411 53 54 412 molecules against SARS-CoV-2 [88-91]. Both compounds were shown to be efficient but 55 56 413 HCQ exhibited a less toxic profile [90]. Of 20 drugs previously demonstrated to have in vitro 57 58 414 antiviral activity against SARS-CoV and MERS-CoV, several were found to be effective in 59 60

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1 2 3 415 vitro on SARS-CoV-2, including antitumoral drugs, which are likely not to be easily 4 5 6 416 applicable to treatment in humans, and antimalarial drugs such as amodiaquine and, again, 7 8 417 CQ and HCQ [91]. Of the tested drugs that are suspected to display a broad enough range of 9 10 418 activity to be active on SARS-CoV-2, there were two antimicrobial agents, ivermectin and 11 12 419 teicoplanin [92, 93] and the antiviral remdesivir [89] (Table 1). In another study using a more 13 14 15 420 systematic approach, testing of anti-SARS-CoV-2 activity in 1,520 approved drugs [94], 16 17 421 identified 90 molecules with potential efficacy. Of these, opipramol, quinidine and 18 For Peer Review Only 19 422 omeprazole showed significant activity, and this was again the case for CQ and HCQ. From 20 21 22 423 the antibacterial agents, these authors also identified several fluoroquinolones and the 23 24 424 macrolide azithromycin (AZ). Interestingly, in a unique study associating AZ with HCQ (both 25 26 425 at 5 µM), a relative viral inhibition of 99% was observed, suggesting a synergistic effect [95]. 27 28 In another study, the authors cloned, tagged and expressed 26 of 29 SARS-CoV-2 proteins 29 426 30 31 427 and studied their interactions with human proteins as inhibitors of these interactions [96]. Of 32 33 428 the 69 suggested compounds, 29 FDA-approved drugs were identified as being able to inhibit 34 35 429 SARS-CoV-2 replication in vitro, including HCQ. In conclusion, many drugs have been 36 37 38 430 identified as efficient, including many FDA-approved and well known drugs that have been in 39 40 431 use for decades. However, it would be illusory to imagine using psychotropic, antitumoral or 41 42 432 anabolic steroids that were identified in high throughput screenings. Therefore, drugs such as 43 44 45 433 CQ, HCQ, antibiotics or antihistamines used for allergies, and proton pumps inhibitors used 46 47 434 to treat gastroduodenal ulcers, alone or in association with other treatments are likely to 48 49 435 represent the most promising drugs to be tested in clinical trials against COVID-19 (Table 1). 50 51 Finally, another approach uses in silico structure-based virtual drug screening. This consists 52 436 53 54 437 of identifying candidate drugs potentially active on SARS-CoV-2, explaining the activity of 55 56 438 drugs or trying to explain observed activities [97, 98]. This approach does not require, as is 57 58 439 the case of in vitro testing of antiviral activity, a cell culture platform, viral strains, or a P3- 59 60

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1 2 3 440 security level laboratory. It only predicts interactions and the inhibition of viral replication 4 5 6 441 using bioinformatic approaches based on structures, biochemical interactions, structural and 7 8 442 molecular modeling analyses, in silico docking models, or protein-protein interaction 9 10 443 networks. For instance, 69 compounds including HCQ were identified as targeting 66 11 12 444 druggable human proteins or host factors [96]. Compounds were also described as interacting 13 14 15 445 with the viral S-protein and angiotensin-converting enzyme 2 (ACE2)-host cell receptor [99] 16 17 446 while three viral polymerase inhibitors, zidovudine, tenofovir and alovudine, were suspected 18 For Peer Review Only 19 447 to inhibit SARS-CoV-2 RNA polymerase [100]. In addition, structural and molecular 20 21 22 448 modeling analyses made it possible to propose a mechanism of action of CQ and HCQ on 23 24 449 SARS-CoV-2, by inhibiting the binding of the viral S protein to gangliosides, which are 25 26 450 present on the host cell surface and are linked with sialic acids that are used by the virus for 27 28 its entry, in addition to ACE2 [101]. 29 451 30 31 452 4.2 Antiviral properties of chloroquine 32 33 453 CQ is a synthetic 4-aminoquinoline related to quinine. As far back as the mid-1960s, it 34 35 454 was demonstrated that CQ inhibited the mouse hepatitis virus 3 and the encephalomyocarditis 36 37 38 455 virus. Since these pioneering results, a large number of in vitro studies have confirmed that 39 40 456 CQ and HCQ appear to be large spectrum bioactive agents, which possess antiviral activities 41 42 457 against numerous viruses including rabies virus, poliovirus, human immunodeficiency virus, 43 44 45 458 hepatitis viruses, influenza viruses, arboviruses and Ebola virus among others [102]. 46 47 459 However, the demonstration of in vitro activity cannot anticipate the in vivo efficacy of CQ. 48 49 460 Although there was evidence of an antiviral effect of CQ/HCQ in humans infected by 50 51 hepatitis C virus, more disappointing results were reported on other infectious diseases, in 52 461 53 54 462 particular in the treatment of Chikungunya virus, where CQ seemed to worsen symptoms 55 56 463 [103]. With regard to human coronaviruses, CQ was reported to inhibit the in vitro replication 57 58 464 of HCoV-229E, SARS-CoV, HCoV-O43 coronavirus, and MERS-CoV coronavirus [104]. 59 60

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1 2 3 465 Recently, it was reported that CQ/HCQ actually inhibited the in vitro replication of SARS- 4 5 6 466 CoV-2 [89]. With HCoV-O43 it was reported that lethal infections in newborn mice could be 7 8 467 prevented by administering CQ through the mother’s milk [105]. However, CQ apparently 9 10 468 failed to cure MERS-CoV in vivo [106]. 11 12 469 CQ is known for its multiple in vitro mechanisms of action on viruses. Coronavirus cell 13 14 15 470 entry occurs mainly through the endolysosomal pathway. A recent study reported the antiviral 16 17 471 activities of CQ/HCQ against SARS-CoV-2 in Vero E6 cells treated for one hour with the 18 For Peer Review Only 19 472 drugs before exposure to the virus [90]. The virus yield in the cell supernatant was quantified 20 21 22 473 by qRT-PCR, while vesicle-containing virion was investigated by confocal microscopy. The 23 24 474 authors reported that CQ/HCQ significantly inhibited viral entry. Indeed, CQ/HCQ may 25 26 475 possibly inhibit at least five steps of SARS-CoV-2 replication. Recently, the results of a 27 28 molecular modeling approach suggesting that CQ binds to sialic acids and gangliosides with 29 476 30 31 477 high affinity were reported [101]. They hypothesized that both CQ and HCQ inhibit the 32 33 478 attachment of the amino acid region 111-158 of the viral spike of SARS-CoV-2 to 34 35 479 gangliosides. A second mechanism of action of CQ/HCQ on the same step requires the drug 36 37 38 480 to enter target cells to modulate the activity of cellular enzymes. CQ is likely to inhibit the 39 40 481 biosynthesis of sialic acid found at the extremity of sugar chains of glycoproteins. The potent 41 42 482 effects of CQ observed in vitro in cultures of Vero cells exposed to SARS-CoV was 43 44 45 483 considered attributable to a deficit in the glycosylation of cell surface receptor ACE2, which 46 47 484 is the first target of the virus [107]. The second step of the SARS-CoV-2 replicative cycle that 48 49 485 could possibly be inhibited by CQ is the pH-dependent viral endocytosis. This was previously 50 51 demonstrated for several viruses. The protonated form of CQ increased the pH of endosomal 52 486 53 54 487 compartments, thereby blocking the release of the infectious nucleic acid and enzymes 55 56 488 necessary for its replication [108, 109]. The third step of the SARS-CoV-2 that could be a 57 58 489 target for CQ/HCQ is transcription. Indeed, CQ could modulate the activity of the SARS- 59 60

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1 2 3 490 CoV-2 RNA-dependent RNA-polymerase through its function as an ionophore [110] favoring 4 5 6 491 the intracellular transport of the mineral zinc, which inhibits the activity of the polymerase 7 8 492 [111, 112]. The fourth step of the SARS-CoV-2 replicative cycle that is likely to be impaired 9 10 493 by CQ/HCQ deals with the posttranslational modifications of viral proteins within the 11 12 494 endoplasmic reticulum and trans-Golgi network vesicles, possibly by impairing the 13 14 15 495 maturation of its M protein [113]. A fifth level of CQ action could be its effect on cell 16 17 496 signaling, in particular through MAPK [114]. 18 For Peer Review Only 19 497 Since CQ/HCQ have well-characterized immunomodulating activities, in particular anti- 20 21 22 498 inflammatory properties [115], the use of these drugs in the treatment of COVID-19 was also 23 24 499 suggested to possibly protect patients from the cytokine storm that marks the most severe 25 26 500 forms of COVID-19 disease. 27 28 4.3 Clinical use of chloroquine derivatives in COVID-19 patients (Table 2) 29 501 30 31 502 An early observational prospective controlled open label trial was performed in France 32 33 503 at the very beginning of the French epidemic and reported a dramatic beneficial effect on viral 34 35 504 shedding [116], although no clinical outcomes were investigated. In this context, we recently 36 37 38 505 sought to clarify its clinical efficacy through a meta-analysis of comparative studies 39 40 506 conducted on COVID-19 patients [117]. The first findings were that no meta-analysis could 41 42 507 be performed due to major discrepancies in the direction of effect. We therefore investigated 43 44 45 508 which moderator variables could explain such significant heterogeneity while in vitro studies 46 47 509 consistently evidenced an activity of the drug on SARS-CoV-2. Several parameters were 48 49 510 tested, including CQ versus HCQ, dosages, duration and timing of treatment, severity of the 50 51 disease (mild versus severe), combination with an antibiotic (notably AZ), design of studies 52 511 53 54 512 (prospective or retrospective, multicentric or monocentric, randomized controlled trials, 55 56 513 clinical studies or big data analyses conducted on medical records, etc.), comparable groups at 57 58 514 baseline, diagnostic approach (PCR, CT scan, clinical), and combination with other antivirals 59 60

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1 2 3 515 (notably oseltamivir, lopinavir/ritonavir, ribavirin, umifenovir and alpha-interferon). 4 5 6 516 Strikingly, we observed that the main parameter determining the direction of effect was a 7 8 517 study design of big data versus clinical studies, and big data studies were associated with 9 10 518 country (USA), a conflict of interest and the absence of a detailed treatment protocol. When 11 12 519 analyzing big data separately (data were extracted from electronic record files by analysts 13 14 15 520 who did not treat COVID-19-infected patients) and clinical studies (performed by physicians 16 17 521 who treated COVID-19-infected patients), heterogeneity was controlled, and the directions of 18 For Peer Review Only 19 522 the effects became consistent in each group. In big data studies, CQ derivatives were 20 21 22 523 associated with either a null [118-121] or a deleterious effect [122]. Strikingly, the latter big 23 24 524 data study reporting a deleterious effect in 96,000 electronic medical records was 25 26 525 subsequently retracted [123], confirming that clinical studies could be more reliable than big 27 28 data studies. 29 526 30 31 527 In clinical studies, a favorable effect was observed for the duration of fever, duration of 32 33 528 cough, clinical cure, death and/or ICU transfer, and viral shedding [117]. In clinical studies, 34 35 529 three [124-126] out of four [124-127] randomized controlled trials reported a significant 36 37 38 530 beneficial effect. Overall, CQ derivatives were beneficial and improved survival in COVID- 39 40 531 19 infection. However, a standardized therapeutic protocol is required with an adequate 41 42 532 dosage (between 400 and 1,000 mg/d). Studies with higher dosages were associated with 43 44 45 533 significant toxicity [128]. In our experience, a dosage of 600 mg/day for 10 days is adequate. 46 47 534 Indeed, we previously proposed this dosage in Q fever endocarditis for at least 18 months and 48 49 535 in Whipple’s disease for 12 months. This has been subsequently recommended by the 50 51 American CDC [129]. We recently updated this meta-analysis [130] confirming a significant 52 536 53 54 537 beneficial effect on mortality and viral shedding including two large observational studies 55 56 538 from the USA [131, 132] and two from China [133]. Since then, two studies [134, 135], 57 58 539 including the RECOVERY trial [134], have been published with a diagnosis not confirmed by 59 60

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1 2 3 540 PCR, which does not allow for a conclusion to be drawn. In the RECOVERY trial, cases 4 5 6 541 could be “clinically suspected” without laboratory confirmation (approximately 10% negative 7 8 542 tests and 10% without any testing), and 2,400 mg of hydroxychloroquine was administered 9 10 543 during the first 24 hours, which corresponds to a toxic dose. Here, we reported a last update of 11 12 544 our meta-analysis after the inclusion of three very recent studies from Brazil (RCT) [136], 13 14 15 545 Spain [137] and Taiwan [138] and focused on mortality (Figure 5) and persistent viral 16 17 546 shedding (Figure 6). Notably, Cavalcanti et al. reported a better effect with the HCQ-AZ 18 For Peer Review Only 19 547 combination than with HCQ alone, demonstrating the importance of the synergy 20 21 22 548 demonstrated in vitro [136]. Despite substantial heterogeneity, a significant beneficial effect 23 24 549 could be confirmed for both mortality and viral shedding. 25 26 550 However, debate is currently rife in the scientific community and among government 27 28 decision makers as to the risk-benefit of using HCQ in the treatment of COVID-19 patients. 29 551 30 31 552 Recently, the WHO decided to ban the use of HCQ for COVID-19 patients, although various 32 33 553 clinical trials suggest that the benefits of using this molecule in combination with 34 35 554 azithromycin outweigh any harmful effects [126]. 36 37 38 555 4.4 Azithromycin and COVID-19 39 40 556 AZ is a well-known and safe macrolide antibiotic with immunomodulatory properties. It 41 42 557 has a long half-life and a large volume of distribution [139]. It has excellent tissue penetration 43 44 45 558 in the lung and is widely prescribed for the treatment of respiratory infections. Its mechanism 46 47 559 of immunomodulation includes decreased production of pro-inflammatory cytokines (IL-6, 48 49 560 IL-8 and TNFα) and inhibition of neutrophil activation [140]. Although AZ has not been 50 51 labeled for the treatment of antiviral infections, it has been studied in vitro and in clinical 52 561 53 54 562 trials for activity against several viruses [139]. Numerous investigations have reported the in 55 56 563 vitro antiviral activity of AZ against viral pathogens, including SARS-Cov-2, at 57 58 564 concentrations that are physiologically achievable with the usual doses used for the treatment 59 60

