key: cord-270049-54t3w94z authors: Campione, Elena; Lanna, Caterina; Cosio, Terenzio; Rosa, Luigi; Conte, Maria Pia; Iacovelli, Federico; Romeo, Alice; Falconi, Mattia; Del Vecchio, Claudia; Franchin, Elisa; Lia, Maria Stella; Minieri, Marilena; Chiaramonte, Carlo; Ciotti, Marco; Nuccetelli, Marzia; Terrinoni, Alessandro; Iannuzzi, Ilaria; Coppeda, Luca; Magrini, Andrea; Moricca, Nicola; Sabatini, Stefano; Rosapepe, Felice; Bartoletti, Pier Luigi; Bernardini, Sergio; Andreoni, Massimo; Valenti, Piera; Bianchi, Luca title: Pleiotropic effect of Lactoferrin in the prevention and treatment of COVID-19 infection: randomized clinical trial, in vitro and in silico preliminary evidences date: 2020-08-17 journal: bioRxiv DOI: 10.1101/2020.08.11.244996 sha: doc_id: 270049 cord_uid: 54t3w94z The current treatments against SARS-CoV-2 have proved so far inadequate. A potent antiviral drug is yet to be discovered. Lactoferrin, a multifunctional glycoprotein, secreted by exocrine glands and neutrophils, possesses an antiviral activity extendable to SARS-Cov-2. We performed a randomized, prospective, interventional study assessing the role of oral and intra-nasal lactoferrin to treat mild-to-moderate and asymptomatic COVID-19 patients to prevent disease evolution. Lactoferrin induced an early viral clearance and a fast clinical symptoms recovery in addition to a statistically significant reduction of D-Dimer, Interleukin-6 and ferritin blood levels. The antiviral activity of lactoferrin related to its binding to SARS-CoV-2 and cells and protein-protein docking methods, provided the direct recognition between lactoferrin and spike S, thus hindering the spike S attachment to the human ACE2 receptor and consequently virus entering into the cells. Lactoferrin can be used as a safe and efficacious natural agent to prevent and treat COVID-19 infection. In December 2019, in Whuan, China, a cluster of pneumonia cases was observed. This cluster was 46 related to a novel member of Betacoronavirus, named SARS-CoV-2, possessing more than 80% identity to SARS-CoV and 50% to the MERS-CoV 1,2 . Coronavirus are spherical, enveloped viruses Then, the efficacy of different concentrations of bLf in inhibiting SARS-CoV-2 infection was tested 161 on Vero E6 and Caco-2 cells according to different experimental procedures: i) control: untreated 162 SARS-CoV-2 and cells; ii) bLf pre-incubated with virus inoculum for 1 h at 37°C before cell 163 infection; iii) cells pre-incubated with bLf for 1 h at 37°C before virus infection; iv) bLf added 164 together with virus inoculum at the moment of infection step; v) virus and cells separately pre-165 incubated with bLf for 1 h at 37°C before infection. 166 The results obtained with Vero E6 cells are shown in Figure 3A (MOI 0.1) and 3B (MOI 0.01). CoV-2 infection (p < 0.001 and p < 0.001, respectively) ( Figure 3A and 3B). 171 On the contrary, the data illustrated in Figure 3A and 3B, independently from the MOI used, Regarding Caco-2 cells, at MOI 0.1, no significant differences were observed in all experimental 179 conditions compared to the control ones when using bLf at 100 µg/ml ( Figure 4A ). At MOI 0.01, an 180 inhibition of viral load in supernatants was observed at 24 hours post-infection (hpi) only when 100 181 µg/ml of bLf was pre-incubated with the viral inoculum and when the cells were pre-incubated with 182 100 µg/ml of bLf compared to the control one (p < 0.05) ( Figure 4B ). At 48 hpi, an inhibition of 183 viral load was observed only when the cells were pre-incubated with bLf (p < 0.05) ( Figure 4B ). When bLf was used at a concentration of 500 µg/ml, a decrease of viral load up to 48 hpi was 185 observed when the viral inoculum was pre-incubated with bLf compared to the control group, 186 independently from the MOI used (p < 0.05) ( Figure 4C, 4D) . When the cells were pre-incubated 187 with bLf, a decrease of viral load up to 24 hpi was observed compared to the control at MOI 0.1 (p 188 < 0.001 after 6 hpi and p < 0.05 after 24hpi) ( Figure 4C ), while at MOI 0.01 the decrease of viral 189 load remained statistically significant up to 48 hpi compared to the control group (p < 0.05) ( Figure 190 4D). When bLf was added together with SARS-CoV-2 inoculum during the adsorption step a 191 decrease of viral load up to 24 hpi was observed compared to untreated SARS-CoV-2 infection, 192 independently from the MOI used (p < 0.001 after 6 hpi and p < 0.05 after 24hpi for MOI 0.1; p < 193 0.05 after 6 and 24 hpi for MOI 0.01) ( Figure 4C, 4D) . When the cells were pre-incubated with bLf and infected with SARS-CoV-2 previously pre-incubated with bLf, a decrease of viral load up to 24 195 hpi was observed for MOI 0.1 compared to untreated SARS-CoV-2 infection (p < 0.001 after 6 hpi 196 and p < 0.05 after 24hpi for MOI 0.1) ( Figure 4C ), while at MOI 0.01 the decrease of