The REinfection in COVID-19 Estimation of Risk (RECOVER) study: Reinfection and serology dynamics in a cohort of Canadian healthcare workers. (preprint)

Background : Understanding the immune response to natural infection by SARS-CoV-2 is key to pandemic management, especially in the current context of emerging variants. Uncertainty remains regarding the efficacy and duration of natural immunity against reinfection. Method : We conducted an observational prospective cohort study in Canadian healthcare workers (HCWs) with a history of PCR-confirmed SARS-CoV-2 infection to : (i) measure the average incidence rate of reinfection and (ii), describe the serological immune response to the primary infection. Results : We detected 5 cases of reinfection over 14 months of follow-up, for a reinfection incidence rate of 3.3 per 100 person-years. Median duration of seropositivity was 420 days in symptomatics at primary infection compared to 213 days in asymptomatics (p<0.0001). Other variables associated with prolonged seropositivity for IgG against the spike protein included age 55 and above, obesity, and non-Caucasian ethnicity. Summary : Among healthcare workers, the incidence of reinfection with SARS-CoV-2 following a primary infection remained rare, although our analysis predates the circulation of the Omicron variant.

Global epidemiology of SARS-CoV-2 infection: a systematic review and meta-analysis of standardized population-based seroprevalence studies, Jan 2020-Oct 2021 (preprint)

Background COVID-19 case data underestimates infection and immunity, especially in low- and middle-income countries (LMICs). We meta-analyzed standardized SARS-CoV-2 seroprevalence studies to estimate global seroprevalence. Objectives/Methods We conducted a systematic review and meta-analysis, searching MEDLINE, Embase, Web of Science, preprints, and grey literature for SARS-CoV-2 seroprevalence studies aligned with the WHO UNITY protocol published between 2020-01-01 and 2021-10-29. Eligible studies were extracted and critically appraised in duplicate. We meta-analyzed seroprevalence by country and month, pooling to estimate regional and global seroprevalence over time; compared seroprevalence from infection to confirmed cases to estimate under-ascertainment; meta-analyzed differences in seroprevalence between demographic subgroups; and identified national factors associated with seroprevalence using meta-regression. PROSPERO: CRD42020183634. Results We identified 396 full texts reporting 736 distinct seroprevalence studies (41% LMIC), including 355 low/moderate risk of bias studies with national/sub-national scope in further analysis. By April 2021, global SARS-CoV-2 seroprevalence was 26.1%, 95% CI [24.6-27.6%]. Seroprevalence rose steeply in the first half of 2021 due to infection in some regions (e.g., 18.2% to 45.9% in Africa) and vaccination and infection in others (e.g., 11.3% to 57.4% in the Americas high-income countries), but remained low in others (e.g., 0.3% to 1.6% in the Western Pacific). In 2021 Q1, median seroprevalence to case ratios were 1.9:1 in HICs and 61.9:1 in LMICs. Children 0-9 years and adults 60+ were at lower risk of seropositivity than adults 20-29. In a multivariate model using data pre-vaccination, more stringent public health and social measures were associated with lower seroprevalence. Conclusions Global seroprevalence has risen considerably over time and with regional variation, however much of the global population remains susceptible to SARS-CoV-2 infection. True infections far exceed reported COVID-19 cases. Standardized seroprevalence studies are essential to inform COVID-19 control measures, particularly in resource-limited regions.

A system biology approach identifies candidate drugs to reduce mortality in severely ill COVID-19 patients (preprint)

Despite the availability of highly efficacious vaccines, Coronavirus Disease 2019 (COVID-19) caused by severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) lacks effective drug treatment which results in a high rate of mortality. To address this therapeutic shortcoming, we applied a system biology approach to the study of patients hospitalized with severe COVID. We show that, at the time of hospital admission, patients who were equivalent on the clinical ordinal scale displayed significant differential monocyte epigenetic and transcriptomic attributes between those who would survive and those who would succumb to COVID-19. We identified mRNA metabolism, RNA splicing, and interferon signaling pathways as key host responses overactivated by patients who would not survive. Those pathways are prime drug targets to reduce mortality of critically ill COVID-19 patients leading us to identify Tacrolimus, Zotatifin, and Nintedanib as three strong candidates for treatment of severely ill patients at the time of hospital admission. TeaserEpigenetics distinguishes COVID-19 survivors already at hospital admission: lessons for drug repurposing.