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1 2 3 565 of bacterial respiratory infections [139]. The precise mechanism of antiviral activity is not 4 5 6 566 known [139]. However, the intracellular accumulation of AZ, a weak base in endosomal 7 8 567 vesicles and lysosomes intracellularly may result in an increase in endosomal and/or 9 10 568 lysosomal pH and limit viral replication, through the lack of an optimal acidic environment in 11 12 569 the intracellular milieu. Interestingly, HCQ is also a weak base, and this could explain how 13 14 15 570 the two drugs work together to inhibit viral replication. Recently, our group demonstrated that 16 17 571 the combination of HCQ and AZ had a synergistic effect in vitro on SARS-CoV-2 at 18 For Peer Review Only 19 572 concentrations compatible with that obtained in the human lung [95]. 20 21 22 573 Many clinical studies on the efficacy of AZ alone or in combination with other drugs 23 24 574 against various viral infections have been observational, single‐arm, non-randomized studies 25 26 575 or retrospective evaluations and have mainly focused on viral load as an end point. 27 28 29 576 Collectively, however, they present preliminary evidence that the inclusion of AZ in various 30 31 577 treatment regimens can influence the course of viral infection and clinical outcomes [139, 32 33 578 141, 142]. 34 35 579 4.5 Clinical use of zinc in COVID-19 patients 36 37 38 580 Zinc has both antiviral and immune properties [143]. Known to inhibit the 39 40 581 multiplication of several viruses, in vitro models have demonstrated that low zinc 41 42 582 concentrations combined with ionophores block the elongation of the SARS-CoV-1 RNA- 43 44 45 583 dependent RNA polymerase. [112]. Interestingly HCQ and CQ are ionophores that enhance 46 47 584 zinc uptake, thereby increasing its concentration into the lysosomes [110]. The potential 48 49 585 synergistic effect of a combination with CQ/HCQ on SARS-CoV-2 replication remains, 50 51 52 586 however, to be demonstrated. Zinc is also involved in antiviral immunity through several 53 54 587 mechanisms including the modulation of interferon response [143]. As zinc supplementation 55 56 588 over a short period is not harmful to health, it has been proposed in combination with HCQ in 57 58 589 COVID-19 patients, with promising results [144]. 59 60

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1 2 3 590 4.6 Clinical uses of remdesivir on COVID-19 patients 4 5 6 591 To date, eight studies (1,773 patients) have reported the use of remdesivir in COVID-19 7 8 592 patients. Case reports showed that remdesivir has no clinically relevant efficacy. A 9 10 593 compassionate uncontrolled study supported and funded by Gilead reported that 68% of 11 12 594 treated patients saw clinical improvements, but there was considerable missing data, and the 13 14 15 595 outcomes of 9/61 patients were still under evaluation at the time of publication and were not 16 17 596 reported [145]. One randomized controlled study (RCT) included 236 patients (158/78) from 18 For Peer Review Only 19 597 10 hospitals in Wuhan. The mean age, sex ratio, delay from onset to enrolment, comorbidities, 20 21 22 598 enrolment criteria (O2<95%), and radiologically confirmed pneumonia, were comparable in 23 24 599 the two arms. The primary clinical endpoint was the time to clinical improvement within 28 25 26 600 days after randomization, and 100% of patients enrolled were evaluated in the intention-to- 27 28 treat analysis. No significant differences were noted between the two groups. Serious adverse 29 601 30 31 602 events or events leading to administration of the drug being stopped were reported in 18% and 32 33 603 12% of patients, respectively, in the remdesivir group compared to 6% and 5%, respectively, 34 35 604 in the placebo group, demonstrating the poor safety profile of the drug [146]. Another RCT 36 37 38 605 conducted on 1,063 patients (541/522) argued for a significant benefit of remdesivir on time- 39 40 606 to-recovery and mortality in the intention-to-treat analysis; however, at the time of 41 42 607 publication, only one-third of enrolled patients had received complete treatment and had been 43 44 45 608 analyzed. Such a loss to follow-up poses serious threats to the validity of this study [147]. The 46 47 609 last released paper compares five days to 10 days of treatment with remdesivir with no 48 49 610 significant difference in mortality rates or improvement in clinical status. Serious adverse 50 51 events were reported in 27.7% of treated patients, including 4.7% acute kidney injuries. In 52 611 53 54 612 7.3% of patients, adverse events led to the treatment being stopped [148]. In addition, 55 56 613 remdesivir is currently only available intravenously, making early treatment at home 57 58 59 60

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1 2 3 614 impossible and exposing patients to the risk of intravenous catheter complications 4 5 6 615 (lymphangitis, thrombophlebitis, endocarditis). 7 8 616 4.7. Use of nonantiviral therapy in COVID-19 patients 9 10 617 During the immunopathogenic phase of COVID-19, serum IL-6 concentrations have 11 12 618 been found to be consistently elevated (90 to 160 pg/ml) and correlated with severity [1, 67]. 13 14 15 619 IL-6 is produced by various cells, mainly in response to IL-1, and is the main inducer of acute 16 17 620 phase-protein synthesis by the liver, notably CRP. It plays a role in B lymphocyte 18 For Peer Review Only 19 621 differentiation and encourages mononuclear cell inflammation and Th-17 response [149, 150]. 20 21 22 622 IL-6 acts through binding to a ligand receptor, IL-6R, then complexes with signaling receptor 23 24 623 gp130. Although gp130 is widely expressed, IL-6R expression is limited to leukocytes and 25 26 624 hepatocytes but can be shed from these cells in a soluble form, which binds to IL-6 and then 27 28 to gp130, rendering IL-6R-negative cells sensitive to IL-6, a mechanism named trans- 29 625 30 31 626 signaling [151]. Through this mechanism, IL-6 has been involved in various inflammatory 32 33 627 diseases, such as rheumatoid arthritis (RA) and lung fibrosis [149] [152]. 34 35 628 Tocilizumab is a humanized anti-IL-6R monoclonal antibody (mAb) that is able to bind 36 37 38 629 both to the membrane and soluble IL-6R and to inhibit IL-6 functions. It is currently used in 39 40 630 the treatment of various chronic diseases, notably rheumatoid arthritis, giant cell arteritis, and 41 42 631 systemic juvenile idiopathic arthritis (sJIA), and has a good safety profile despite its long half- 43 44 45 632 life [149]. sJIA is often complicated by macrophage activation syndrome characterized by a 46 47 633 cytokine storm, and tocilizumab has been shown to be effective in the treatment of this 48 49 634 disease [153]. Moreover, cancer treatments using CAR-T cells may be complicated by a 50 51 cytokine release syndrome, which is efficiently treated by tocilizumab [154]. Tocilizumab 52 635 53 54 636 was, therefore, used to treat 21 severe or critical COVID-19 patients in China and was 55 56 637 retrospectively reported to be effective [155]. The patients received a single 400 mg infusion. 57 58 638 Their body temperature returned to normal within 24 hours, oxygen saturation improved 59 60

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1 2 3 639 rapidly in 75% of cases, and CRP returned to normal in 80% of the patients after five days. 4 5 6 640 This observation was confirmed in Italy [156]. Tolerance to treatment appeared to be good in 7 8 641 all studies, but acute hypertriglyceridemia and pancreatitis were recently reported in two 9 10 642 patients [157]. 11 12 643 Together, these results are promising and have justified several ongoing trials in Italy, 13 14 15 644 France and the US (using sarilumab, another anti-IL-6R mAb) [158]. However, other 16 17 645 therapeutic strategies may be effective in order to treat the cytokine storm associated with 18 For Peer Review Only 19 646 COVID-19, such as IL-1 receptor antagonists (anakinra), which act upstream of IL-6 and have 20 21 22 647 a short half-life, anti-IFNg (emapalumab), NLRP3 pharmacological inhibitors (dapansutrile) 23 24 648 or JAK kinase inhibitors, which may block IL-6 and IFNs signaling downstream of their 25 26 649 receptors. Recent retrospective case series or small cohorts conducted in Italy [159] and in 27 28 France [160] have shown that anakinra may be an effective treatment in arresting 29 650 30 31 651 inflammatory respiratory deterioration. 32 33 652 In addition, preliminary results from the RECOVERY trial suggest that dexamethasone 34 35 653 either by mouth or by intravenous injection improves prognosis in patients on oxygen and 36 37 38 654 those in intensive care [161]. However, final detailed results are not published. Moreover, no 39 40 655 benefit was observed among those patients who did not require respiratory support. In this 41 42 656 context, hydroxychloroquine, with its strong anti-IL6 activity [162], remains an attractive 43 44 45 657 option as an early nonantiviral therapy. 46 47 658 4.8 Prophylactic use of hydroxychloroquine against COVID-19 48 49 659 4.8.1 Pre-exposure prophylaxis 50 51 In a case-controlled study in India on symptomatic healthcare workers (HCWs) 52 660 53 54 661 including 378 positive cases (symptomatic HCWs with SARS-CoV-2 positive PCR, including 55 56 662 172 with HCQ prophylaxis) and 373 controls (symptomatic HCWs with SARS-CoV-2 57 58 663 negative PCR including 193 with HCQ prophylaxis), the administration of at least four HCQ 59 60

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1 2 3 664 doses was associated with a significant, fifty percent lower risk of COVID-19 (adjusted odds 4 5 6 665 ratio [aOR] 0.44, 95% confidence interval 0.22–0.88) [163]. A dose-dependent relationship 7 8 666 was observed with an adjusted odds ratio decreasing from 0.44 (0.22–0.88, p = .02) for four– 9 10 667 five doses and 0.04 (0.01–0.16, p < .001) for six doses and more. One to three doses had no 11 12 668 effect while six or more prophylactic doses had a remarkably high (>80%) protective effect. 13 14 15 669 Of the 365 HCWs who reported taking HCQ, nausea, headache and diarrhea were reported in 16 17 670 approximately 5%; only individual case reported palpitations. 18 For Peer Review Only 19 671 4.8.2 Postexposure prophylaxis 20 21 22 672 In South Korea, a COVID-19 exposure event occurred in a long-term care hospital, and 23 24 673 HCQ postexposure prophylaxis was provided to 211 individuals [164]. None of them 25 26 674 developed the disease, and none had a positive PCR during follow-up. However, since no 27 28 subjects who did not receive prophylaxis became positive, effectiveness could not be truly 29 675 30 31 676 evaluated. In the USA, a double-blinded randomized controlled trial was performed with 414 32 33 677 subjects receiving HCQ and 407 receiving folate as a placebo within four days of exposure 34 35 678 [165]. No arrhythmias or deaths occurred. Furthermore, 49 (11.8%) developed a disease 36 37 38 679 compatible with COVID-19 in the HCQ group compared to 58 (14.3%) in the control group 39 40 680 (p = 0.35). The authors thus concluded that HCQ did not prevent the disease. However, most 41 42 681 of the people included did not have a definitive diagnosis of COVID-19 by PCR. In addition, 43 44 45 682 the delay in postexposure chemoprophylaxis strongly impacts the efficacy, with a fifty percent 46 47 683 decrease in the risk of infection when HCQ was administered on day 1 and decreasing 48 49 684 efficacy over time. For influenza, postexposure chemoprophylaxis has only been 50 51 recommended when antivirals can be initiated within 48 hours of exposure [166]. This 52 685 53 54 686 highlights the need to initiate chemoprophylaxis rapidly after exposure. 55 56 687 4.9. Global care of COVID-19 patients 57 58 688 4.9.1 Outpatient care 59 60

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1 2 3 689 In the context of a brutal and deadly pandemic (global mortality rate of approximately 4 5 6 690 6% and up to 18% in France), no randomized trial investigating early versus delayed 7 8 691 treatment has been conducted. Barbosa Esper et al. reported that early telemedicine treatment 9 10 692 with HCQ and AZ was associated with a significant decrease in hospitalization rates [167]. 11 12 693 Zev Zelenko, a general practitioner in the suburbs of New York, USA, reported on the early 13 14 15 694 treatment of 405 patients with HCQ 200 mg 2/d, AZ 500 mg 1/d and zinc sulphate 220 mg 16 17 695 1/d for five days at a total cost of $20 [168]. All patients with dyspnea or with risk factors 18 For Peer Review Only 19 696 (regardless of clinical status) were treated. As of 26 April, he had reported one death in a 20 21 22 697 patient with leukemia (1/405 (0.2%)). The only adverse events reported were nausea and 23 24 698 diarrhea in 10% of cases but no cardiac toxicity. Guerin et al. reported on a French study on 25 26 699 outpatient treatment of 88 patients with HCQ-AZ, HCQ and controls [169]. Early HCQ or 27 28 HCQ-AZ treatment was associated with a significantly shorter time to clinical recovery. There 29 700 30 31 701 was no significant difference between HCQ alone and HCQ-AZ. No cardiac toxicity was 32 33 702 observed. 34 35 703 Finally, massive screening and early treatment have been implemented in our center 36 37 38 704 (Marseille, France), with more than three-quarters of the 1,061 reported cases managed in a 39 40 705 day care hospital for initial evaluation and treatment [141]. The therapeutic protocol included 41 42 706 at least an ECG, potassium measurement, D-dimers, low-dose CT scan, HCQ (200 mg three 43 44 45 707 times per day in the absence of ECG repolarization disorder and abnormal kalemia), and AZ 46 47 708 (500 mg on the first day then 250 mg per day for four additional days). Correction of 48 49 709 hypokalemia was needed in approximately 15% of our cohort and was associated with 50 51 severity [170]. This is critical since hypokalemia could increase the risk of cardiac 52 710 53 54 711 arrhythmia. After confirming in our cohort that zinc deficiency was associated with an 55 56 712 unfavorable prognosis, and in view of a comparative study in favor of zinc [144], 15 mg of 57 58 713 zinc three times daily was added. Any patient with positive D-dimers was evaluated for 59 60

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1 2 3 714 pulmonary embolism, and treatment with low molecular weight heparin was systematically 4 5 6 715 discussed on an outpatient or inpatient basis. At least one patient was diagnosed with a 7 8 716 pulmonary embolism in our day care hospital after systematic D-dimer assessment without 9 10 717 any clinical signs. In the presence of a clinical (NEWS score ≥ 5) or biological sign of 11 12 718 severity, or if the treatment became difficult, patients were systematically hospitalized. 13 14 15 719 4.9.2 Inpatient care 16 17 720 Under this global management strategy, based on early massive nonselective screening, 18 For Peer Review Only 19 721 day care hospitals, inpatient and early resuscitation care, no deaths under the age of 60 years 20 21 22 722 have been observed. In the 60+ age group, the mortality regardless of treatment was 5.0%, 23 24 723 which was similar to the mortality in the same age group reported in China (6.0%) but lower 25 26 724 than that reported in Italy (12.3%) [171]. On the other hand, among patients aged 60 years and 27 28 over who had at least three days of dual HCQ-AZ therapy in our center, the mortality was 29 725 30 31 726 3.1%, which was much lower than that reported in China and Italy for the same age group. 32 33 727 Based on the currently available literature, we would recommend measuring D-dimers, 34 35 728 prothrombin time, and platelet count in all patients with COVID-19 infection. The optimal 36 37 38 729 dosage of low molecular weight heparin is currently the subject of much discussion within the 39 40 730 medical community. Although the majority suggests prophylactic daily LMWH, a minority 41 42 731 considers intermediate or therapeutic doses [83]. In all cases, the optimal doses for each 43 44 45 732 patient need to take account specific patient thrombotic risk factors, such as active cancer 46 47 733 (treatment within the last six months), recent personal history of thromboembolic events 48 49 734 ( 30 kg/m2) [172]. 50 51 Regarding possible drug interactions, oral anticoagulants prescribed for long-term use should 52 735 53 54 736 be replaced by curative heparin therapy. 55 56 737 4.9.3 Intensive care 57 58 59 60

