DHFR Inhibitors Display a Pleiotropic Anti-viral Activity Against SARS-CoV-2: Insights Into the Mechanisms of Action (preprint)

During COVID-19 pandemic, drug repurposing represented an effective strategy to obtain quick answers to medical emergencies. Basing on previous data on Methotrexate (MTX), we evaluated the anti-viral activity of several DHFR inhibitors in two cell lines. We observed that this class of compounds showed a significant influence on the virus-induced cytopathic effect (CPE) partly associated to the intrinsic antimetabolic activity of these drugs, but also to a specific antiviral function. To elucidate the molecular mechanisms, we took advantage of our EXSCALATE platform for in-silico molecular modelling and further validated the influence of these inhibitors on nsp13 and viral entry. Interestingly, Pralatrexate and Trimetrexate showed superior effects in counteracting the viral infection compared to other DHFR inhibitors. Our results indicate that their higher activity is due to their polypharmacological and pleiotropic profile. These compounds can thus potentially give a clinical advantage in the management of SARS-CoV-2 infection in patients already treated with this class of drugs.

Genetic and structural data on the SARS-CoV-2 Omicron BQ.1 variant reveal its low potential for epidemiological expansion (preprint)

The BQ.1 SARS-CoV-2 variant, also known as Cerberus, is one of the most recent Omicron descendant lineages. Compared to its direct progenitor BA.5, BQ.1 carries out some additional spike mutations in some key antigenic site which confer it further immune escape ability over other circulating lineage. In such a context, here we performed a genome-based survey aimed to obtain an as complete as possible nuance of this rapidly evolving Omicron subvariant. Genetic data suggests that BQ.1 represents an evolutionary blind background, lacking of the rapid diversification which is typical of a dangerous lineage. Indeed, the evolutionary rate of BQ.1 is very similar to that of BA.5 (7.6 x 10-4 and 7 x 10-4 subs/site/year, respectively), which is circulating by several months. Bayesian Skyline Plot reconstruction, indicates low level of genetic variability, suggesting that the peak has been reached around September 3, 2022. Structure analyses performed by comparing the properties of BQ.1 and BA.5 RBD indicated that the impact of the BQ.1 mutations on the affinity for ACE2 may be modest. Likewise, immunoinformatic analyses showed modest differences between the BQ.1 and the BA5 potential B-cells epitope. In conclusion, genetic and structural analysis on SARS-CoV-2 BQ.1 suggest that, it does not show evidence about its particular dangerous or high expansion capability. The monitoring genome-based must continue uninterrupted for a better understanding of its descendant and all other lineages.

Potential Benefit of Early Treatment with Sotrovimab in Patients With High Risk for Severe COVID-19 Carrying BA.2 Infection (preprint)

Coronavirus disease 19 (COVID-19) continues to spread worldwide as a severe pandemic. The Omicron BA.2 became the predominant variant and the protagonist of the ongoing surge. As the virus continues to mutate, using of approved drugs or developing new therapeutic or prophylactic therapies against COVID-19 could be more complex. Sotrovimab is a monoclonal antibody (mAb) targeting the conserved epitope on the spike protein receptor; the most recent studies observed that it has substantially decreased in vitro activity against the Omicron BA.2 subvariant, but real-life data are still scarce. We describe the outcome of a case series of outpatients with BA.1 or BA.2 infection treated with sotrovimab. We conducted a retrospective observational study including all non-hospitalized adult patients treated with sotrovimab, for which a Sanger sequencing of SARS-CoV-2 was performed within a regional genomic surveillance program. Eleven (50%) patients with BA.1 infection and eleven (50%) with BA.2 infection were considered. Most patients were immunocompromised. During the follow-up period, no patient died and only one with BA.1 infection was hospitalized for severe COVID-19 pneumonia onset. One month after treatment, 90.9% of patients were completely asymptomatic in each group. We demonstrated that patients carrying the BA.2 variant treated with sotrovimab did not evolve to severe COVID-19, showing a similar outcome to BA.1 infected patients. Further studies are needed to prove that vaccination or the presumably high doses of mAbs used can protect this group of patients at high risk of progression.