Specificity of SARS-CoV-2 antibody-detection assays against S and N protein among pre-COVID-19 sera from patients with protozoan and helminth parasitic infections (preprint)

BackgroundWe aimed to assess the specificity of SARS-CoV-2 antibody detection assays among people with known tissue-borne parasitic infections. MethodsWe tested three SARS-CoV-2 antibody-detection assays (cPass SARS-CoV-2 Neutralization Antibody Detection Kit, Abbott SARS-CoV-2 IgG assay, and STANDARD Q COVID-19 IgM/IgG Combo Rapid Test) among 559 pre-COVID-19 sera. ResultsThe specificity of assays was 95-98% overall. However, lower specificity was observed among sera from patients with protozoan infections of the reticuloendothelial system, such as human African trypanosomiasis (Abbott Architect; 88% [95%CI 75-95]), visceral leishmaniasis (SD RDT IgG; 80% [95%CI 30-99]), and from patients with recent malaria from a holoendemic area of Senegal (ranging from 91% for Abbott Architect and SD RDT IgM to 98-99% for cPass and SD RDT IgG). For specimens from patients with evidence of past or present helminth infection overall, test specificity estimates were all [≥] 96%. Sera collected from patients clinically suspected of parasitic infections that tested negative for these infections yielded a specificity of 98-100%. The majority (>85%) of false-positive results were positive by only one assay. ConclusionsThe specificity of SARS-CoV-2 serological assays among sera from patients with tissue-borne parasitic infections was below the threshold required for decisions about individual patient care. Specificity is markedly increased by the use of confirmatory testing with a second assay. Finally, the SD RDT IgG proved similarly specific to laboratory-based assays and provides an option in low-resource settings when detection of anti-SARS-CoV-2 IgG is indicated.

Evaluation of a Commercial Culture-free Neutralization Antibody Detection Kit for Severe Acute Respiratory Syndrome-Related Coronavirus-2 and Comparison with an Anti-RBD ELISA Assay (preprint)

BackgroundSARS-CoV-2 surrogate neutralization assays that obviate the need for viral culture offer substantial advantages regarding throughput and cost. The cPass SARS-CoV-2 Neutralization Antibody Detection Kit (Genscript) is the first such commercially available assay, detecting antibodies that block RBD/ACE-2 interaction. We aimed to evaluate cPass to inform its use and assess its added value compared to anti-RBD ELISA assays. MethodsSerum reference panels comprising 205 specimens were used to compare cPass to plaque-reduction neutralization test (PRNT) and a pseudotyped lentiviral neutralization (PLV) assay for detection of neutralizing antibodies. We assessed the correlation of cPass with an ELISA detecting anti-RBD IgG, IgM, and IgA antibodies at a single timepoint and across intervals from onset of symptoms of SARS-CoV-2 infection. ResultsCompared to PRNT-50, cPass sensitivity ranged from 77% - 100% and specificity was 95% - 100%. Sensitivity was also high compared to the pseudotyped lentiviral neutralization assay (93% [95%CI 85-97]), but specificity was lower (58% [95%CI 48-67]). Highest agreement between cPass and ELISA was for anti-RBD IgG (r=0.823). Against the pseudotyped lentiviral neutralization assay, anti-RBD IgG sensitivity (99% [95%CI 94-100]) was very similar to that of cPass, but overall specificity was lower (37% [95%CI 28-47]). Against PRNT-50, results of cPass and anti-RBD IgG were nearly identical. ConclusionsThe added value of cPass compared to an IgG anti-RBD ELISA was modest.

Global seroprevalence of SARS-CoV-2 antibodies: a systematic review and meta-analysis (preprint)