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1 2 3 738 Approximately 15% of COVID-19 patients require admission to an ICU. The biggest 4 5 6 739 reason for admission is acute respiratory failure [173]. Gattinoni et al. recently described two 7 8 740 phenotypes of ICU patients: phenotype L and phenotype H. Phenotype L corresponds to a 9 10 741 clinical picture of ARDS with low elastance, which is unexpected in this syndrome. 11 12 742 Phenotype H for high elastance is in line with the common picture of ARDS, representing 20- 13 14 15 743 30% of COVID-19 patients [174]. A CT scan is a perfect tool to differentiate the two 16 17 744 phenotypes: the nonaerated lung is close to 0 in phenotype L patients, while posterior 18 For Peer Review Only 19 745 condensations are found in phenotype H patients [174]. Most patients evolve from phenotype 20 21 22 746 L to phenotype H, which may be due to COVID-19 pneumonia evolution or to the adverse 23 24 747 effects of high-pressure mechanical ventilation. The hospital mortality rate of COVID-19 25 26 748 patients admitted to the ICU ranged from 26% [20] to 50% [175] and was even higher in 27 28 elderly patients [173]. However, in our center, the in-ICU mortality was approximately 18% 29 749 30 31 750 (unpublished data) in a context of a global strategy including early testing, early care and 32 33 751 early treatment [141]. 34 35 752 While COVID-19 was initially documented as isolated acute respiratory failure, patients 36 37 38 753 admitted to the ICU develop multiple organ failure in approximately 50% of cases [173]. 39 40 754 Among organ failures, an exacerbated activation of coagulation in COVID-19 patients 41 42 755 resulted in a large number of thromboses and pulmonary embolisms. The activation of 43 44 45 756 coagulation is a well-known host response in patients with septic shock. Specific treatments 46 47 757 were developed to counteract this response, but successive studies failed to demonstrate 48 49 758 efficiency. However, disruptions of coagulation strongly affect the outcomes of ICU patients 50 51 with COVID-19. In a cohort of 184 ICU patients, 27% of patients developed deep vein 52 759 53 54 760 thrombosis, and pulmonary embolism was found in 80% of cases [84]. Anticoagulation 55 56 761 treatment should be widely administered to reduce the risk of pulmonary embolism, and 57 58 762 protocols should include curative anticoagulation in most mechanically ventilated patients. 59 60

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1 2 3 763 Monitoring coagulation factors, including D-dimers and fibrinogens, provides relevant 4 5 6 764 information to decide between prophylactic or curative anticoagulation. Those with an 7 8 765 increased body mass index are at a very high risk of thrombosis. 9 10 766 In ICU patients, an intense inflammatory response has been described in the late phase 11 12 767 of the disease. The production of pro-inflammatory cytokines has been reported to be 13 14 15 768 exacerbated in patients with negative SARS-CoV-2 PCR. This response profile encouraged a 16 17 769 few teams to use steroids, notably for patients developing ARDS [68]. In a cohort series, the 18 For Peer Review Only 19 770 use of steroids in ARDS patients was associated with improved outcomes, within the 20 21 22 771 limitations of this study based on multivariate analysis [176]. However, the pro-inflammatory 23 24 772 profile of septic patients in the ICU was previously described, while recent trends revealed 25 26 773 profound immunosuppression. Recent data do not confirm the pro-inflammatory response in 27 28 COVID-19 patients in the ICU (personal data) as the profile is time dependent. Introducing 29 774 30 31 775 steroids to patients with viral infections and immunosuppressed patients can be high risk, 32 33 776 resulting in an increase in superinfections. Thus, it seems prudent to restrict the use of steroids 34 35 777 in those severe patients who usually have a severe lymphopenia. 36 37 38 778 4.10 Traditional Medicine 39 40 779 Traditional Chinese Medicine (TCM) therapy has played a role in treating epidemic 41 42 780 diseases in China's long history [177]. In combination with Western medicine, TCM therapy 43 44 45 781 was widely used in China for the management of COVID-19, with up to 91.5% of patients 46 47 782 with a confirmed diagnosis having reportedly received TCM [178]. Several studies support 48 49 783 that the current practice of TCM has a favorable impact in the management of COVID-19 and 50 51 can shorten of the course of fever, course of the disease, and length of hospital stay, and 52 784 53 54 785 reduce the number of severe patients and death rate [177, 179]. Three patented TCM 55 56 786 medicines (Lianhua Qingwen capsules, Jinhua Qinggan granules, and Xuebijing injection) 57 58 787 and three TCM decoctions (Qingfei Paidu, Huashi Baidu, and Xuanfei Baidu) have mainly 59 60

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1 2 3 788 been highlighted for the management of COVID-19 [177, 180]. Lianhua Qingwen is a 4 5 6 789 formulation prepared from the classic compounds of ancient China, commonly used in the 7 8 790 treatment of colds and flu. Jinhua Qinggan is a formulation developed for the treatment of 9 10 791 influenza A (H1N1) in 2009. The use of Jinhua Qinggan and Lianhua Qingwen is suggested 11 12 792 for medical observation of COVID-19 [181]; they could also be used for patients with mild 13 14 15 793 and moderate symptoms. Xuebijing injection was developed for the treatment of SARS in 16 17 794 China and is indicated for systemic inflammatory response syndrome induced by infections 18 For Peer Review Only 19 795 [180]. Xuebijing is used for severe and critical COVID-19 infection [181]. Qingfei Paidu, 20 21 22 796 Huashi Baidu, and Xuanfei Baidu decoctions are three new prescriptions specifically designed 23 24 797 for COVID-19 [181]. Their use is suggested in the prevention and mild and moderate 25 26 798 infections of SARS-CoV-2 [181]. The action mechanisms of Chinese medicines are still 27 28 unclear. Most of them are founded on pharmacology-based predictions [177]. The 29 799 30 31 800 composition, potential active ingredients, predicted targets, signaling pathways and 32 33 801 mechanisms of these Chinese herbal medicines have been recently detailed by Huang et al. in 34 35 802 a review article [177]. The use of TCM at different stages of COVID-19 is among the 36 37 38 803 recommendations in China and South Korea [177, 182]. Ho et al. have summarized the 39 40 804 guidelines of the National Health Commission and the National Administration of Traditional 41 42 805 Chinese Medicine of the People's Republic of China for the management of COVID-19, 43 44 45 806 which, in addition to the abovementioned formulas, includes compositions adapted according 46 47 807 to the patient’s condition [182]. The reported duration of COVID-19 treatment with herbal 48 49 808 medicines usually varied from 5 to 15 days [179]. Regarding potential side effects, no severe 50 51 discomfort or abnormal liver or kidney function has been identified [179]. Although basic and 52 809 53 54 810 clinical research is still needed to understand the mechanisms of action and lead to evidence- 55 56 811 based medicine, it is worth paying attention to TCM. Indeed, it must be remembered that the 57 58 812 use of herbal medicine against malaria has allowed to the discovery of artemisinin, a plant 59 60

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1 2 3 813 extract of Artemisia annua used as a standard treatment against Plasmodium falciparum 4 5 6 814 worldwide [183]. 7 8 815 5. Conclusions 9 10 816 COVID-19 is a newly emerging disease that requires us to adjust our medical approach as we 11 12 817 receive new observational evidence about the virus and its behaviors. Indeed, the observation 13 14 15 818 of symptoms usually rarely associated with respiratory infections, such as anosmia or a lack 16 17 819 of perceived dyspnea with documented hypoxemia and severe radiological impairment on CT 18 For Peer Review Only 19 820 scan, and viral thrombotic disease with up to 30% of critical cases developing pulmonary 20 21 22 821 embolism, led us to adapt our clinical approach toward SARS-CoV-2-associated pneumonia. 23 24 822 Early treatment of patients through the rapid implementation of specific PCR diagnostic tools 25 26 823 has been, in many cases, key to successfully controlling the disease. In most affected 27 28 countries, the epidemic appears to spontaneously resolve over time, but COVID-19 morbidity 29 824 30 31 825 and mortality indicators are, paradoxically, worse in the rich countries of Western Europe and 32 33 826 the US than in developing countries. The debate about the usefulness of chloroquine 34 35 827 derivative-based treatment, initially proposed by Chinese physicians, and that of new 36 37 38 828 antivirals such as the orphan drug remdesivir, has reached unprecedented levels of 39 40 829 aggressiveness. This culminated in the recent retraction of COVID-19 publications from the 41 42 830 world’s two most prestigious medical journals, The Lancet and The New England Journal of 43 44 45 831 Medicine. Thus, lessons from the COVID-19 pandemic go far beyond the disease itself and 46 47 832 question our responsiveness toward a disease with medical, societal, economic, and political 48 49 833 consequences that may lead to major editorial pitfalls challenging the credibility of the main 50 51 actors in the field of medical literature. 52 834 53 54 835 6. Expert opinion (Figure 7) 55 56 836 The COVID-19 epidemic is an unprecedented crisis, not in terms of mortality, but in terms of 57 58 837 emotion and the measures taken to fight it. It is not the worst epidemic we have experienced 59 60

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1 2 3 838 in recent decades, but it has led to changes in the organization of care, including lockdown 4 5 6 839 measures, which are unprecedented. However, it is likely that in most countries, COVID- 7 8 840 related mortality will go unnoticed in the annual mortality statistics. This is particularly 9 10 841 because it has mostly affected older people. The epidemic has also revealed the 11 12 842 disorganization of political decisions with totally different strategies from one country to 13 14 15 843 another and paradoxically higher mortality in the richest countries, especially those in 16 17 844 Western Europe and the US. No consensus has been reached around the implementation of 18 For Peer Review Only 19 845 basic strategies including diagnostic tests, isolation of contagious patients, patient care and 20 21 22 846 potentially safe and effective therapies. Finally, unsupported decisions taken by governments 23 24 847 and the WHO have increased confusion. This has led to doubtful publications being retracted 25 26 848 from the best journals in the world and has given rise to a previously unknown state of 27 28 nervousness. When studying the disease seriously, a few things are worth noting. It does not 29 849 30 31 850 present itself as common flu but commonly presents with anosmia. The patient may be free of 32 33 851 fever, cough, and shortness of breath, despite significant hypoxia and CT lesions. This 34 35 852 warrants an expanded strategy of diagnostic testing, measuring oxygen saturation and 36 37 38 853 performing low-dose CT scans to detect specific lesions. Early therapeutic management in 39 40 854 terms of care (anticoagulants, oxygen therapy) and drugs that have proven or will prove to be 41 42 855 effective at this stage (HCQ and AZ and possibly remdesivir in the early stages) will prevent 43 44 45 856 progression to respiratory failure, resuscitation and death. A number of markers have been 46 47 857 associated with this pejorative evolution including a lymphopenia below 500 being be the 48 49 858 most predictive, decreased zinc levels, eosinopenia, increase in polymorphonuclear cells and 50 51 increase in D-dimers. Subjects with hypertension are more at risk as a result of hypertension 52 859 53 54 860 or antihypertensive drug use. The disease appears to unfold in three stages: a purely 55 56 861 virological stage, a stage associating the virus with the immune response, and a final stage in 57 58 862 the most severe forms, which appears essentially as an immune response without the virus. 59 60

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1 2 3 863 This explains why antiviral treatment attempts in patients with very severe disease are usually 4 5 6 864 ineffective, and samples taken to look for the viral load at this stage are often negative. A 7 8 865 cytokine storm occurs against which no drug has proven to be effective. This is something 9 10 866 that is common in adult acute respiratory distress syndromes, where treating the cause is less 11 12 867 critical than properly managing it. Immunity as evidenced by antibodies against the virus 13 14 15 868 appear around the tenth day and is extremely marked in the most seriously affected subjects. 16 17 869 In our experience, deceased subjects had the highest antibody levels. Concerning the 18 For Peer Review Only 19 870 epidemiology of the disease, it presents in the form of a bell curve, which is highly typical of 20 21 22 871 respiratory viral infections. It appears that part of the population is naturally immune, which 23 24 872 may be related to endemic circulating coronaviruses, which cause 10 to 20% of respiratory 25 26 873 infections, especially in children. Indeed, it is remarkable that the incidence of both the virus 27 28 and the disease in children has been low. Under these conditions, the use of a vaccination 29 874 30 31 875 before this point is likely to present more risks than benefits. The future of this disease is 32 33 876 unknown. 34 35 877 What lessons has this crisis taught us? First, in 2020, infectious disease diagnosis can no 36 37 38 878 longer be based solely on clinical criteria without microbiological testing. Indeed, at the peak 39 40 879 of the epidemic in our center, only 22% of symptomatic patients were PCR positive. Many 41 42 880 viruses have cocirculated, including endemic coronaviruses. Second, general 43 44 45 881 recommendations cannot be made until the disease is observed and known. It is only recently 46 47 882 that the four stages of the disease have been identified: 1) viral, 2) viral and dysimmune, 3) 48 49 883 dysimmune, and 4) lesional (ARDS) stages. Each stage corresponds to a specific treatment 50 51 (Figure 3). Then, when evaluating the efficacy of a treatment, the molecule should not be 52 884 53 54 885 considered in isolation. A treatment is not a molecule, it is a therapeutic protocol with one or 55 56 886 more molecules (synergistic effect of the HCQ+AZ+zinc combination [144]), indications, 57 58 887 contraindications, precautions for use, and a precise dosage and duration. For example, for 59 60