Peptide-antibody Fusions Engineered by Phage Display Exhibit Ultrapotent and Broad Neutralization of SARS-CoV-2 Variants (preprint)

The COVID-19 pandemic has been exacerbated by the emergence of variants of concern (VoCs). Many VoC mutations are found in the viral spike protein (S-protein), and are thus implicated in host infection and response to therapeutics. Bivalent neutralizing antibodies (nAbs) targeting the S-protein receptor-binding domain (RBD) are promising therapeutics for COVID-19, but are limited due to low potency and vulnerability to RBD mutations found in VoCs. To address these issues, we used naive phage-displayed peptide libraries to isolate and optimize 16-residue peptides that bind to the RBD or the N-terminal domain (NTD) of the S-protein. We fused these peptides to the N-terminus of a moderate affinity nAb to generate tetravalent peptide-IgG fusions, and showed that both classes of peptides were able to improve affinities for the S-protein trimer by >100-fold (apparent KD <1 pM). Critically, cell-based infection assays with a panel of six SARS-CoV-2 variants demonstrate that an RBD-binding peptide was able to enhance the neutralization potency of a high-affinity nAb >100-fold. Moreover, this peptide-IgG was able to neutralize variants that were resistant to the same nAb in the bivalent IgG format. To show that this approach is general, we fused the same peptide to a clinically approved nAb drug, and showed that it rescued neutralization against a resistant variant. Taken together, these results establish minimal peptide fusions as a modular means to greatly enhance affinities, potencies, and breadth of coverage of nAbs as therapeutics for SARS-CoV-2.

Ultrapotent and Broad Neutralization of SARS-CoV-2 Variants by Modular, Tetravalent, Bi-paratopic Antibodies (preprint)

Neutralizing antibodies (nAbs) that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (S-protein) are promising therapeutics for COVID-19. However, natural bivalent nAbs suffer from limited potency and are vulnerable to SARS-CoV-2 variants with mutated RBDs. We report a novel format that enables modular assembly of bi-paratopic, tetravalent nAbs with antigen-binding sites from two distinct nAbs. The diabody-Fc-Fab format consists of a central Fc with a bivalent diabody fused to its N-terminus and two Fabs fused to its C-terminus. The diabody and Fab modules do not interfere with each other, and thus, any diabody can be combined with any Fab in a facile manner. We engineered a diabody-Fc-Fab that contained the paratopes of two distinct nAbs derived from a phage-displayed library of synthetic Abs. The tetravalent nAb was purified in high yields with methods used to produce conventional IgGs, and it exhibited favorable biophysical characteristics comparable to those of approved therapeutic antibodies. The tetravalent nAb bound to the S-protein trimer at least 100-fold more tightly than the bivalent IgGs (apparent KD <1 pM). Most importantly, the tetravalent nAb exhibited extremely high potencies in neutralization assays across a panel of pseudoviruses representing seven natural SARS-CoV-2 variants (IC50 <5 ng/mL), including several that resisted IgGs and are known to evade approved IgG drugs. Taken together, our results showed that the tetravalent diabody-Fc-Fab is a robust, modular platform for rapid production of drug-grade nAbs with potencies and breadth of coverage that far exceed those of conventional bivalent IgGs.

Phylogenetic analysis and in silico studies link spike Q675H mutation to SARS-CoV-2 adaptive evolution (preprint)