BackgroundStudies reporting estimates of the seroprevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies have rapidly emerged. We aimed to synthesize seroprevalence data to better estimate the burden of SARS-CoV-2 infection, identify high-risk groups, and inform public health decision making. MethodsIn this systematic review and meta-analysis, we searched publication databases, preprint servers, and grey literature sources for seroepidemiological study reports, from January 1, 2020 to August 28, 2020. We included studies that reported a sample size, study date, location, and seroprevalence estimate. Estimates were corrected for imperfect test accuracy with Bayesian measurement error models. We conducted meta-analysis to identify demographic differences in the prevalence of SARS-CoV-2 antibodies, and meta-regression to identify study-level factors associated with seroprevalence. We compared region-specific seroprevalence data to confirmed cumulative incidence. PROSPERO: CRD42020183634. FindingsWe identified 338 seroprevalence studies including 2.3 million participants in 50 countries. Seroprevalence was low in the general population (median 3.2%, IQR 1.0-6.4%) and slightly higher in at-risk populations (median 5.4%, IQR 1.5-18.4%). Median seroprevalence varied by WHO Global Burden of Disease region (p < 0.01), from 1.0% in Southeast Asia, East Asia and Oceania to 18.8% in South Asia. National studies had lower seroprevalence estimates than local (p = 0.02) studies. Compared to White persons, Black persons (prevalence ratio [RR] 2.34, 95% CI 1.60-3.43) and Asian persons (RR 1.56, 95% CI 1.22-2.01) were more likely to be seropositive. Seroprevalence was higher among people ages 18-64 compared to 65 and over (RR 1.26, 95% CI 1.04-1.52). Health care workers had a 1.74x (95% CI: 1.18-2.58) higher risk compared to the general population. There was no difference in seroprevalence between sexes. There were 123 studies (36%) at low or moderate risk of bias. Seroprevalence estimates from national studies were median 11.9 (IQR 8.0 - 16.6) times higher than the corresponding SARS-CoV-2 cumulative incidence. InterpretationMost of the population remains susceptible to SARS-CoV-2 infection. Public health measures must be improved to protect disproportionately affected groups, including non-White people and adults. Measures taken in SE Asia, E Asia and Oceania, and Latin America and Caribbean may have been more effective in controlling virus transmission than measures taken in other regions. FundingPublic Health Agency of Canada through the COVID-19 Immunity Task Force.

Sequential ER stress and inflammatory responses are induced by SARS-CoV-2 ORF3 through ERphagy (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections have resulted in a number of severe cases of COVID-19 and deaths worldwide. However, knowledge of SARS-CoV-2 infection, diseases and therapy remains limited, underlining the urgency of fundamental studies and drug development. Studies have shown that induction of autophagy and hijacking of autophagic machinery are essential for infection and replication of SARS-CoV-2; however, the mechanism of this manipulation and function of autophagy during SARS-CoV-2 infection remain unclear. In the present study, we identified ORF3 as an inducer of autophagy and revealed that ORF3 localizes to the ER and induces FAM134B-related ERphagy through the HMGB1-Beclin1 pathway. As a consequence, ORF3 induces ER stress and inflammatory responses through ERphagy and sensitizes cells to ER stress-induced cell death, suggesting that SARS-CoV-2 ORF3 hijacks ERphagy and then harms ER homeostasis to induce inflammatory responses through excessive ER stress. These findings reveal a sequential induction of ERphagy, ER stress and acute inflammatory responses during SARS-CoV-2 infection and provide therapeutic potential for ERphagy and ER stress-related drugs for COVID-19 treatment and prevention. ImportanceSARS-CoV-2 infection and replication require autophagosome-like double-membrane vacuoles. Inhibition of autophagy suppresses viral replication, indicating the essential role of autophagy in SARS-CoV-2 infection. However, how SARS-CoV-2 hijacks autophagy and the function of autophagy in the disease progression remain unknown. Here, we reveal that SARS-CoV-2 ORF3 induces ERphagy and consequently induces ER stress to trigger acute inflammatory responses and enhance sensitivity to ER stress-induced apoptosis. Our studies uncover ERphagy-induced inflammatory responses during SARS-CoV-2 infection and provide a promising therapeutic approach for treating SARS-CoV-2 infection and inflammatory responses in COVID-19 by manipulating autophagy and ER stress.

An assessment of efficacy of Iodine complex (Renessans) against SARS-CoV-2 in non-human primates (Rhesus macaque) (preprint)

Renessans is an iodine complex which has proven in vitro antiviral activity including Anti-SARS-CoV-2 activity. The present study was designed to determine its efficacy against SARS-CoV-2 in monkeys (Rhesus macaque). A total of 14 monkeys were divided into four groups: A) Prophylactic group (n=03), (B) Treatment group (n=03), (C) infection control group (n=04) and (D) negative control group (n=04) and were housed in BSL-3 Animal facility while group D was housed at another animal house. Group A was administered with Renessans @ 2.85 mg/7 kg from 5 days prior to the infection to 08 days post infections (DPI). Group B was administered with Renessans from 03-08 DPI @ 2.85 mg/7 kg. Group C was administered with WIF only. The infection @ 2 x 106 TCID of SARS-CoV-2 was given to all group monkeys through intranasal and oral route under anesthesia. Nasal swab samples (at different times) and fecal matter on daily basis were collected for the detection of SARS-CoV-2 through real-time quantitative PCR. Three monkeys (one from each of group A, B and C) were euthanized at 07 DPI to determine the gross pathological lesions and SARS-CoV-2 detection from internal tissues. Nasal swabs from all the monkeys from group A, B and C were positive for SARS-CoV-2 at 02 and 07 DPI (Day 05 of treatment). At 14 DPI, all (100%) nasal swabs from group A were negative for SARS-CoV-2 while 50% and 100% were positive from group B and C, respectively. At 21 DPI, monkeys from group B were negative and all in group C were still positive for SARS-CoV-2. Similarly, fecal matter of monkeys in group A and B was returned negative in significantly lesser time as compared to monkeys from infection control group. Based on these research findings it is concluded that the Renessans has in-vivo SARS-CoV-2 activity and may result in early clearance of SARS-CoV-2. Therefore, a clinical trial of the drug in COVID-19 patients may reveal its anti-COVID-19 potential.