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1 2 3 888 several decades we have been administering HCQ at 200 mg three times a day for 18 to 24 4 5 6 889 months in Coxiella burnetii endocarditis in association with doxycycline [184]. This dosage is 7 8 890 necessary to achieve therapeutic blood levels [184]. This protocol has been used according to 9 10 891 the recommendations of the American CDC for the same indication [129] and in the most 11 12 892 well-known professional sites (https://www.uptodate.com/contents/q-fever-endocarditis). In 13 14 15 893 rheumatic diseases such as lupus or rheumatoid arthritis, dosages of 400 to 600 mg/d are used 16 17 894 (www.uptodate.com). In COVID-19, extreme dosages of 2,400 mg/d were used in the 18 For Peer Review Only 19 895 RECOVERY trial, i.e., four times the usual dose, while in the Discovery trial, low doses of 20 21 22 896 400 mg/d were used. Overall, standardized therapeutic protocols associated with a 23 24 897 comprehensive strategy including early massive screening would appear to be critical in 25 26 898 responding to the COVID-19 pandemic. 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 899 Declaration of interest statement 4 5 6 900 The authors have no relevant affiliations or financial involvement with any organization or 7 8 901 entity with a financial interest in or financial conflict with the subject matter or materials 9 10 902 discussed in the manuscript. This includes employment, consultancies, honoraria, stock 11 12 903 ownership or options, expert testimony, grants or patents received or pending, or royalties. 13 14 15 904 CD declares owning SANOFI shares. 16 17 18 905 Financial supportFor Peer Review Only 19 20 21 906 This study was supported by the Institut Hospitalo-Universitaire (IHU) Méditerranée 22 23 907 Infection, the National Research Agency under the program « Investissements d’avenir », 24 25 26 908 reference ANR-10-IAHU-03, the Région Provence Alpes Côte d’Azur. 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 909 References 4 5 6 910 1. Zhou F, Yu T, Du R, et al. Clinical course and risk factors for mortality of adult 7 8 9 911 inpatients with COVID-19 in Wuhan, China: a retrospective cohort study. Lancet 10 11 912 395(10229):1054-1062 (2020). 12 13 14 913 2. Wang D, Hu B, Hu C, et al. Clinical Characteristics of 138 Hospitalized Patients With 15 16 17 914 2019 Novel Coronavirus-Infected Pneumonia in Wuhan, China. JAMA 323:1061-9 18 For Peer Review Only 19 915 (2020). 20 21 22 916 3. Lagier JC, Million M, Gautret P, Cortaredona S, Giraud-Gatineau A. Outcomes of 23 24 917 3,737 COVID-19 patients treated with hydroxychloroquine/azithromycin and other 25 26 27 918 regimens in Marseille, France: a retrospective analysis. Travel Med Infect Dis 28 29 919 36:101791 (2020). 30 31 32 920 4. Liao X, Wang B, Kang Y. Novel coronavirus infection during the 2019-2020 33 34 35 921 epidemic: preparing intensive care units-the experience in Sichuan Province, China. 36 37 922 Intensive Care Med 46:357-60 (2020). 38 39 40 923 5. Menni C, Sudre CH, Steves CJ, Ourselin S, Spector TD. Quantifying additional 41 42 43 924 COVID-19 symptoms will save lives. Lancet 395(10241):e107-e108 (2020). 44 45 46 925 6. Couzin-Frankel J. The mystery of the pandemic's 'happy hypoxia'. Science 368:455-6 47 48 926 (2020). 49 50 51 927 7. Middeldorp S, Coppens M, van Haaps TF, et al. Incidence of venous 52 53 54 928 thromboembolism in hospitalized patients with COVID-19. J Thromb Haemost 55 56 929 (2020). 57 58 59 60

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1 2 3 930 8. Leonard-Lorant I, Delabranche X, Severac F, et al. Acute Pulmonary Embolism in 4 5 6 931 COVID-19 Patients on CT Angiography and Relationship to D-Dimer Levels. 7 8 932 Radiology201561 (2020). 9 10 11 933 9. Wichmann D, Sperhake JP, Lütgehetmann M, et al. Autopsy Findings and Venous 12 13 934 Thromboembolism in Patients With COVID-19. Ann Intern Med M20-2003 (2020). 14 15 16 17 935 10. Qin C, Zhou L, Hu Z, et al. Dysregulation of immune response in patients with 18 For Peer Review Only 19 936 COVID-19 in Wuhan, China. Clin Infect Dis 71(15):762-768 (2020). 20 21 22 937 11. Wang J, Wang BJ, Yang JC, et al. [Advances in the research of mechanism of 23 24 25 938 pulmonary fibrosis induced by Corona Virus Disease 2019 and the corresponding 26 27 939 therapeutic measures]. Zhonghua Shao Shang Za Zhi 36:E006 (2020). 28 29 30 940 12. Ye Z, Zhang Y, Wang Y, Huang Z, Song B. Chest CT manifestations of new 31 32 coronavirus disease 2019 (COVID-19): a pictorial review. Eur Radiol 30(8):4381- 33 941 34 35 942 4389 (2020). 36 37 38 943 13. Kim H. Outbreak of novel coronavirus (COVID-19): What is the role of radiologists? 39 40 944 Eur Radiol 30:3266-7 (2020). 41 42 43 44 945 14. Shi H, Han X, Jiang N, et al. Radiological findings from 81 patients with COVID-19 45 46 946 pneumonia in Wuhan, China: a descriptive study. Lancet Infect Dis 20:425-34 (2020). 47 48 49 947 15. Li K, Wu J, Wu F, et al. The Clinical and Chest CT Features Associated With Severe 50 51 52 948 and Critical COVID-19 Pneumonia. Invest Radiol55:327-31 (2020). 53 54 55 949 16. Bai HX, Hsieh B, Xiong Z, et al. Performance of radiologists in differentiating 56 57 950 COVID-19 from viral pneumonia on chest CT. Radiology 296(2):E46-E54 (2020). 58 59 60

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1 2 3 951 17. Ai T, Yang Z, Hou H, et al. Correlation of Chest CT and RT-PCR Testing in 4 5 6 952 Coronavirus Disease 2019 (COVID-19) in China: A Report of 1014 Cases. Radiology 7 8 953 296(2):E32-E40 (2020). 9 10 11 954 18. Li L, Qin L, Xu Z, et al. Using Artificial Intelligence to Detect COVID-19 and 12 13 955 Community-acquired Pneumonia Based on Pulmonary CT: Evaluation of the 14 15 16 956 Diagnostic Accuracy. Radiology 296(2):E65-E71 (2020) 17 18 For Peer Review Only 19 957 19. Wang B, Li R, Lu Z, Huang Y. Does comorbidity increase the risk of patients with 20 21 958 COVID-19: evidence from meta-analysis. Aging (Albany NY) 12:6049-57 (2020). 22 23 24 959 20. Grasselli G, Zangrillo A, Zanella A, et al. Baseline Characteristics and Outcomes of 25 26 27 960 1591 Patients Infected With SARS-CoV-2 Admitted to ICUs of the Lombardy Region, 28 29 961 Italy. JAMA 323:1574-81 (2020). 30 31 32 962 21. Faggiano P, Bonelli A, Paris S, et al. Acute pulmonary embolism in COVID-19 33 34 35 963 disease: Preliminary report on seven patients. Int J Cardiol 313:129-131 (2020). 36 37 38 964 22. Spagnolo P, Balestro E, Aliberti S, et al. Pulmonary fibrosis secondary to COVID-19: 39 40 965 a call to arms? Lancet Respir Med 2600(20)30222-8 (2020). 41 42 43 44 966 23. Schanzer DL, Langley JM, Dummer T, Viboud C, Tam TW. A composite epidemic 45 46 967 curve for seasonal influenza in Canada with an international comparison. Influenza 47 48 968 Other Respir Viruses 4:295-306 (2010). 49 50 51 969 24. Luo J. Predictive Monitoring of COVID-19. White paper 52 53 54 970 2020;https://www.newsbeast.gr/files/1/2020/05/COVID19PredictionPaper.pdf. 55 56 57 58 59 60

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1 2 3 971 25. Wu Z, McGoogan JM. Characteristics of and Important Lessons From the Coronavirus 4 5 6 972 Disease 2019 (COVID-19) Outbreak in China: Summary of a Report of 72314 Cases 7 8 973 From the Chinese Center for Disease Control and Prevention. JAMA (2020). 9 10 11 974 26. Lee PI, Hu YL, Chen PY, et al. Are children less susceptible to COVID-19? J 12 13 975 Microbiol Immunol Infect 53:371-2 (2020). 14 15 16 17 976 27. Huang C, Wang Y, Li X, et al. Clinical features of patients infected with 2019 novel 18 For Peer Review Only 19 977 coronavirus in Wuhan, China. Lancet395:497-506 (2020). 20 21 22 978 28. Hong H, Wang Y, Chung HT, et al. Clinical characteristics of novel coronavirus 23 24 25 979 disease 2019 (COVID-19) in newborns, infants and children. Pediatr Neonatol 61:131- 26 27 980 2 (2020). 28 29 30 981 29. Dong Y, Mo X, Hu Y, et al. Epidemiology of COVID-19 Among Children in China. 31 32 Pediatrics 145(6):e20200702 (2020). 33 982 34 35 36 983 30. Colson P, Tissot-Dupont H, Morand A, et al. Children account for a small proportion 37 38 984 of diagnoses of SARS-CoV-2 infection and do not exhibit greater viral loads than 39 40 985 adults. Pre-print IHU 2020;https://www.mediterranee-infection.com/children-account- 41 42 43 986 for-a-small-proportion-of-diagnoses-of-sars-cov-2-infection-and-do-not-exhibit- 44 45 987 greater-viral-loads-than-adults/. 46 47 48 988 31. Gudbjartsson DF, Helgason A, Jonsson H, et al. Spread of SARS-CoV-2 in the 49 50 51 989 Icelandic Population. N Engl J Med 38:2302-15 (2020) * A large study in Iceland 52 53 990 population that identified that children are rarely infected by SARS-CoV-2. 54 55 56 991 32. Nickbakhsh S, Ho A, Marques DFP, et al. Epidemiology of seasonal coronaviruses: 57 58 Establishing the context for COVID-19 emergence. J Infect Dis 222(1):17-25 (2020). 59 992 60

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1 2 3 993 33. Grifoni A, Weiskopf D, Ramirez SI, et al. Targets of T Cell Responses to SARS-CoV- 4 5 6 994 2 Coronavirus in Humans with COVID-19 Disease and Unexposed Individuals. Cell 7 8 995 181(7):1489-1501 (2020)** This study detected circulating SARS-CoV-2 specific 9 10 996 CDA+and CD8+ T cells in 20-60% of SARS-CoV-2-unexposed individuals, which 11 12 997 suggest croos-immune reactivities between SARS-CoV-2 and endemic 13 14 15 998 coronaviruses. 16 17 For Peer Review Only 18 999 34. Ng K, Faulkner N, Cornish G, et al. Pre-existing and de novo humoral immunity to 19 20 1000 SARS-CoV-2 in humans. bioRxiv (2020) https://doi.org/10.1101/2020.05.14.095414. 21 22 23 24 1001 35. de Assis RR, Jain A, Nakajima R, et al. Analysis of SARS-CoV-2 Antibodies in 25 26 1002 COVID-19 Convalescent Blood using a Coronavirus Antigen Microarray. bioRxiv 27 28 1003 (2020) https://doi.org/10.1101/2020.04.15.043364. 29 30 31 36. Wang W, Xu Y, Gao R, et al. Detection of SARS-CoV-2 in Different Types of 32 1004 33 34 1005 Clinical Specimens. JAMA 323(18):1843-1844 (2020). 35 36 37 1006 37. Zhai P, Ding Y, Wu X, et al. The epidemiology, diagnosis and treatment of COVID- 38 39 1007 19. Int J Antimicrob Agents 55(5):105955 2020. 40 41 42 43 1008 38. To KK, Tsang OT, Chik-Yan YC, et al. Consistent detection of 2019 novel 44 45 1009 coronavirus in saliva. Clin Infect Dis 71(15):841-843 (2020). 46 47 48 1010 39. Liu Z, Sun CB. Conjunctiva is not a preferred gateway of entry for SARS-CoV-2 to 49 50 51 1011 infect respiratory tract. J Med Virol 10.1002/jmv.25859 (2020). 52 53 54 1012 40. Peng L, Liu J, Xu W, et al. SARS-CoV-2 can be detected in urine, blood, anal swabs, 55 56 1013 and oropharyngeal swabs specimens. J Med Virol 10.1002/jmv.25936 (2020). 57 58 59 60

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1 2 3 1014 41. Ong SWX, Tan YK, Chia PY, et al. Air, Surface Environmental, and Personal 4 5 6 1015 Protective Equipment Contamination by Severe Acute Respiratory Syndrome 7 8 1016 Coronavirus 2 (SARS-CoV-2) From a Symptomatic Patient. JAMA 323(16):1610- 9 10 1017 1612 (2020). 11 12 13 1018 42. Rothe C, Schunk M, Sothmann P, et al. Transmission of 2019-nCoV Infection from an 14 15 16 1019 Asymptomatic Contact in Germany. N Engl J Med 382:970-1 (2020). 17 18 For Peer Review Only 19 1020 43. van DN, Bushmaker T, Morris DH, et al. Aerosol and Surface Stability of SARS- 20 21 1021 CoV-2 as Compared with SARS-CoV-1. N Engl J Med 382(16):1564-1567 (2020). 22 23 24 25 1022 44. Cai J, Sun W, Huang J, et al. Indirect Virus Transmission in Cluster of COVID-19 26 27 1023 Cases, Wenzhou, China, 2020. Emerg Infect Dis 26(6):1343-1345 (2020). 28 29 30 1024 45. Guo ZD, Wang ZY, Zhang SF, et al. Aerosol and Surface Distribution of Severe 31 32 Acute Respiratory Syndrome Coronavirus 2 in Hospital Wards, Wuhan, China, 2020. 33 1025 34 35 1026 Emerg Infect Dis 26(7):1583-1591 (2020). 36 37 38 1027 46. Bouvet M, Debarnot C, Imbert I, et al. In vitro reconstitution of SARS-coronavirus 39 40 1028 mRNA cap methylation. PLoS Pathog 6(4):e1000863 (2010). 41 42 43 44 1029 47. Fung TS, Liu DX. Coronavirus infection, ER stress, apoptosis and innate immunity. 45 46 1030 Front Microbiol 5:296 (2014). 47 48 49 1031 48. Perlman S, Netland J. Coronaviruses post-SARS: update on replication and 50 51 52 1032 pathogenesis. Nat Rev Microbiol 7:439-50 (2009). 53 54 55 1033 49. Hoffmann M, Kleine-Weber H, Schroeder S, et al. SARS-CoV-2 Cell Entry Depends 56 57 1034 on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. 58 59 Cell 181:271-80 (2020). 60 1035

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1 2 3 1036 50. Harzallah I, Debliquis A, Drénou B. Lupus anticoagulant is frequent in patients 4 5 6 1037 with Covid-19. J Thromb Haemost (2020). 7 8 9 1038 51. Wu F, Wang A, Liu M, et al. Neutralizing antibody responses to SARS-CoV-2 in a 10 11 1039 COVID-19 recovered patient cohort and their implications. MedRxiv (2020). 12 13 14 1040 52. Hadjadj J, Yatim N, Barnabei L, et al. Impaired type I interferon activity and 15 16 17 1041 inflammatory responses in severe COVID-19 patients. Science (2020). 18 For Peer Review Only 19 20 1042 53. Xu Z, Shi L, Wang Y, et al. Pathological findings of COVID-19 associated with acute 21 22 1043 respiratory distress syndrome. Lancet Respir Med 8:420-2 (2020). 23 24 25 26 1044 54. Feng Z, Diao B, Wang R, et al. The Novel Severe Acute Respiratory Syndrome 27 28 1045 Coronavirus 2 (SARS-CoV-2) Directly Decimates Human Spleens and Lymph Nodes. 29 30 1046 MedRxiv (2020). 31 32 33 55. Giamarellos-Bourboulis EJ, Netea MG, Rovina N, et al. Complex Immune 34 1047 35 36 1048 Dysregulation in COVID-19 Patients with Severe Respiratory Failure. Cell Host 37 38 1049 Microbe 27(6):992-1000.e3 (2020): **A study of immune responses to SARS-CoV- 39 40 1050 2 in 54 patients identifying two distinct immune profiles associated with severe 41 42 43 1051 respiratory failure: macrophage activation syndrome and a typical immune 44 45 1052 dysregulation pattern with increased IL-6, decreased macrophage antigen 46 47 1053 presentation and hyperinflammation. 48 49 50 51 1054 56. Wang X, Xu W, Hu G, et al. SARS-CoV-2 infects T lymphocytes through its spike 52 53 1055 protein-mediated membrane fusion. Cell Mol Immunol 1-3 (2020). 54 55 56 57 58 59 60

