With the lack of effective therapy, chemoprevention and vaccination [1] , focusing on the immediate repurposing of existing drugs gives hope of curbing the pandemic. Importantly, a most recent genomics-guided tracing of the SARS-CoV-2 targets in human cells identified vitamin D among the three top-scoring molecules manifesting potential infection mitigation patterns through their effects on gene expression [2] . In particular, by activating or repressing several genes in the promoter region of which it binds to the vitamin D response element [3] , vitamin D may theoretically prevent or improve COVID-19 adverse outcomes by regulating (i) the renin-angiotensin system (RAS), (ii) the innate and adaptive cellular immunity, and (iii) the physical barriers [4] . Consistently, epidemiology shows that hypovitaminosis D is more common from October to March at northern latitudes above 20 degrees [3] , which corresponds precisely to the latitudes with the highest lethality rates of COVID-19 during the first months of winter 2020 [1] . In line with this, significant inverse associations were found in 20 European countries between serum 25-hydroxyvitamin D (25(OH)D) concentration and the number of COVID-19 cases, as well as with COVID-19 mortality [5] . This suggests that increasing serum 25(OH)D concentration may improve the prognosis of COVID-19.
However, no randomized controlled trial (RCT) has tested the effect of vitamin D supplements on COVID-19 outcomes yet. We had the opportunity to examine the association between the use of vitamin D3 supplements and COVID-19 mortality in a sample of frail elderly nursing-home residents infected with SARS-CoV-2. The main objective of this quasiexperimental study was to determine whether bolus vitamin D3 supplementation taken during or just before COVID-19 was effective in improving survival among frail elderly COVID-19 J o u r n a l P r e -p r o o f patients living in nursing-home. The secondary objective was to determine whether this intervention was effective in limiting the clinical severity of the infection.
The study consisted in a quasi-experimental study in a middle-sized nursing-home in Rhône, South-East of France, the residents of which were largely affected by COVID-19 in March-April 2020 (N=96, including n=66 with COVID-19). Data were retrospectively collected from the residents' records of the nursing-home.
The nursing-home is dedicated to residents with physical disabilities, major neurocognitive 
All residents in the nursing-home receive chronic vitamin D supplementation with regular maintenance boluses (single oral dose of 80,000 IU vitamin D3 every 2-3 months), without systematically performing serum control test as recommended in French nursing-homes [6] due to the very high prevalence of hypovitaminosis D reaching 90-100% in this population [7] . Here, the "Intervention group" was defined as all COVID-19 residents who received an oral bolus of 80,000 IU vitamin D3 either in the week following the suspicion or diagnosis of COVID-19, or during the previous month. The "Comparator group" corresponded to all other COVID-19 residents who did not receive any recent vitamin D supplementation. None received D2 or intramuscular supplements. All medications were dispended and supervised by a nurse.
The primary outcome was mortality of COVID-19 residents during follow-up. Follow-up started from the day of COVID-19 diagnosis for each patient, and continued until May 15, 2020, or until death if applicable.
The secondary outcome was the score on the World Health Organization's Ordinal Scale for Clinical Improvement (OSCI) for COVID-19 [8] . The score was calculated by the geriatrician of the nursing-home during the most severe acute phase of COVID-19 for each patient. The OSCI distinguishes between several levels of COVID-19 clinical severity according to the outcomes and dedicated treatments required, with a score ranging from 0 (benign) to 8 (death). A score of 4 corresponds to the introduction of oxygen (nasal oxygen catheter or oral nasal mask), and a score of 6 to intubation and invasive ventilation [8] .
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Age, gender, number of drugs daily taken, functional abilities, nutritional status, COVID-19 treatment with corticosteroids and/or hydroxychloroquine and/or dedicated antibiotics, and hospitalization for COVID-19 were used as potential confounders. The number of drugs usually taken per day was recovered from prescriptions in the nursing-home and served as a measure of the burden of disease, as previously reported [9] . The use of corticosteroids and/or hydroxychloroquine and/or dedicated antibiotics (i.e., azithromycin or rovamycin) were noted from prescriptions in the nursing-home or during hospitalization, as appropriate. Functional abilities prior to COVID-19 were measured from 1 to 6 (best) with the Iso-Resources Groups (GIR) [10] . Finally, the prognosis related to the nutritional status prior to COVID-19 was evaluated using the last measure of serum albumin concentration during the past semester, as appropriate [11] .