viral load 197 remains statistically significant up to 48 hpi compared to untreated SARS-CoV-2 infection (p < 198 0.05) ( Figure 4D ). Computational results 200 The molecular docking simulation suggests a potential interaction of the bLf structure with the 201 spike glycoprotein CDT1 domain in the up conformation (Fig. 5A ). The first three solutions 202 obtained by Frodock clustering procedure account for more than 60% of the total generated (Table S2A , supplemental data). A detailed analysis of the interaction network reveals the presence of 28 different interactions, 213 which persist for more than 25% of the simulation time, in agreement with the high interaction 214 energy calculated. In detail, we found 3 salt bridges, 5 hydrogen bonds and 20 residue pairs 215 involved in hydrophobic contacts (Table S3 left side, supplemental data). To check if some of the Spike residues targeted by the bLf protein are involved in the binding with 217 ACE2, we have compared the average structure extracted from the simulation with the ACE2/CDT1 218 domain complex structure (PDB ID: 6LZG 33 Fig. 6) . Surprisingly, only two Spike residues (Gly502 219 and Tyr505) are shared between the complexes interfaces (Table S3 left side, supplemental data), as 220 evaluated from the inspection of the superimposed structures and from the paper analysis 33 . Despite 221 this, Lf holds the same position assumed by the ACE2 enzyme, i.e. above the up CDT1 domain. 222 We performed the same analysis over the evaluated human lactoferrin (hLF)-Spike complex, 223 obtaining a binding pose superimposable to that observed for the bovine protein (Fig. 5B) . Besides supplemental data), we observed that also for the hLf only two residues (Thr500 and Tyr505) are 233 shared between the complexes interfaces (Table S3 right side, supplemental data). These results allow us to hypothesize that, in addition to the HSPGs binding 12 , both bLf and hLf 235 should be able to hinder the spike glycoprotein attachment to the ACE2 receptor, consequently 236 blocking the virus from entering into the cells. The current treatment approaches to COVID-19 have so far proved to be inadequate, and a potent 240 antiviral drug or effective vaccine are yet to be discovered and eagerly awaited The immediate 241 priority is to harness innate immunity in order to accelerate early antiviral immune responses. to-moderate disease and in COVID-19 asymptomatic patients. 256 We focused our research on asymptomatic and mild-to-moderate COVID-19 patients, considering 257 them a transmission reservoir with possible evolution to the most severe disease form 36 . Li et al, 258 analyzing the viral shedding dynamics in asymptomatic and mildly symptomatic patients infected 259 with SARS-CoV-2, observed a long-term viral shedding, also in the convalescent phase of the disease, where specific antibody production to SARS-CoV-2 may not guarantee viral clearance 261 after hospital discharge. In their study, the median duration of viral shedding appeared to be shorter 262 in pre-symptomatic patients (11.5 days) than in asymptomatic (28 days) and mild symptomatic 263 cases (31 days) 37 . In our study, Lf induced an early viral clearance just after 15 days from the 264 beginning of the treatment in 31% of patients, and after 30 days of treatment in the rest of our Liver function is known to be deranged in COVID-19 and a meta-analysis showed that 16% and 308 20% of patients with COVID-19 had ALT and AST levels higher than the normal range 51 . Liver and SARS-CoV-2 were previously pre-incubated with bLf ( Figure 4D ). Our experimental results indicate that bLf exerts its antiviral activity either by direct attachment to Taken together these results reveal that, even if the definitive mechanism of action still has to be 380 explored, the antiviral properties of Lf are also extendable to SARS-CoV-2 virus. Considering the risk of COVID19 relapse 80 , we also suggest additional long-term studies to 388 evaluate the maintenance of viral clearance with Lf continuous administration. Finally, due to ethical reasons, we could not include placebo arms in our study and therefore we 390 could not evaluate properly the different disease evolution in treated and not-treated patients. However, considering the reported natural disease course 37 we can state Lf induced an early RT-392 PCR negative conversion and a fast clinical symptoms recovery. This study is part of the GEFACOVID2.0 research program coordinated by the Tor Vergata 394 University of Rome. Clinical trial 397 We performed a randomized, prospective, interventional study to assess the efficacy of a liposomal Blood parameters obtained at T0 in COVID-19 group and control group were compared using t-test. Data were then analyzed with a significant two-tailed p-value <= 0.05. 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