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1 2 3 1056 57. Tay MZ, Poh CM, Rénia L, et al. The trinity of COVID-19: immunity, inflammation 4 5 6 1057 and intervention. Nat Rev Immunol 20(6):363-374 (2020): *A comprehensive review 7 8 1058 of SARS-CoV-2 induced immune responses. 9 10 11 1059 58. Grifoni A, Sidney J, Zhang Y, et al. A Sequence Homology and Bioinformatic 12 13 1060 Approach Can Predict Candidate Targets for Immune Responses to SARS-CoV-2. 14 15 16 1061 Cell Host Microbe 27:671-80 (2020). 17 18 For Peer Review Only 19 1062 59. Braun J, Loyal L, Frentsch M, et al. Presence of SARS-CoV-2-reactive T cells in 20 21 1063 COVID-19 patients and healthy 2 donors. MedRxiv (2020) 22 23 24 1064 https://doi.org/10.1101/2020.04.17.20061440.t. 25 26 27 1065 60. Lv H, Wu NC, Tak-Yin TO, et al. Cross-reactive antibody response between SARS- 28 29 1066 CoV-2 and SARS-CoV infections. Cell Rep 31(9): 107725 (2020). 30 31 32 61. Long QX, Liu BZ, Deng HJ, et al. Antibody responses to SARS-CoV-2 in patients 33 1067 34 35 1068 with COVID-19. Nat Med 26(6):845-848 (2020). 36 37 38 1069 62. Edouard S, Colson P, Melenotte C, et al. Evaluating the serological status of COVID- 39 40 1070 19 patients using an indirect immunofluorescent assay, France. IHU Médirranée 41 42 43 1071 Infection (2020) https://www.mediterranee-infection.com/evaluating-the-serological- 44 45 1072 status-of-covid-19-patients-using-an-indirect-immunofluorescent-assay-france/. 46 47 48 1073 63. Ni L, Ye F, Cheng ML, et al. Detection of SARS-CoV-2-Specific Humoral and 49 50 51 1074 Cellular Immunity in COVID-19 Convalescent Individuals. Immunity 2(6):971-977 52 53 1075 (2020). 54 55 56 1076 64. Liu L, Wei Q, Lin Q, et al. Anti-spike IgG causes severe acute lung injury by skewing 57 58 macrophage responses during acute SARS-CoV infection. JCI Insight 4 (2019). 59 1077 60

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1 2 3 1078 65. Zhang Y, Xiao M, Zhang S, et al. Coagulopathy and Antiphospholipid Antibodies in 4 5 6 1079 Patients with Covid-19. N Engl J Med 382:e38 (2020). 7 8 9 1080 66. Cao B, Wang Y, Wen D, et al. A Trial of Lopinavir-Ritonavir in Adults Hospitalized 10 11 1081 with Severe Covid-19. N Engl J Med 382:1787-99 (2020). 12 13 14 1082 67. Chen G, Wu D, Guo W, et al. Clinical and immunological features of severe and 15 16 17 1083 moderate coronavirus disease 2019. J Clin Invest 130:2620-9 (2020). 18 For Peer Review Only 19 20 1084 68. Mehta P, McAuley DF, Brown M, et al. COVID-19: consider cytokine storm 21 22 1085 syndromes and immunosuppression. Lancet 395:1033-4 (2020). 23 24 25 26 1086 69. Herold T, Jurinovic V, Amreich C, et al. Level of IL-6 predicts respiratory failure in 27 28 1087 hospitalized symptomatic COVID-19 patients. MedRxiv (2020). 29 30 1088 https://doi.org/10.1101/2020.04.01.20047381. 31 32 33 70. Yue Y, Nabar NR, Shi CS, et al. SARS-Coronavirus Open Reading Frame-3a drives 34 1089 35 36 1090 multimodal necrotic cell death. Cell Death Dis 9:904 (2018). 37 38 39 1091 71. Channappanavar R, Fehr AR, Vijay R, et al. Dysregulated Type I Interferon and 40 41 1092 Inflammatory Monocyte-Macrophage Responses Cause Lethal Pneumonia in SARS- 42 43 44 1093 CoV-Infected Mice. Cell Host Microbe 19:181-93 (2016). 45 46 47 1094 72. Zalinger ZB, Elliott R, Weiss SR. Role of the inflammasome-related cytokines Il-1 48 49 1095 and Il-18 during infection with murine coronavirus. J Neurovirol 23:845-54 (2017). 50 51 52 53 1096 73. Shi CS, Nabar NR, Huang NN, et al. SARS-Coronavirus Open Reading Frame-8b 54 55 1097 triggers intracellular stress pathways and activates NLRP3 inflammasomes. Cell Death 56 57 1098 Discov 5:101 (2019). 58 59 60

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1 2 3 1099 74. van de Veerdonk FL, Netea MG, Dinarello CA, et al. Inflammasome activation and 4 5 6 1100 IL-1β and IL-18 processing during infection. Trends Immunol 32:110-6 (2011). 7 8 9 1101 75. Lu W, Zheng BJ, Xu K, et al. Severe acute respiratory syndrome-associated 10 11 1102 coronavirus 3a protein forms an ion channel and modulates virus release. Proc Natl 12 13 1103 Acad Sci U S A 103:12540-5 (2006). 14 15 16 17 1104 76. Dinarello CA. Interleukin-1 in the pathogenesis and treatment of inflammatory 18 For Peer Review Only 19 1105 diseases. Blood 117:3720-32 (2011). 20 21 22 1106 77. Kaplanski G. Interleukin-18: Biological properties and role in disease pathogenesis. 23 24 25 1107 Immunol Rev 281:138-53 (2018). 26 27 28 1108 78. Slaats J, Ten OJ, van de Veerdonk FL, et al. IL-1β/IL-6/CRP and IL-18/ferritin: 29 30 1109 Distinct Inflammatory Programs in Infections. PLoS Pathog 12:e1005973 (2016). 31 32 33 79. Lippi G, Favaloro EJ. D-dimer is Associated with Severity of Coronavirus Disease 34 1110 35 36 1111 2019: A Pooled Analysis. Thromb Haemost 120:876-8 (2020): **Elevated D-Dimer 37 38 1112 are associated with poor outcome : a pooled analysis. 39 40 41 1113 80. Tang N, Li D, Wang X, Sun Z. Abnormal coagulation parameters are associated with 42 43 44 1114 poor prognosis in patients with novel coronavirus pneumonia. J Thromb Haemost 45 46 1115 18:844-7 (2020). 47 48 49 1116 81. Engelmann B, Massberg S. Thrombosis as an intravascular effector of innate 50 51 52 1117 immunity. Nat Rev Immunol 13:34-45 (2013). 53 54 55 1118 82. Gupta N, Zhao YY, Evans CE. The stimulation of thrombosis by hypoxia. Thromb 56 57 1119 Res 181:77-83 (2019). 58 59 60

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1 2 3 1120 83. Connors JM, Levy JH. COVID-19 and its implications for thrombosis and 4 5 6 1121 anticoagulation. Blood 135(23):2033-2040 (2020). 7 8 9 1122 84. Klok FA, Kruip MJHA, van der Meer NJM, et al. Incidence of thrombotic 10 11 1123 complications in critically ill ICU patients with COVID-19. Thromb Res 191:145-147 12 13 1124 (2020). 14 15 16 17 1125 85. Poissy J, Goutay J, Caplan M, et al. Pulmonary Embolism in COVID-19 Patients: 18 For Peer Review Only 19 1126 Awareness of an Increased Prevalence. Circulation 142(2):184-186 (2020). 20 21 22 1127 86. Wang T, Chen R, Liu C, et al. Attention should be paid to venous thromboembolism 23 24 25 1128 prophylaxis in the management of COVID-19. Lancet Haematol 7:e362-e363 (2020). 26 27 28 1129 87. Tang N, Bai H, Chen X, et al. Anticoagulant treatment is associated with decreased 29 30 1130 mortality in severe coronavirus disease 2019 patients with coagulopathy. J Thromb 31 32 Haemost 18:1094-9 (2020). 33 1131 34 35 36 1132 88. Yao X, Ye F, Zhang M, et al. In Vitro Antiviral Activity and Projection of Optimized 37 38 1133 Dosing Design of Hydroxychloroquine for the Treatment of Severe Acute Respiratory 39 40 1134 Syndrome Coronavirus 2 (SARS-CoV-2). Clin Infect Dis 71(15):732-739 (2020). 41 42 43 44 1135 89. Wang M, Cao R, Zhang L, et al. Remdesivir and chloroquine effectively inhibit the 45 46 1136 recently emerged novel coronavirus (2019-nCoV) in vitro. Cell Res 30:269-71(2020). 47 48 49 1137 90. Liu J, Cao R, Xu M, et al. Hydroxychloroquine, a less toxic derivative of chloroquine, 50 51 52 1138 is effective in inhibiting SARS-CoV-2 infection in vitro. Cell Discov 6:16 (2020). 53 54 55 1139 91. Weston S, Coleman CM, Haupt R, et al. Broad anti-coronaviral activity of FDA 56 57 1140 approved drugs against 2 SARS-CoV-2 in vitro and SARS-CoV in vivo. bioRxiv 58 59 (2020) https://doi.org/10.1101/2020.03.25.008482. 60 1141

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1 2 3 1142 92. Zhang J, Ma X, Yu F, et al. Teicoplanin potently blocks the cell entry of 2019-nCoV. 4 5 6 1143 bioRxiv (2020) https://doi.org/10.1101/2020.02.05.935387. 7 8 9 1144 93. Caly L, Druce JD, Catton MG, et al. The FDA-approved drug ivermectin inhibits the 10 11 1145 replication of SARS-CoV-2 in vitro. Antiviral Res 178:104787 (2020). 12 13 14 1146 94. Touret F, Gilles M, Barral K, et al. In vitro screening of a FDA approved chemical 15 16 17 1147 library reveals potential inhibitors of SARS-CoV-2 replication. bioRxiv (2020) 18 For Peer Review Only 19 1148 https://doi.org/10.1101/2020.04.03.023846. 20 21 22 1149 95. Andreani J, Le BM, Duflot I, et al. In vitro testing of combined hydroxychloroquine 23 24 25 1150 and azithromycin on SARS-CoV-2 shows synergistic effect. Microb Pathog 26 27 1151 145:104228 (2020). 28 29 30 1152 96. Gordon DE, Jang GM, Bouhaddou M, et al. A SARS-CoV-2 protein interaction map 31 32 reveals targets for drug repurposing. Nature 583(7816):459-468 (2020):** This large 33 1153 34 35 1154 study identified 69 compounds, including FDA-approved drugs, that target 36 37 1155 druggable human proteins of host factos, then performed screening in viral 38 39 1156 assays.. 40 41 42 43 1157 97. Cava C, Bertoli G, Castiglioni I. In Silico Discovery of Candidate Drugs against 44 45 1158 Covid-19. Viruses 12:v12040404 (2020). 46 47 48 1159 98. Shah B, Modi P, Sagar SR. In silico studies on therapeutic agents for COVID-19: 49 50 51 1160 Drug repurposing approach. Life Sci 252:117652 (2020) doi: 52 53 1161 10.1016/j.lfs.2020.117652.:117652. 54 55 56 57 58 59 60

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1 2 3 1162 99. Choudary S, Malik YS, Tomar S. Identification of SARS-CoV-2 Cell Entry Inhibitors 4 5 6 1163 by Drug Repurposing Using in Silico Structure-Based Virtual Screening Approach. 7 8 1164 ChemRxiv (2020) https://doi.org/10.26434/chemrxiv.12005988.v2. 9 10 11 1165 100. Chien M, Anderson TK, Jockusch S, et al. Nucleotide Analogues as Inhibitors of 12 13 1166 SARS-CoV-2 Polymerase. bioRxiv (2020) 14 15 16 1167 https://doi.org/10.1101/2020.03.18.997585. 17 18 For Peer Review Only 19 1168 101. Fantini J, Di SC, Chahinian H, et al. Structural and molecular modelling studies reveal 20 21 1169 a new mechanism of action of chloroquine and hydroxychloroquine against SARS- 22 23 24 1170 CoV-2 infection. Int J Antimicrob Agents 55(5):105960 (2020). 25 26 27 1171 102. Devaux CA, Rolain JM, Colson P, et al. New insights on the antiviral effects of 28 29 1172 chloroquine against coronavirus: what to expect for COVID-19? Int J Antimicrob 30 31 Agents 55(5):105938 (2020). 32 1173 33 34 35 1174 103. Touret F, de L, X. Of chloroquine and COVID-19. Antiviral Res 177:104762 (2020). 36 37 38 1175 104. Keyaerts E, Vijgen L, Maes P, et al. In vitro inhibition of severe acute respiratory 39 40 1176 syndrome coronavirus by chloroquine. Biochem Biophys Res Commun 323:264-8 41 42 43 1177 (2004). 44 45 46 1178 105. Keyaerts E, Li S, Vijgen L, et al. Antiviral activity of chloroquine against human 47 48 1179 coronavirus OC43 infection in newborn mice. Antimicrob Agents Chemother 49 50 51 1180 53:3416-21 (2009). 52 53 54 1181 106. Mo Y, Fisher D. A review of treatment modalities for Middle East Respiratory 55 56 1182 Syndrome. J Antimicrob Chemother 71:3340-50 (2016). 57 58 59 60

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1 2 3 1183 107. Vincent MJ, Bergeron E, Benjannet S, et al. Chloroquine is a potent inhibitor of SARS 4 5 6 1184 coronavirus infection and spread. Virol J 2:69 (2005). 7 8 9 1185 108. Cassell S, Edwards J, Brown DT. Effects of lysosomotropic weak bases on infection 10 11 1186 of BHK-21 cells by Sindbis virus. J Virol 52:857-64 (1984). 12 13 14 1187 109. Yang ZY, Huang Y, Ganesh L, et al. pH-dependent entry of severe acute respiratory 15 16 17 1188 syndrome coronavirus is mediated by the spike glycoprotein and enhanced by 18 For Peer Review Only 19 1189 dendritic cell transfer through DC-SIGN. J Virol 78:5642-50 (2004). 20 21 22 1190 110. Xue J, Moyer A, Peng B, et al. Chloroquine is a zinc ionophore. PLoS ONE 23 24 25 1191 9:e109180 (2014). 26 27 28 1192 111. Kaushik N, Subramani C, Anang S, et al. Zinc Salts Block Hepatitis E Virus 29 30 1193 Replication by Inhibiting the Activity of Viral RNA-Dependent RNA Polymerase. J 31 32 Virol 91(21):e00754-17 (2017). 33 1194 34 35 36 1195 112. te Velthuis AJ, van den Worm SH, Sims AC, et al. Zn(2+) inhibits coronavirus and 37 38 1196 arterivirus RNA polymerase activity in vitro and zinc ionophores block the replication 39 40 1197 of these viruses in cell culture. PLoS Pathog6:e1001176 (2010). 41 42 43 44 1198 113. Perrier A, Bonnin A, Desmarets L, et al. The C-terminal domain of the MERS 45 46 1199 coronavirus M protein contains a trans-Golgi network localization signal. J Biol Chem 47 48 1200 294:14406-21 (2019). 49 50 51 52 1201 114. Kono M, Tatsumi K, Imai AM, et al. Inhibition of human coronavirus 229E infection 53 54 1202 in human epithelial lung cells (L132) by chloroquine: involvement of p38 MAPK and 55 56 1203 ERK. Antiviral Res 77:150-2 (2008). 57 58 59 60