Participants' characteristics were summarized using means and standard deviations (SD) or frequencies and percentages, as appropriate. As the number of observations was higher than 40, comparisons were not affected by the shape of the error distribution and no transform was applied [12] . First, comparisons between participants separated into Intervention and Comparator groups were performed using Mann-Whitney U test or the Chi-square test or Fisher test, as appropriate; and then according to mortality. Secondly, a full-adjusted Cox regression was used to examine the associations of mortality (dependent variable) with bolus vitamin D3 supplements and covariables (independent variables). The model produces a survival function that provides the probability of death at a given time for the characteristics supplied for the independent variables. Third, the elapsed time to death was studied by survival curves computed according to Kaplan-Meier method and compared by log-rank test.
Finally, univariate and multiple linear regressions were used to examine the association of J o u r n a l P r e -p r o o f bolus vitamin D3 supplementation (independent variable) with OSCI score (dependent variable), while adjusting for potential confounders. P-values<0.05 were considered significant. All statistics were performed using SPSS (v23.0, IBM Corporation, Chicago, IL) and SAS® version 9.4 software (Sas Institute Inc).
The study was conducted in accordance with the ethical standards set forth in the Helsinki Declaration (1983) . No participant or relatives objected to the use of anonymized clinical and biological data for research purposes. The study was approved by the Ethical Committee of Angers University Hospital (2020/67). The study protocol was also declared to the National Commission for Information Technology and civil Liberties (CNIL).
Sixty-six participants (mean±SD age 87.7±9.0years, range 63-103years; 77.3% women) were infected with SARS-CoV-2 and included in this quasi-experimental study. The mean followup was 36±17days. Fifty-one people survived COVID-19, while 15 died. The two groups were comparable at baseline with no significant difference regarding the age (P=0.699), gender (P=0.731), the mean number of drugs usually taken per day (P=0.053), the GIR score (P=0.209) and the serum albumin concentration (P=0.263) ( Table 1) . Regarding care dedicated to COVID-19, only the proportion of patients who received a bolus of vitamin D3 during or just before COVID-19 differed between deceased participants and survivors, with a higher prevalence in survivors (respectively 92.2% versus 66.7%, P=0.023). In contrast, there was no between-group difference in the proportion of patients treated with corticosteroids, hydroxychloroquine or dedicated antibiotics, or hospitalized for COVID-19. Table 1 indicates the characteristics of participants separated into Intervention (n=57) and
Comparator (n=9) groups. Their baseline characteristics (age, gender, albuminemia) did not J o u r n a l P r e -p r o o f differ between groups, with the exception of the number of drugs usually taken (which involved the use of vitamin D supplements) and the disability score (Table 1) Finally, the linear regression model illustrated in Table 2 showed that bolus vitamin D3 supplementation during or just before COVID-19 was inversely associated with the OSCI score for COVID-19 in acute phase. Similar results were found before (β=-2.96 [95%CI: -4.79;-1.12], P=0.002) and after adjusting the analyses for potential confounders (β=-3.84
[95%CI: -6.07;-1.62], P=0.001).
The main finding of this nursing-home-based quasi-experimental study is that, J o u r n a l P r e -p r o o f irrespective of all measured potential confounders, bolus vitamin D3 supplementation during or just before COVID-19 was associated with less severe COVID-19 and better survival rate in frail elderly. No other treatment showed protective effect. This novel finding provides a scientific basis for vitamin D replacement trials attempting to improve COVID-19 prognosis.
To our knowledge we provide here the first quasi-experimental data examining the effect of vitamin D supplementation on the survival rate of COVID-19 patients. To date, only rare observational data, all of which are consistent, are available on the link between vitamin D and COVID-19. The first reports in COVID-19 patients indicated that hypovitaminosis D is highly prevalent in this population, reaching 85% [13] , and that serum 25(OH)D concentrations are lower in COVID-19 patients compared to controls [14] . Similarly, significant inverse correlations were found in 20 European countries between the mean serum 25(OH)D concentrations and the number of COVID-19 cases/1M, as well as with mortality/1M [5] . The severity of hypovitaminosis D appears to relate to the prognosis of COVID-19 since the mortality rate was multiplied by 7.6 among people with hypovitaminosis D <75nmol/L, and by 10.1 among those with hypovitaminosis D <50nmol/L (P<0.001) [15] .