HLA class I genotypes customize vaccination strategies in immune simulation to combat COVID-19 (preprint)

Memory CD8+ T cells are associated with a better outcome in Coronavirus Disease 2019 (COVID-19) and recognized as promising vaccine targets against viral infections. This study determined the efficacy of population-dominant and infection-relevant human leukocyte antigens (HLA) class I proteins to present severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) peptides through calculating binding affinities and simulating CD8+ T cell responses. As a result, HLA class I proteins distinguished or shared various viral peptides derived from viruses. HLA class I supertypes clustered viral peptides through recognizing anchor and preferred residues. SARS-CoV-2 peptides overlapped significantly with SARS but minimally with common human coronaviruses. Immune simulation of CD8+ T cell activation using predicted SARS-CoV-2 peptide antigens depended on high-affinity peptide binding, anchor residue interaction, and synergistic presentation of HLA class I proteins in individuals. Results demonstrated that multi-epitope vaccination, employing a strong binding affinity, viral adjuvants, and heterozygous HLA class I genes, induced potent immune responses. Therefore, optimal CD8+ T cell responses can be achieved and customized contingent on HLA class I genotypes in human populations, supporting a precise vaccination strategy to combat COVID-19.

The expression of PDCD1 and CD274 in T cells and macrophages correlated positively with COVID-19 severity (preprint)

The immune responses underlying the infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain unclear. To help understand the pathology of coronavirus disease 2019 (COVID-19) pandemics, public data were analyzed and the expression of PDCD1 (encoding PD-1) and CD274 (encoding PD-L1) in T cells and macrophages were identified to correlate positively with COVID-19 severity.

Universally available herbal teas based on sage and perilla elicit potent antiviral activity against SARS-CoV-2 in vitro (preprint)

The current SARS-CoV-2/COVID-19 pandemic represents an unprecedented medical and socioeconomic crisis. Highly efficient treatment options preventing morbidity and mortality are not broadly available and approved drugs are hardly affordable in developing countries. Even after vaccine approvals, it will take several months until the vaccinated and convalescent individuals establish herd immunity. Meanwhile, non-pharmaceutical interventions and antiviral treatments are indispensable to curb the death toll of the pandemic. To identify cost-effective and ubiquitously available options, we tested common herbs consumed worldwide as herbal teas. We found that aqueous infusions prepared by boiling leaves of the Lamiaceae plants perilla and sage elicit potent antiviral activity against SARS-CoV-2 in human cells. Sustained antiviral activity was evident even when cells were treated for only half an hour, and in therapeutic as well as prophylactic regimens. Given the urgency, such inexpensive and broadly available substances might provide help during the pandemic - especially in low-income regions.

Single-cell Transcriptome of Bronchoalveolar Lavage Fluid Reveals Dynamic Change of Macrophages During SARS-CoV-2 Infection in Ferrets (preprint)