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1 2 3 1204 115. Savarino A, Boelaert JR, Cassone A, et al. Effects of chloroquine on viral infections: 4 5 6 1205 an old drug against today's diseases? Lancet Infect Dis 3:722-7 (2003). 7 8 9 1206 116. Gautret P, Lagier JC, Parola P, et al. Hydroxychloroquine and azithromycin as a 10 11 1207 treatment of COVID-19: results of an open-label non-randomized clinical trial. Int J 12 13 1208 Antimicrob Agents 105949 (2020). 14 15 16 17 1209 117. Million M, Gautret P, Colson P, et al. Clinical Efficacy of Chloroquine Derivatives in 18 For Peer Review Only 19 1210 COVID-19 Infection: Comparative Meta-analysis between the Big Data and the Real 20 21 1211 World. New Microbes and New Infection (2020) 22 23 24 1212 https://doi.org/10.1016/j.nmni.2020.100709. 25 26 27 1213 118. Geleris J, Sun Y, Platt J, et al. Observational Study of Hydroxychloroquine in 28 29 1214 Hospitalized Patients with Covid-19. N Engl J Med 382(25):2411-2418 (2020). 30 31 32 119. Rosenberg ES, Dufort EM, Udo T, et al. Association of Treatment With 33 1215 34 35 1216 Hydroxychloroquine or Azithromycin With In-Hospital Mortality in Patients With 36 37 1217 COVID-19 in New York State. JAMA 323(24):2493-2502 (2020). 38 39 40 1218 120. Singh S, Khan A, Chowdhry M, Chatterjee A. Outcomes of Hydroxychloroquine 41 42 43 1219 Treatment Among Hospitalized COVID-19 Patients in the United States- Real-World 44 45 1220 Evidence From a Federated Electronic Medical Record Network. MedRxiv (2020) 46 47 1221 https://doi.org/10.1101/2020.05.12.20099028. 48 49 50 51 1222 121. Magagnoli J, Narendran S, Pereira F, et al. Outcomes of hydroxychloroquine usage in 52 53 1223 United States veterans hospitalized with Covid-19. MedRxiv (2020) 54 55 1224 https://doi.org/10.1101/2020.04.16.20065920. 56 57 58 59 60

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1 2 3 1225 122. Mehra MR, Desai SS, Ruschitzka F, et al. Hydroxychloroquine or chloroquine with or 4 5 6 1226 without a macrolide for treatment of COVID-19: a multinational registry analysis. 7 8 1227 Lancet 2020. 9 10 11 1228 123. Mehra MR, Ruschitzka F, Patel AN. Retraction-Hydroxychloroquine or chloroquine 12 13 1229 with or without a macrolide for treatment of COVID-19: a multinational registry 14 15 16 1230 analysis. Lancet 395:1820 (2020). 17 18 For Peer Review Only 19 1231 124. Chen Z, Hu J, Zhang Zo, et al. Efficacy of hydroxychloroquine in patients with 20 21 1232 COVID-19: results of a randomized clinical trial. MedRxiv (2020) 22 23 24 1233 https://doi.org/10.1101/2020.03.22.20040758. 25 26 27 1234 125. Huang M, Tang T, Pang P, et al. Treating COVID-19 with Chloroquine. J Mol Cell 28 29 1235 Biol 12:322-5 (2020). 30 31 32 126. Tang W, Cao Z, Han M, et al. Hydroxychloroquine in patients mainly with mild to 33 1236 34 35 1237 moderate COVID–19: an open–label, randomized, controlled trial. MedRxiv (2020) 36 37 1238 https://doi.org/10.1101/2020.04.10.20060558. 38 39 40 1239 127. Chen J, Liu D, Liu L, et al. A pilot study of hydroxychloroquine in treatment of 41 42 43 1240 patients with common coronavirus disease-19 (COVID-19). J of Zjeijang University 44 45 1241 (2020). 46 47 48 1242 128. Borba MGS, Val FFA, Sampaio VS, et al. Effect of High vs Low Doses of 49 50 51 1243 Chloroquine Diphosphate as Adjunctive Therapy for Patients Hospitalized With 52 53 1244 Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection: A 54 55 1245 Randomized Clinical Trial. JAMA Netw Open 3:e208857 (2020). 56 57 58 59 60

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1 2 3 1246 129. Anderson A, Bijlmer H, Fournier PE, et al. Diagnosis and Management of Q fever- 4 5 6 1247 United States, 2013. Centers for Disease Control and Prevention 62:1-23 (2013). 7 8 9 1248 130. Raoult D, Million M, Gautret P, et al. Hydroxychloroquine and Azithromycin as a 10 11 1249 Treatment of COVID-19: Results of an Open-Label Non-Randomized Clinical Trial: 12 13 1250 Response to David Spencer (Elsevier). IHU Preprints (2020) 14 15 16 1251 https://doi.org/10.35088/bjjr-cy47. 17 18 For Peer Review Only 19 1252 131. Arshad S, Kilgore P, Chaudhry ZS, et al. Treatment with hydroxychloroquine, 20 21 1253 azithromycin, and combination in patients hospitalized with COVID-19. Int J Infect 22 23 24 1254 Dis 97:396-403 (2020). 25 26 27 1255 132. Mikami T, Miyashita H, Yamada T, et al. Risk Factors for Mortality in Patients with 28 29 1256 COVID-19 in New York City. J Gen Intern Med 1-10 (2020). 30 31 32 133. Chen L, Zhang ZY, Fu JG, et al. Efficacy and safety of chloroquine or 33 1257 34 35 1258 hydroxychloroquine in moderate type of COVID-19: a prospective open-label 36 37 1259 randomized controlled study. MedRxiv (2020) 38 39 1260 https://doi.org/10.1101/2020.06.19.20136093. 40 41 42 43 1261 134. Horby P, Mafham M, Linsell L, et al. Effect of Hydroxychloroquine in Hospitalized 44 45 1262 Patients with COVID-19: Preliminary results from a multi-centre, randomized, 46 47 1263 controlled trial. MedRxiv (2020) https://doi.org/10.1101/2020.07.15.20151852. 48 49 50 51 1264 135. Skipper CP, Pastick KA, Engen NW, et al. Hydroxychloroquine in Nonhospitalized 52 53 1265 Adults With Early COVID-19: A Randomized Trial. Ann Intern Med M20-4207 54 55 1266 (2020). 56 57 58 59 60

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1 2 3 1267 136. Cavalcanti AB, Zampieri FG, Rosa RG, et al. Hydroxychloroquine with or without 4 5 6 1268 Azithromycin in Mild-to-Moderate Covid-19. N Engl J Med (2020). 7 8 9 1269 137. Bernaola N, Mena R, Bernaola A, et al. Observational Study of the Efficiency of 10 11 1270 Treatments in Patients Hospitalized with Covid-19 in Madrid. MedRxiv (2020) 12 13 1271 https://doi.org/10.1101/2020.07.17.20155960. 14 15 16 17 1272 138. Chen CP, Lin YC, Chen TC, et al. A Multicenter, randomized, open-label, controlled 18 For Peer Review Only 19 1273 trial to evaluate the efficacy and tolerability of hydroxychloroquine and a retrospective 20 21 1274 study in adult patients with mild to moderate Coronavirus disease 2019 (COVID-19). 22 23 24 1275 MedRxiv (2020) https://doi.org/10.1101/2020.07.08.20148841. 25 26 27 1276 139. Damle B, Vourvahis M, Wang E, et al. Clinical Pharmacology Perspectives on the 28 29 1277 Antiviral Activity of Azithromycin and Use in COVID-19. Clin Pharmacol Ther 30 31 108(2):201-211 (2020). 32 1278 33 34 35 1279 140. Zimmermann P, Ziesenitz VC, Curtis N, et al. The Immunomodulatory Effects of 36 37 1280 Macrolides-A Systematic Review of the Underlying Mechanisms. Front Immunol 38 39 1281 9:302 (2018). 40 41 42 43 1282 141. Million M, Lagier JC, Gautret P, et al. Early treatment of COVID-19 patients with 44 45 1283 hydroxychloroquine and azithromycin: A retrospective analysis of 1061 cases in 46 47 1284 Marseille, France. Travel Med Infect Dis 35:101738 (2020). 48 49 50 51 1285 142. Gautret P, Lagier JC, Parola P, et al. Clinical and microbiological effect of a 52 53 1286 combination of hydroxychloroquine and azithromycin in 80 COVID-19 patients with 54 55 1287 at least a six-day follow up: A pilot observational study. Travel Med Infect Dis 56 57 34:101663 (2020). 58 1288 59 60

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1 2 3 1289 143. Read SA, Obeid S, Ahlenstiel C, et al. The Role of Zinc in Antiviral Immunity. Adv 4 5 6 1290 Nutr 10:696-710 (2019). 7 8 9 1291 144. Carlucci PM, Ahuja T, Petrilli C, et al. Hydroxychloroquine and azithromycin plus 10 11 1292 zinc vs hydroxychloroquine and azithromycin alone: outcomes in hospitalized 12 13 1293 COVID-19 patients. MedRxiv (2020) https://doi.org/10.1101/2020.05.02.20080036. 14 15 16 17 1294 145. Grein J, Ohmagari N, Shin D, et al. Compassionate Use of Remdesivir for Patients 18 For Peer Review Only 19 1295 with Severe Covid-19. N Engl J Med 382(24):2327-2336 (2020). 20 21 22 1296 146. Wang Y, Zhang D, Du G, et al. Remdesivir in adults with severe COVID-19: a 23 24 25 1297 randomised, double-blind, placebo-controlled, multicentre trial. Lancet 395:1569-78 26 27 1298 (2020). 28 29 30 1299 147. Beigel JH, Tomashek KM, Dodd LE, et al. Remdesivir for the Treatment of Covid-19 31 32 - Preliminary Report. N Engl J Med (2020). 33 1300 34 35 36 1301 148. Goldman JD, Lye DCB, Hui DS, et al. Remdesivir for 5 or 10 Days in Patients with 37 38 1302 Severe Covid-19. N Engl J Med (2020). 39 40 41 1303 149. Choy EH, De BF, Takeuchi T, et al. Translating IL-6 biology into effective treatments. 42 43 44 1304 Nat Rev Rheumatol 16(6):335-345 (2020). 45 46 47 1305 150. Kaplanski G, Marin V, Montero-Julian F, et al. IL-6: a regulator of the transition from 48 49 1306 neutrophil to monocyte recruitment during inflammation. Trends Immunol 24:25-9 50 51 52 1307 (2003). 53 54 55 1308 151. Garbers C, Heink S, Korn T, et al. Interleukin-6: designing specific therapeutics for a 56 57 1309 complex cytokine. Nat Rev Drug Discov 17:395-412 (2018). 58 59 60

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1 2 3 1310 152. Le TT, Karmouty-Quintana H, Melicoff E, et al. Blockade of IL-6 Trans signaling 4 5 6 1311 attenuates pulmonary fibrosis. J Immunol 193:3755-68 (2014). 7 8 9 1312 153. De BF, Brunner HI, Ruperto N, et al. Randomized trial of tocilizumab in systemic 10 11 1313 juvenile idiopathic arthritis. N Engl J Med 367:2385-95 (2012). 12 13 14 1314 154. Brudno JN, Kochenderfer JN. Toxicities of chimeric antigen receptor T cells: 15 16 17 1315 recognition and management. Blood 127:3321-30 (2016). 18 For Peer Review Only 19 20 1316 155. Xu X, Han M, Li T, et al. Effective Treatment of Severe COVID-19 Patients with 21 22 1317 Tocilizumab. ChinaXiv (2020) chinaXiv:202003.00026v1. 23 24 25 26 1318 156. Toniati P, Piva S, Cattalini M, et al. Tocilizumab for the treatment of severe COVID- 27 28 1319 19 pneumonia with hyperinflammatory syndrome and acute respiratory failure: A 29 30 1320 single center study of 100 patients in Brescia, Italy. Autoimmun Rev 19:102568 31 32 (2020). 33 1321 34 35 36 1322 157. Morrison AR, Johnson JM, Ramesh M, et al. Letter to the Editor: Acute 37 38 1323 hypertriglyceridemia in patients with COVID-19 receiving tocilizumab. J Med Virol 39 40 1324 (2020). 41 42 43 44 1325 158. Buonaguro FM, Puzanov I, Ascierto PA. Anti-IL6R role in treatment of COVID-19- 45 46 1326 related ARDS. J Transl Med 18:165 (2020). 47 48 49 1327 159. Cavalli G, De LG, Campochiaro C, et al. Interleukin-1 blockade with high-dose 50 51 52 1328 anakinra in patients with COVID-19, acute respiratory distress syndrome, and 53 54 1329 hyperinflammation: a retrospective cohort study. Lancet Rheumatol 2:e325-e331. 55 56 1330 (2020) 57 58 59 60

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1 2 3 1331 160. Huet T, Beaussier H, Voisin O, Jouveshomme S, Dauriat G, Lazareth I. Anakinra for 4 5 6 1332 severe forms of COVID-19: a cohort study. Lancet Rheumatol (2020) 7 8 1333 https://doi.org/10.1016/S2665-9913(20)30164-8. 9 10 11 1334 161. Low-cost dexamethasone reduces death by up to one third in hospitalised patients 12 13 1335 with severe respiratory complications of COVID-19. University of Oxford (2020) 14 15 16 1336 http://www.ox.ac.uk/news/2020-06-16-low-cost-dexamethasone-reduces-death-one- 17 For Peer Review Only 18 1337 third-hospitalised-patients-severe. 19 20 21 1338 162. Yu B, Li C, Chen P, et al. Low dose of hydroxychloroquine reduces fatality of 22 23 24 1339 critically ill patients with COVID-19. Sci China Life Sci 1-7 (2020). 25 26 27 1340 163. Chatterjee P, Anand T, Singh KJ, et al. Healthcare workers & SARS-CoV-2 infection 28 29 1341 in India: A case-control investigation in the time of COVID-19. Indian J Med Res 30 31 (2020) http://www.ijmr.org.in/preprintarticle.asp?id=285520. 32 1342 33 34 35 1343 164. Lee SH, Son H, Peck KR. Can post-exposure prophylaxis for COVID-19 be 36 37 1344 considered as an outbreak response strategy in long-term care hospitals? Int J 38 39 1345 Antimicrob Agents 55(6):105988 (2020). 40 41 42 43 1346 165. Boulware DR, Pullen MF, Bangdiwala AS, et al. A Randomized Trial of 44 45 1347 Hydroxychloroquine as Postexposure Prophylaxis for Covid-19. N Engl J Med (2020). 46 47 48 1348 166. Fiore AE, Fry A, Shay D, et al. Antiviral agents for the treatment and 49 50 51 1349 chemoprophylaxis of influenza --- recommendations of the Advisory Committee on 52 53 1350 Immunization Practices (ACIP). MMWR Recomm Rep 60:1-24 (2011). 54 55 56 1351 167. Barbosa-Esper R, Souza da Silva R, Teiichi Costa Oikawa F, et al. Empirical 57 58 treatment with hydroxychloroquine and azithromycin for suspected cases of COVID- 59 1352 60