Similarly, another observational study in 212 COVID-19 cases showed that, for each standard deviation increase in serum 25(OH)D, the probability of mild rather than severe COVID-19 was multiplied by 7.9 (P<0.001), while the probability of mild rather than critical COVID-19 was multiplied by 19.6 (P<0.001) [16] . These results suggest that increasing serum 25(OH)D concentration may improve the prognosis of COVID-19. Interventional studies dedicated to COVID-19 are yet warranted for investigating the role of vitamin D supplementation on COVID-19 outcomes. Interestingly, previous meta-analyses found that high-dose prophylactic vitamin D supplementation was able to reduce the risk of respiratory tract infections [17] .
Based on this observation, we and others are conducting an RCT designed to test the effect of high-dose versus standard-dose vitamin D3 on 14-day mortality in COVID-19 older patients J o u r n a l P r e -p r o o f (https://clinicaltrials.gov/ct2/show/NCT04344041). While waiting for the recruitment of this RCT to be completed, the findings of the present quasi-experimental study strongly suggest a benefit of bolus vitamin D3 supplementation on COVID-19 outcomes and survival.
How vitamin D supplementation may improve COVID-19 outcomes and survival is not fully elucidated. Three mechanisms are possible: regulation of (i) the RAS, (ii) the innate and adaptive cellular immunity, and (iii) the physical barriers [4] . First, vitamin D reduces pulmonary permeability in animal models of acute respiratory distress syndrome (ARDS) by modulating the activity of RAS and the expression of the angiotensin-2 converting enzyme (ACE2) [18] . This action is crucial since SARS-CoV-2 reportedly uses ACE2 as a receptor to infect host cells [19] and downregulates ACE2 expression [20] . ACE2 is expressed in many organs, including the endothelium and the pulmonary alveolar epithelial cells, where it has protective effects against inflammation [21] . During COVID-19, downregulation of ACE2 results in an inflammatory chain reaction, the cytokine storm, complicated by ARDS [22] . In contrast, a study in rats with chemically-induced ARDS showed that the administration of vitamin D increased the levels of ACE2 mRNA and proteins [23] . Rats supplemented with vitamin D had milder ARDS symptoms and moderate lung damage compared to controls.
Second, many studies have described the antiviral effects of vitamin D, which works either by induction of antimicrobial peptides with direct antiviral activity against enveloped and nonenveloped viruses, or by immunomodulatory and anti-inflammatory effects [24] . These are potentially important during COVID-19 to limit the cytokine storm. Vitamin D can prevent ARDS [25] by reducing the production of pro-inflammatory Th1 cytokines, such as TNFα and interferon γ [24] . It also increases the expression of anti-inflammatory cytokines by macrophages [24] . Third, vitamin D stabilizes physical barriers [4] . These barriers are made up of closely linked cells to prevent outside agents (such as viruses) from reaching tissues susceptible to viral infection. Although viruses alter the integrity of the cell junction, vitamin J o u r n a l P r e -p r o o f D contributes to the maintenance of functional tight junctions via E-cadherin [4] . All these antiviral effects could potentiate each other and explain our results.
We also found that none of the other dedicated drugs used in this quasi-experimental study was associated with better survival rate in COVID-19 patients. The interest of these drugs in COVID-19 is still debated, whether for corticosteroids [26] , hydroxychloroquine [27] or azithromycin [28] . However, it should be noted that these drugs were given here as part of patient care in the most severe clinical situations, which could have biased and masked their effectiveness (if any).
The strengths of the present study include i) the originality of the research question on an emerging infection for which there is no scientifically validated treatment, ii) the detailed description of the participants' characteristics allowing the use of multivariate models to measure adjusted associations, and iii) the standardized collection of data from a single research center.
Regardless, a number of limitations also existed. First, the study cohort was restricted to a limited number of nursing-home residents who might be unrepresentative of all older adults. Second, although we were able to control for the important characteristics that could modify the association, residual potential confounders might still be present such as the serum concentration of 25(OH)D at baseline -a low level classically ensuring the effectiveness of the supplementation [29] . As this analysis was not initially planned, no concerted efforts were made to systematically measure the serum 25(OH)D concentration before and after supplementation. Third, the quasi-experimental design of our study is less robust than an RCT. Participants in the Comparator group did not receive vitamin D placebo, and there was no randomization. It should yet be noted that the characteristics of the two groups did not differ at baseline, which allows interpreting the survival difference as linked to the vitamin D3 supplementation.
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In conclusion, we were able to report among frail elderly residents that bolus vitamin D3 supplementation taken during or just before COVID-19 was associated with less severe 