Although the profile of immune cells changes during the natural course of SARS-CoV-2 inflection in human patients, few studies have used a longitudinal approach to reveal their dynamic features. Here, we performed single-cell RNA sequencing of bronchoalveolar lavage fluid cells longitudinally obtained from SARS-CoV-2-infected ferrets. Landscape analysis of the lung immune microenvironment showed dynamic changes in cell proportions and characteristics in uninfected control, at 2 days post-infection (dpi) (early stage of SARS-CoV-2 infection with peak viral titer), and 5 dpi (resolution phase). NK cells and CD8+ T cells exhibited activated subclusters with interferon-stimulated features, which were peaked at 2 dpi. Intriguingly, macrophages were classified into 10 distinct subpopulations, and their relative proportions changed over the time. We observed prominent transcriptome changes among monocyte-derived infiltrating macrophages and differentiated M1/M2 macrophages, especially at 2 dpi. Moreover, trajectory analysis revealed gene expression changes from monocyte-derived infiltrating macrophages toward M1 or M2 macrophages and identified the distinct macrophage subpopulation that had rapidly undergone SARS-CoV-2-mediated activation of inflammatory responses. Finally, we found that different spectrums of M1 or M2 macrophages showed distinct patterns of gene modules downregulated by immune-modulatory drugs. Overall, these results elucidate fundamental aspects of the immune response dynamics provoked by SARS-CoV-2 infection.

Generation and Characterization of recombinant SARS-CoV-2 expressing reporter genes (preprint)

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogen responsible of coronavirus disease 2019 (COVID-19), has devastated public health services and economies worldwide. Despite global efforts to contain the COVID-19 pandemic, SARS-CoV-2 is now found in over 200 countries and has caused an upward death toll of over 1 million human lives as of November 2020. To date, only one Food and Drug Administration (FDA)-approved therapeutic drug (Remdesivir) and a monoclonal antibody, MAb (Bamlanivimab), but no vaccines, are available for the treatment of SARS-CoV-2. As with other viruses, studying SARS-CoV-2 requires the use of secondary approaches to detect the presence of the virus in infected cells. To overcome this limitation, we have generated replication-competent recombinant (r)SARS-CoV-2 expressing fluorescent (Venus or mCherry) or bioluminescent (Nluc) reporter genes. Vero E6 cells infected with reporter-expressing rSARS-CoV-2 can be easily detected via fluorescence or luciferase expression and display a good correlation between reporter gene expression and viral replication. Moreover, rSARS-CoV-2 expressing reporter genes have comparable plaque sizes and growth kinetics to those of wild-type virus, rSARS-CoV-2/WT. We used these reporter-expressing rSARS-CoV-2 to demonstrate their feasibility to identify neutralizing antibodies (NAbs) or antiviral drugs. Our results demonstrate that reporter-expressing rSARS-CoV-2 represent an excellent option to identify therapeutics for the treatment of SARS-CoV-2, where reporter gene expression can be used as valid surrogates to track viral infection. Moreover, the ability to manipulate the viral genome opens the feasibility of generating viruses expressing foreign genes for their use as vaccines for the treatment of SARS-CoV-2 infection. ImportanceSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogen that causes coronavirus disease 2019 (COVID-19), has significantly impacted the human health and economic status worldwide. There is an urgent need to identify effective prophylactics and therapeutics for the treatment of SARS-CoV-2 infection and associated COVID-19 disease. The use of fluorescent- or luciferase-expressing reporter expressing viruses has significantly advanced viral research. Here, we generated recombinant (r)SARS-CoV-2 expressing fluorescent (Venus and mCherry) or luciferase (Nluc) reporter genes and demonstrate that they represent an excellent option to track viral infections in vitro. Importantly, reporter-expressing rSARS-CoV-2 display similar growth kinetics and plaque phenotype that their wild-type counterpart (rSARS-CoV-2/WT), demonstrating their feasibility to identify drugs and/or neutralizing antibodies (NAbs) for the therapeutic treatment of SARS-CoV-2. Henceforth, these reporter-expressing rSARS-CoV-2 can be used to interrogate large libraries of compounds and/or monoclonal antibodies (MAb), in high-throughput screening settings, to identify those with therapeutic potential against SARS-CoV-2.

Replicating bacterium-vectored vaccine expressing SARS-CoV-2 Membrane and Nucleocapsid proteins protects against severe COVID-19 disease in hamsters (preprint)

An inexpensive readily manufactured COVID-19 vaccine that protects against severe disease is needed to combat the pandemic. We have employed the LVS {Delta}capB vector platform, previously used successfully to generate potent vaccines against the Select Agents of tularemia, anthrax, plague, and melioidosis, to generate a COVID-19 vaccine. The LVS {Delta}capB vector, a replicating intracellular bacterium, is a highly attenuated derivative of a tularemia vaccine (LVS) previously administered to millions of people. We generated vaccines expressing SARS-CoV-2 structural proteins and evaluated them for efficacy in the golden Syrian hamster, which develops severe COVID-19 disease. Hamsters immunized intradermally or intranasally with a vaccine co-expressing the Membrane (M) and Nucleocapsid (N) proteins, then challenged 5-weeks later with a high dose of SARS-CoV-2, were protected against severe weight loss and lung pathology and had reduced viral loads in the oropharynx and lungs. Protection by the vaccine, which induces murine N-specific interferon-gamma secreting T cells, was highly correlated with pre-challenge serum anti-N TH1-biased IgG. This potent vaccine against severe COVID-19 should be safe and easily manufactured, stored, and distributed, and given the high homology between MN proteins of SARS-CoV and SARS-CoV-2, has potential as a universal vaccine against the SARS subset of pandemic causing {beta}-coronaviruses.