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1 2 3 1353 19 followed up by telemedicine. Dropbox (2020) 4 5 6 1354 https://www.dropbox.com/s/5qm58cd4fneeci2/2020.04.15%20journal%20manuscript 7 8 1355 %20final.pdf?dl=0.. 9 10 11 1356 168. Zelenko V. Zelenko Letter Sent To 5000 Shluchim In 6 Languages. (2020) 12 13 1357 https://crownheights info/chabad-news/706267/shluchim-office-sends-zelenko-letter- 14 15 16 1358 to-5000-shluchim-in-6-languages/ 2020. 17 18 For Peer Review Only 19 1359 169. Guerin V, Levy P, Thomas J, et al. Azithromycin and Hydroxychloroquine Accelerate 20 21 1360 Recovery of Outpatients with Mild/Moderate COVID-19. Preprint (2020) doi: 22 23 24 1361 10.20944/preprints202005.0486.v1. 25 26 27 1362 170. Lippi G, South AM, Henry BM. Electrolyte imbalances in patients with severe 28 29 1363 coronavirus disease 2019 (COVID-19). Ann Clin Biochem 57:262-5 (2020). 30 31 32 171. Onder G, Rezza G, Brusaferro S. Case-Fatality Rate and Characteristics of Patients 33 1364 34 35 1365 Dying in Relation to COVID-19 in Italy. JAMA (2020). 36 37 38 1366 172. Bikdeli B, Madhavan MV, Jimenez D, et al. COVID-19 and Thrombotic or 39 40 1367 Thromboembolic Disease: Implications for Prevention, Antithrombotic Therapy, and 41 42 43 1368 Follow-up. J Am Coll Cardiol 75(23):2950-2973 (2020). 44 45 46 1369 173. Richardson S, Hirsch JS, Narasimhan M, et al. Presenting Characteristics, 47 48 1370 Comorbidities, and Outcomes Among 5700 Patients Hospitalized With COVID-19 in 49 50 51 1371 the New York City Area. JAMA (2020). 52 53 54 1372 174. Gattinoni L, Chiumello D, Caironi P, et al. COVID-19 pneumonia: different 55 56 1373 respiratory treatments for different phenotypes? Intensive Care Med 46(6):1099-1102 57 58 (2020). 59 1374 60

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1 2 3 1375 175. Bhatraju PK, Ghassemieh BJ, Nichols M, et al. Covid-19 in Critically Ill Patients in 4 5 6 1376 the Seattle Region - Case Series. N Engl J Med 382:2012-22 (2020). 7 8 9 1377 176. Wu C, Chen X, Cai Y, et al. Risk Factors Associated With Acute Respiratory Distress 10 11 1378 Syndrome and Death in Patients With Coronavirus Disease 2019 Pneumonia in 12 13 1379 Wuhan, China. JAMA Intern Med (2020). 14 15 16 17 1380 177. Huang YF, Bai C, He F, et al. Review on the potential action mechanisms of Chinese 18 For Peer Review Only 19 1381 medicines in treating Coronavirus Disease 2019 (COVID-19). Pharmacol Res 20 21 1382 158:104939 (2020). 22 23 24 25 1383 178. Zhang D, Zhang B, Lv JT, et al. The clinical benefits of Chinese patent medicines 26 27 1384 against COVID-19 based on current evidence. Pharmacol Res 157:104882 (2020). 28 29 30 1385 179. Xiong X, Wang P, Su K, et al. Chinese herbal medicine for coronavirus disease 2019: 31 32 A systematic review and meta-analysis. Pharmacol Res160:105056 (2020). 33 1386 34 35 36 1387 180. Li Q, Wang H, Li X, et al. The role played by traditional Chinese medicine in 37 38 1388 preventing and treating COVID-19 in China. Front Med 1-8 (2020). 39 40 41 1389 181. Li C, Wang L, Ren L. Antiviral mechanisms of candidate chemical medicines and 42 43 44 1390 traditional Chinese medicines for SARS-CoV-2 infection. Virus Res 286:198073 45 46 1391 (2020). 47 48 49 1392 182. Ho LTF, Chan KKH, Chung VCH, et al. Highlights of traditional Chinese medicine 50 51 52 1393 frontline expert advice in the China national guideline for COVID-19. Eur J Integr 53 54 1394 Med 36:101116 (2020). 55 56 57 1395 183. Tu Y. Artemisinin-A Gift from Traditional Chinese Medicine to the World (Nobel 58 59 Lecture). Angew Chem Int Ed Engl 55:10210-26 (2016). 60 1396

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1 2 3 1397 184. Eldin C, Melenotte C, Mediannikov O, et al. From Q Fever to Coxiella burnetii 4 5 6 1398 Infection: a Paradigm Change. Clin Microbiol Rev 30:115-90 (2017). 7 8 9 1399 185. Pizzorno A, Padey B, Dubois J, et al. In vitro evaluation of antiviral activity of single 10 11 1400 and combined repurposable drugs against SARS-CoV-2. Antiviral Res 181:104878 12 13 1401 (2020). 14 15 16 17 1402 186. Tang W, Cao Z, Han M, et al. Hydroxychloroquine in patients with mainly mild to 18 For Peer Review Only 19 1403 moderate coronavirus disease 2019: open label, randomised controlled trial. BMJ 20 21 1404 369:m1849 (2020). 22 23 24 25 1405 187. Gao J, Tian Z, Yang X. Breakthrough: Chloroquine phosphate has shown apparent 26 27 1406 efficacy in treatment of COVID-19 associated pneumonia in clinical studies. Biosci 28 29 1407 Trends 14(1):72-73 (2020). 30 31 32 188. Dong L, Hu S, Gao J. Discovering drugs to treat coronavirus disease 2019 (COVID- 33 1408 34 35 1409 19). Drug Discov Ther 14:58-60 (2020). 36 37 38 1410 189. Nicastri E, Petrosillo N, Bartoli TA, et al. National Institute for the Infectious Diseases 39 40 1411 "L. Spallanzani", IRCCS. Recommendations for COVID-19 clinical management. 41 42 43 1412 Infect Dis Rep 12:8543 (2020). 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 1414 Figure Legends 4 5 6 1415 Figure 1: LDCT image showing ground glass opacities and consolidation in A. The deep 7 8 1416 learning algorithm provides automatic segmentation of the consolidation in B (yellow label), 9 10 1417 ground glass opacities in C (green label) and the lung in D (purple label). 11 12 1418 Figure 2: Number of newly diagnosed SARS-CoV-2 patients per week at the IHU 13 14 15 1419 Méditerranée Infection by reverse transcription PCR 16 17 1420 Figure 3: Natural history of COVID-19 and treatment options (adapted from reference [3]) 18 For Peer Review Only 19 1421 Figure 4: Schematic representation of SARS-CoV-2 replication cycle. ACE2: angiotensin- 20 21 22 1422 converting enzyme 2, NSPs: nonstructural proteins, ERGIC: endoplasmic reticulum Golgi 23 24 1423 intermediate compartment 25 26 1424 Figure 5: Meta-analysis of chloroquine and COVID-19 mortality 27 28 CI: confidence interval, HCQ: hydroxychloroquine, CQ: chloroquine, (H)CQ: chloroquine 29 1425 30 31 1426 derivatives (hydroxychloroquine (HCQ) or chloroquine (CQ)). This meta-analysis was 32 33 1427 performed with a random-effects model using Comprehensive Meta-Analysis v3 (Biostat, 34 35 1428 Englewood, NJ, USA). 36 37 38 1429 Figure 6: Meta-analysis of chloroquine derivatives and SARS-CoV-2 persistent shedding 39 40 1430 CI: confidence interval, HCQ: hydroxychloroquine, CQ: chloroquine, RCT: randomized 41 42 1431 controlled trial, (H)CQ: chloroquine derivatives (hydroxychloroquine (HCQ) or chloroquine 43 44 45 1432 (CQ)). This meta-analysis was performed with a random-effects model using Comprehensive 46 47 1433 Meta-Analysis v3 (Biostat, Englewood, NJ, USA). 48 49 1434 Figure 7: Schematic representation of previous findings by multiple correspondence analysis. 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 1435 Table Legend 4 5 6 1436 Table 1: Level of evidence for the efficacy of a combination of hydroxychloroquine and 7 8 1437 hydroxychloroquine-azithromycin against COVID-19 based on clinical studies with a clear 9 10 1438 description of the therapeutic protocol. 11 12 13 14 15 16 17 18 For Peer Review Only 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 1439 Table 1. Anti-COVID-19 potential drugs according to in vitro results and chemical structure 4 5 6 EC50 (µM) CC50 (µM) Selectivity Index Reference 7 4-aminoquinoleinesa 8 Chloroquine 1.13 (EC90 : 6.9) >100 >88.5 [89] 9 2.71-7.36 273 37-100 [90] 10 42-46 >50 >1.07-1.19 [91] 11 (Inhibition index 1.35) [94] 12 5.5 For Peer Review Only [88] 13 1.38 238 172 [185] 14 Hydroxychloroquineb 4.06-13.00 249 14-61 [90] 15 9-11 >>50 >>2.81 [91] 16 4 (EC90 : 25) >40 >10 [94] 17 <10 [96] 18 0.72 [88] 19 Mefloquine 7-8 18 2.3-2.6 [91] 20 Amiodaquine 2-5 >38 >6->13 [91] 21 Phenothiazine Chlorpromazine 3-4 12 3-4 [91] 22 Fluphenazine 6-9 20 2-3 [91] 23 Promethazine 9-10 >42 >4 [91] 24 Thiethylperazine 7-8 18 2-3 [91] 25 Tricyclic antidepressant 26 Clomipramine 5-7 >30 >4-5 [91] 27 Ribonucleic analogs 28 Ribavirin 109 >400 >3.65 [89] 29 >10 >100 [185] 30 Remdesivir 0.77 (EC90 : 1.76) >100 >129 [89] 31 1.6 [94] 32 0.99 275 278 [185] 33 Penciclovir 95 >400 >4.17 [89] 34 Other chemical classes 35 Azithromycinb 2 (EC90 : 8) >40 >19 [94] 36 Cycloserine (inhibition index 1.4) [94] 37 Umifenovir 10.7 (EC90 : 15.2) >40 >3.7 [94] 38 3.5 75 21 [185] 39 Omeprazolea 17 (EC90 : 38) >40 >2 [94] 40 41 42 43 URL: https://mc.manuscriptcentral.com/erm Email: [email protected] 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Expert Review of Clinical Immunology Page 66 of 77

1 2 3 Teicoplanin 1.66 [92] 4 Levofloxacin (inhibition index 1.18) [94] 5 Ivermectin 2.8 [93] 6 Favipiravir 62 >400 >6.46 [89] 7 >100 631 <6.3 [185] 8 Nafamostat 22 >100 >4.44 [89] 9 Nitazoxanide 2.12 >35.53 >16.76 [89] 10 1440 EC50: drug concentration needed to inhibit 50% of viral spread, CC50: Cytotoxic concentration causing death to 50% of viable cells, SI: selectivity index. A molecule is a b 11 1441 considered potentially effective when EC50<20 µM [94]. Molecules demonstrated to increase the pH of the endosomal pathway. Synergy demonstrated in vitro [95]. 12 For Peer Review Only 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 URL: https://mc.manuscriptcentral.com/erm Email: [email protected] 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 67 of 77 Expert Review of Clinical Immunology

1 2 3 1442 Table 2. Level of evidence for efficacy of hydroxychloroquine and hydroxychloroquine-azithromycin combination against COVID-19 based on 4 5 1443 clinical studies with a clear description of the therapeutic protocol. 6 1444 7 Level of Type of evidence * Available studies References 8 evidence 9 10 Ia Systematic review (with homogeneity) A meta-analysis of four RCTs conducted in China evidenced a significantly favorable effect of [117, 130] 11 of RCTs hydroxychloroquine in three out of four studies. An update of this meta-analysis including a new 12 For Peer Review Only Brazilian RCT confirmed such an effect (Figure 5 & 6). 13 14 An RCT conducted on 62 COVID-19 patients showed significantly shortened body temperature [124] 15 recovery time and cough remission time and a larger proportion of improved pneumonia as assessed 16 by CT scan in patients treated with 400 mg hydroxychloroquine per day over five days (N=31) than 17 18 in controls (N=31). In addition, a nonsignificant favorable direction effect was observed regarding 19 overall clinical improvement. 20 21 An RCT conducted on 150 COVID-19 patients showed a significant effect on the alleviation of [126, 186] 22 symptoms in post hoc analysis and C-reactive protein reduction in patients treated with 23 hydroxychloroquine 1,200 mg per day for three days then 800 mg for two to three weeks (N=75) than 24 in controls (N=75). It is notable that these results were not included in the version published in the 25 British Medical Journal because these endpoints were not prespecified in the study protocol and due 26 to an underpowered sample size, arguments that we consider fallacious. In addition, a nonsignificant 27 favorable direction effect was observed regarding lymphocyte count decrease and shortened viral 28 shedding duration. 29 30 An RCT conducted on 373 COVID-19 patients showed a significantly shortened length of hospital [125] 31 stay in patients treated with chloroquine 500 mg or 1,000 mg per day for no more than 10 days 32 (N=197) than in controls (N=176). In addition, a nonsignificant favorable direction effect was 33 34 observed regarding clinical and radiological improvement and shortened viral shedding duration. 35 An RCT conducted on 48 COVID-19 patients who were randomized to chloroquine (N = 18), [133] 36 hydroxychloroquine (N = 18) or controls (N = 12). The chloroquine and the hydroxychloroquine 37 groups achieved shorter time to clinical recovery (TTCR) than the control group. The time to reach 38 viral RNA negativity was significantly faster in the chloroquine group and the hydroxychloroquine 39 group than in the control group. 40 41 42 43 URL: https://mc.manuscriptcentral.com/erm Email: [email protected] 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Expert Review of Clinical Immunology Page 68 of 77