Safety of Hydroxychloroquine among Outpatient Clinical Trial Participants for COVID-19 (preprint)

IntroductionUse of hydroxychloroquine in hospitalized patients with COVID-19, especially in combination with azithromycin, has raised safety concerns. Here, we report safety data from three outpatient randomized clinical trials. MethodsWe conducted three randomized, double-blind, placebo-controlled trials investigating hydroxychloroquine as pre-exposure prophylaxis, post-exposure prophylaxis and early treatment for COVID-19. We excluded individuals with contraindications to hydroxychloroquine. We collected side effects and serious adverse events. We report descriptive analyses of our findings. ResultsWe enrolled 2,795 participants. The median age of research participants was 40 (IQR 34-49) years, and 59% (1633/2767) reported no chronic medical conditions. Overall 2,324 (84%) participants reported side effect data, and 638 (27%) reported at least one medication side effect. Side effects were reported in 29% with daily, 36% with twice weekly, 31% with once weekly hydroxychloroquine compared to 19% with placebo. The most common side effects were upset stomach or nausea (25% with daily, 18% with twice weekly, 16% with weekly, vs. 10% for placebo), followed by diarrhea, vomiting, or abdominal pain (23% for daily, 16% twice weekly, 12% weekly, vs. 6% for placebo). Two individuals were hospitalized for atrial arrhythmias, one on placebo and one on twice weekly hydroxychloroquine. No sudden deaths occurred. ConclusionData from three outpatient COVID-19 trials demonstrated that gastrointestinal side effects were common but mild with the use of hydroxychloroquine, while serious side effects were rare. No deaths occurred related to hydroxychloroquine. Randomized clinical trials can safely investigate whether hydroxychloroquine is efficacious for COVID-19. Short SummaryData from three randomized clinical trials using hydroxychloroquine for the prevention and treatment of COVID-19 did not suggest significant safety concerns. Gastrointestinal side effects were common but arrhythmias were rare. There were no sudden deaths in any trial.

Post-exposure Prophylaxis or Preemptive Therapy for SARS-Coronavirus-2: Study Protocol for a Pragmatic Randomized Controlled Trial (preprint)

Background: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in December 2019 causing the coronavirus disease 2019 (COVID-19) pandemic. Currently, there are a lack of evidence-based therapies to prevent COVID-19 following exposure, or to prevent worsening of symptoms following confirmed infection. We describe the design of a clinical trial of hydroxychloroquine for post-exposure prophylaxis and pre-emptive therapy for COVID-19. Methods: We will conduct two nested multicenter international double-blind randomized placebo-controlled clinical trials of hydroxychloroquine for: 1) post-exposure prophylaxis (PEP) of asymptomatic household contacts or healthcare workers exposed to COVID-19 within the past four days, and 2) pre-emptive therapy (PET) for symptomatic outpatients with COVID-19 with a total symptom duration of less than 4 days. We will recruit 1500 patients for each the PEP and PET trials. Participants will be randomized 1:1 to receive 5 days of hydroxychloroquine or placebo. The primary PEP trial outcome will be the incidence of symptomatic COVID-19 disease. The primary PET trial outcome will be an ordinal scale of disease severity (not hospitalized; hospitalized without intensive care, hospitalization with intensive care, or death). Participant screening, informed consent, and follow up will be exclusively internet-based with appropriate regulatory and research ethics board approvals in Canada and the United States. Discussion: These complementary randomized control trials are innovatively designed and adequately powered to rapidly answer urgent questions regarding the effectiveness of hydroxychloroquine to reduce transmission and disease severity of COVID-19 during a pandemic. In-person participant follow-up will not be conducted in order to facilitate social distancing strategies and reduce risks of exposure to study personnel. Innovative trial approaches are needed to urgently assess therapeutic options to mitigate the global impact of this pandemic. Trials Registration: clinicaltrials.gov (NCT04308668); 16 March 2020.