1 2 3 4 An RCT conducted in Brazil on 504 patients with PCR-confirmed COVID-19 infection reported no [136] 5 significant effect, but mortality was lower in patients treated by the HCQ-AZ combination (1.7%) 6 versus control (2.9%) or patients treated by HCQ alone (3.1%). This correspond to a prevented 7 fraction of 40% in the treated population (odds ratio = 0.60). 8 9 By contrast, a Chinese RCT conducted on 30 COVID-19 patients showed no significant differences [127] 10 between patients treated with 400 mg hydroxychloroquine per day for five days (N=15) and controls 11 (N=15) regarding the pharyngeal carriage of viral RNA at day 7, clinical and radiological 12 For improvement;Peer however, Review patients received multiple additional Only treatments, including antivirals. 13 14 15 Ib Individual RCT (with narrow A preliminary French (nonrandomized) clinical trial conducted on 36 COVID-19 patients showed a [116] 16 confidence interval) significant reduction in viral nasopharyngeal carriage at day 6 in patients treated with 17 hydroxychloroquine at 600 mg per day for 10 days (N=20, 70% testing negative) compared with 18 untreated controls (N=16, 12.5% testing negative). In addition, of the twenty patients who were 19 treated with hydroxychloroquine, six received azithromycin for five days (for the purposes of 20 preventing bacterial superinfection) and all (100%) were virologically cured at day 6, compared to 21 57.1% of the remaining 14 patients. 22 23 24 25 Ic All or none study - 26 27 2a Systematic review (with homogeneity) A meta-analysis of ten cohort studies conducted in Iran, Brazil, France, China, South Korea, Spain [117] 28 of cohort studies and Saudi Arabia evidenced (depending on studies) a significant favorable effect of 29 hydroxychloroquine on death or transfer to an intensive care unit, the need for hospitalization, overall 30 clinical cure, body temperature recovery time, shortened viral shedding duration, length of hospital 31 stay, cough remission time and interleukin-6 levels. In addition, a nonsignificant favorable direction 32 33 effect was observed regarding death and/or transfer to an intensive care unit, overall clinical cure, 34 length of hospital stay, radiological improvement, and body temperature recovery time. 35 36 By contrast, a nonsignificant deleterious direction effect was observed on overall clinical cure, length 37 of hospital stay, and shortened viral shedding duration in three of these studies. 38 39 2b Individual cohort study (including low Clinical results were reported in a news briefing by the Chinese government revealing that the [187] 40 41 42 43 URL: https://mc.manuscriptcentral.com/erm Email: [email protected] 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 69 of 77 Expert Review of Clinical Immunology

1 2 3 quality RCT; e.g., <80% follow-up) treatment of over 100 patients with chloroquine phosphate in China had resulted in significant 4 improvements of pneumonia and lung imaging, with reductions in the duration of illness. 5 6 7 8 An uncontrolled French noncomparative observational study was conducted on a cohort of 80 [142] 9 relatively mildly infected inpatients treated with a combination of hydroxychloroquine and 10 azithromycin over a period of at least three days; all patients improved clinically with the exception 11 of one 86-year-old patient who died, and one 74-year-old patient who remains in intensive care. A 12 For Peer Review Only rapid fall in nasopharyngeal viral load was noted, with 83% negative at day 7, and 93% at day 8. 13 14 Virus cultures from patients’ respiratory samples were negative in 97.5% of patients at day 5. 15 Consequently, patients were able to be rapidly discharged with a mean length of stay of five days. 16 17 18 Another similar study by the same team was conducted on 1,061 patients under the same therapeutic [117] 19 protocol. Good clinical outcome and virological cure were obtained in 973 patients within 10 days 20 (91.7%). A poor clinical outcome was observed for 46 patients (4.3%) and eight died (0.75%) (74–95 21 years old). All deaths resulted from respiratory failure and not from cardiac toxicity. 22 23 24 A large study by the same team involved 3,119 patients treated with the same therapeutic protocol [3] 25 and 628 with other regimens. Combined treatment was associated with decreased risk of transfer to 26 an ICU or death (Hazard ratio (HR) 0.18 0.11-0.27), decreased risk of hospitalization ≥10 days (odds 27 ratios 95% CI 0.38 0.27-0.54) and shorter duration of viral shedding (time to negative PCR: HR 1.29 28 1.17-1.42). QTc prolongation (>60 ms) was observed in 25 patients (0.67%) leading to the cessation 29 of treatment in 12 cases including three cases with QTc> 500 ms. No cases of torsade de pointe or sudden death were observed. 30 2c “Outcomes” research; ecological Three studies have demonstrated that chloroquine phosphate inhibits SARS-CoV-2 and two have [88-90, 188] 31 32 studies [88] demonstrated that hydroxychloroquine sulfate inhibits SARS-CoV-2 in vitro. In addition, one 33 study showed that the combination of hydroxychloroquine and azithromycin inhibits SARS-CoV-2 in 34 vitro. 35 36 3c Systematic review (with homogeneity) - 37 of case-control studies 38 39 40 41 42 43 URL: https://mc.manuscriptcentral.com/erm Email: [email protected] 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Expert Review of Clinical Immunology Page 70 of 77

1 2 3 3b Individual case-control study - 4 5 4 Case-series (and poor quality cohort - 6 and case-control studies) 7 8 5 Expert opinion without explicit critical The National Health Commission of the People’s Republic of China published their recommendation [188] 9 appraisal, or based on physiology, mid-February, suggesting treating patients with 500 mg chloroquine phosphate twice a day, for a 10 bench research or “first principles” maximum of 10 days. 11 12 For Peer Review Only 13 14 In Italy, the L. Spallanzani National Institute for Infectious Diseases published their [189] 15 recommendations for treatment on 17 March, which included the provision of 400 mg of HCQ per 16 day or 500 mg CQ per day, in combination with another antiviral agent. 17 1445 * https://www.cebm.net/2009/06/oxford-centre-evidence-based-medicine-levels-evidence-march-2009/ 18 19 1446 20 21 1447 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 URL: https://mc.manuscriptcentral.com/erm Email: [email protected] 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 71 of 77 Expert Review of Clinical Immunology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 For Peer Review Only 17 18 Figure 1 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 URL: https://mc.manuscriptcentral.com/erm Email: [email protected] Expert Review of Clinical Immunology Page 72 of 77

1 2 3 4 2000 5 6 1800 7 8 9 1600 For Peer Review Only 10 11 1400 12 13 14 1200 15 16 1000 17 18 800 19 20 21 600 22 23 400 24 25 200 26 Number of patients newly-diagnosed per week 27 28 0 29 30 31 32 24/02/202002/03/202009/03/202016/03/202023/03/202030/03/202006/04/202013/04/202020/04/202027/04/202004/05/202011/05/202018/05/202025/05/202001/06/2020 33 34 35 36 37 38 39 URL: https://mc.manuscriptcentral.com/erm Email: [email protected] 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 73 of 77 Expert Review of Clinical Immunology

1 2 3 Chemical prophylaxis 4 Hydroxychloroquine? 29,9 kbase long single-stranded linear + RNA 5 13 ORFs 6 TRANSMISSION: 7 Respiratory fluids, essentially 8 Aerosols, hands Fomites Coss-protection conferred 9 by pre-existing immunity to endemic coronaviruses ? VirusFor viability 3 h, up to 72Peer h Review Only 10 11 12 Case isolation 13 CARE: Testing Treatment + Monitoring (Ambulatory; Day-care hospital; Hospitalisation) 14 Preventive therapy Curative therapy Supportive care 15 Hydroxychloroquine? Hydroxychloroquine + Azithromycin Hydroxychloroquine + Azithromycin Hydroxychloroquine? No evidence of drug efficacy Remdesivir? Lopinavir/ritonavir? Remdesivir? Lopinavir/ritonavir? Tocilizumab? Quality of ICU care (respiratory 16 Arbidol? Arbidol? support) Hyperimmune gammaglobulins? Anti-coagulant therapy 17 Anti-coagulant therapy Anti-coagulant therapy 18 SEVERAL EVOLUTIONARY STAGES: 19 INCUBATION: 2-14 d 1ST PHASE: 2ND PHASE: 3RD PHASE: 4TH PHASE: 20 Viral shedding 21 Inflammatory response 22 Antibody response 23 Risk factor for severe outcome:  age, comorbidities: high blood pressure, diabetes, coronary heart disease 24 25 Early lung lesions 3-6 d post-symptom onset 7-10 d post-infection PleiomorphicPleiomorphic clinical clinical presentation: presentation Sudden deterioration Alveolar epithelial cells necrosis Acute respiratory distress syndrom 26 Asymptomatic• Asymptomatic / Symptomatic: mild / Inflammation Pulmonary embolism • severeSymptomatic: mild / severe Cytokine dysregulation Phenotypes L and H 27 Autoimmune manifestations Multiple organ failure Upper respiratory tract infection Thrombotic manifestations "Cytokine storm" 28 Anosmia, agueusia, rhinitis Abnormal coagulation Mostly young people  fibrinogen and D-dimers Mostly good clinical outcome   Major hypercoagulability 29 Lymphocytes D-dimers 30 Lower respiratory tract infection Eosinophils Troponin Pneumonia symptoms, cough, fever, Zinc CRP, LDH, CPK 31 frequent thoracic pain "happy" hypoxia 32 33 Cure 34 Death 35 Pulmonary fibrosis? 36 37 38 39 URL: https://mc.manuscriptcentral.com/erm Email: [email protected] 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Expert Review of Clinical Immunology Page 74 of 77

1 2 3 4 5 6 7 8 9 For Peer Review Only 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 URL: https://mc.manuscriptcentral.com/erm Email: [email protected] 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 75 of 77 Expert Review of Clinical Immunology

1 2 3 Country Study name Statistics for each study Odds ratio and 95% CI 4 Odds Lower Upper 5 6 ratio limit limit p-Value 7 8 USA Arshad, Int J Infect Dis, 2020 0.61 0.43 0.88 0.008954 9 Brazil Cavalcanti, N EnglFor J Med, 2020 - PeerHCQ alone RCT 1.09 Review0.31 3.84 0.892228 Only 10 Brazil Cavalcanti, N Engl J Med, 2020 – HCQ-AZ RCT 0.60 0.14 2.54 0.484016 11 France Guerin, Asian J Med Health, 2020 0.54 0.02 14.01 0.713884 12 France Lagier, Travel Med Infect Dis, 2020 0.64 0.47 0.86 0.002990 13 France Mahevas, MedRxiv, 2020 0.72 0.16 3.30 0.668282 14 Spain Membrillo, Preprints, 2020 0.29 0.14 0.61 0.001115 15 Clinical studies 16 France Paccoud, Clin Infect Dis, 2020 0.71 0.33 1.52 0.378609 17 USA Scholz, Preprints, 2020 0.20 0.03 1.54 0.122632 18 China Yu, MedRxiv, 2020 0.27 0.13 0.58 0.000647 19 0.55 0.44 0.70 0.000000 20 Spain Bernaola, MedRxiv, 2020 0.46 0.34 0.62 0.000001 21 USA Mikami, J Gen Intern Med, 2020 0.70 0.60 0.82 0.000006 22 USA Rosenberg, JAMA, 2020 - HCQ alone 1.04 0.76 1.44 0.789895 23 USA Rosenberg, JAMA, 2020 - HCQ-AZ 1.15 0.88 1.51 0.309873 24 25 France Sbidian, MedRxiv, 2020 - HCQ-AZ 1.11 0.81 1.53 0.502133 26 France Sbidian, MedRxiv, 2020 - HCQ alone 0.77 0.62 0.96 0.021751 studies 27 Big data USA Singh, MedRxiv, 2020 0.95 0.71 1.26 0.715438 28 0.84 0.68 1.05 0.123244 29 0.69 0.59 0.81 0.000004 30 31 0.1 0.2 0.5 1 2 5 10 32 33 34 Favours H(CQ) Favours No (H)CQ 35 36 37 38 39 URL: https://mc.manuscriptcentral.com/erm Email: [email protected] 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Expert Review of Clinical Immunology Page 76 of 77

1 2 3 Country Study name Statistics for each study Odds ratio and 95% CI 4 5 Odds Lower Upper 6 ratio limit limit p-Value 7 8 Taiwan Chen CP, MedRxiv, 2020 RCT 0.71 0.13 3.87 0.688187 9 Taiwan Chen CP, MedRxiv, 2020For - Retrospective Peer study 1.67 0.37Review7.57 0.508116 Only 10 11 China Chen L, MedRxiv, 2020 - CQ RCT 0.13 0.03 0.55 0.005506 12 China Chen L, MedRxiv, 2020 - HCQ RCT 0.15 0.04 0.63 0.009252 13 14 China Chen, J Zheijang Univ, 2020 RCT 2.15 0.17 26.67 0.550103 15 16 China Huang, J Mol Cell Biol, 2020 RCT 0.37 0.01 9.98 0.550511 17 China Huang, MedRxiv, 2020 0.13 0.07 0.25 0.000000 18 19 South Korea Kim, MedRxiv, 2020 0.24 0.09 0.65 0.004518 20 France Lagier, Travel Med Infect Dis, 2020 0.67 0.57 0.78 0.000000 21 22 Saudi Arabia Shabrawishi, MedRxiv, 2020 - HCQ/CQ + AZ 1.00 0.42 2.40 1.000000 23 Saudi Arabia Shabrawishi, MedRxiv, 2020 - HCQ/CQ 1.00 0.35 2.86 1.000000 24 25 Saudi Arabia Shabrawishi, MedRxiv, 2020 - HCQ/CQ + antivirals 1.85 0.35 9.88 0.470789 26 China Tang, MedRxiv, 2020 RCT 0.75 0.42 1.35 0.339175 27 28 0.52 0.32 0.84 0.007291 29 30 31 0.1 0.2 0.5 1 2 5 10 32 33 Favours (H)CQ Favours No (H)CQ 34 35 36 37 38 39 URL: https://mc.manuscriptcentral.com/erm Email: [email protected] 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 77 of 77 Expert Review of Clinical Immunology

1 2 3 4 Obesity 5 6 Upper Lower 7 respiratory respiratory 8 tract tract 9 infection Forinfection Peer ReviewHypertension Only 10 Diabetes 11 Chest pain Dyspnea Cancer 12 diseases 13 Ageusia Fever Chronic heart Age > 74 y 14 Anosmia diseases 15 NEWS NEWS score >7 score 5-6 Pulmonary CT scan 16 Rhinitis severe 17 Von Willebrand factor  18 NEWS score ≤4 Platelets , Fibrinogen , D- 19 dimers , Lymphocytes , Zinc 20 21 , Troponin  22 IgG to SARS-CoV-2 , 23 No chest pain, no ageusia, Lymphocytes , Platelets Intensive 24 no anosmia, no rhinitis, , Fibrinogen , CRP 25 care nodyspnea, no cough , LDH , CPK  26 27 28 29 Eosinophils  Other 30 treatments 31 32 33 HCQ+AZ ≥ 3 34 35 d 36 37Evolutionary Mild Severe Very severe (ICU, 38 Mild 39stages URL: https://mc.manuscriptcentral.com/erm Email: [email protected] ≥10 d, 40 41 death) 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60