Genotype screening was implemented in Italy and showed a significant prevalence of new SARS-CoV-2 mutants carrying Q675H mutation, near the furin cleavage site of spike protein. Currently, this mutation, which is expressed on different SARS-CoV-2 lineages circulating worldwide, has not been thoughtfully investigated. Therefore, we performed phylogenetic and biocomputational analysis to better understand SARS-CoV-2 Q675H mutants evolutionary relationships with other circulating lineages and Q675H function in its molecular context. Our studies reveal that Q675H spike mutation is the result of parallel evolution because it arose independently in separate evolutionary clades. In silico data show that the Q675H mutation gives rise to a hydrogen-bonds network in the spike polar region delimiting the conformational space of the highly flexible loop containing the furin cleavage site. This results in an optimized directionality of arginine residues involved in interaction of spike with the furin binding pocket, thus improving proteolytic exposure of the viral protein. Furin was found to have a greater affinity for Q675H than Q675 substrate conformations. As a consequence, Q675H mutation is likely to confer a fitness advantage to SARS-CoV-2 by promoting a more efficient viral entry. Interestingly, here we show an ongoing increase in the occurrence of Q675H spike mutation in the most common SARS-CoV-2 variants of concern (VOC). This finding highlights that, VOC are still evolving and start acquiring the Q675H mutation. At the same time, it suggests that our hypothesis of fitness advantage prompted by Q675H could be concrete.

Characterization of raloxifene as potential pharmacological agent against SARS-CoV-2 and its variants (preprint)

The new coronavirus that emerged, called SARS-CoV-2, is the causative agent of the COVID-19 pandemic. The identification of potential drug candidates that can rapidly enter clinical trials for the prevention and treatment of COVID-19 is an urgent need, despite the recent introduction of several new vaccines for the prevention and protection of this infectious disease which in many cases becomes severe. Drug repurposing (DR), a process for studying existing pharmaceutical products for new therapeutic indications, represents one of the most effective potential strategies employed to increase the success rate in the development of new drug therapies. We identified raloxifene, a known Selective Estrogen Receptor modulator (SERM), as a potential pharmacological agent for the treatment of COVID-19 patients. Following a virtual screening campaign on the most relevant viral protein targets, in this work we report the results of the first pharmacological characterization of raloxifene in relevant cellular models of COVID-19 infection. The results obtained on all the most common viral variants originating in Europe, United Kingdom, Brazil, South Africa and India, currently in circulation, are also reported, confirming the efficacy of raloxifene and, consequently, the relevance of the proposed approach. Taken together, all the information gathered supports the clinical development of raloxifene and confirms that the drug can be proposed as a viable new option to fight the pandemic in at least some patient populations. The results obtained so far have paved the way for a first clinical study to test the safety and efficacy of raloxifene, just concluded in patients with COVID-19 paucisymptomatic.

Doxycycline inhibition of a pseudotyped virus transduction does not translate to inhibition of SARS-CoV-2 infectivity (preprint)

The pandemic caused by the SARS-CoV-2 has created the need of compounds able to interfere with the biological processes exploited by the virus. Doxycycline, with its pleiotropic effects, including anti-viral activity, has been proposed as a therapeutic candidate for COVID-19 and about twenty clinical trials have started since the beginning of the pandemic. To gain information on the activity of doxycycline against SARS-CoV-2 infection and clarify some of the conflicting clinical data published, we designed in vitro binding tests and infection studies with a pseudotyped virus expressing the spike protein, as well as a clinically isolated SARS-CoV-2 strain. Doxycycline inhibited the transduction of the pseudotyped virus in Vero E6 and HEK-293 T cells stably expressing human receptor angiotensin-converting enzyme 2 but did not affect the entry and replication of SARS-CoV-2. Although this conclusion is apparently disappointing, it is paradigmatic of an experimental approach aimed at developing an integrated multidisciplinary platform. To avoid wasting precious time and resources we believe very stringent experimental criteria are needed in the preclinical phase, including infectious studies with SARS-CoV-2 in the platform before moving on to [failed] clinical trials. Author Summary The pandemic caused by the SARS-CoV-2 virus has created a completely unusual situation in rapidly searching for compounds able to interfere with the biological processes exploited by the virus. This new scenario has substantially changed the timing of drug development which has also resulted in the generation of controversial results, proving that the transition from computational screening to the clinical application requires great caution and careful studies. It is therefore necessary to establish new paradigms for evaluating the efficacy of a potential active molecule. We set up a preclinical platform aimed at identifying molecules active against SARS-CoV-2 infection developing a multidisciplinary approach based on very stringent experimental criteria, comprising in-silico studies, in vitro binding tests and infection studies with pseudovirus expressing the spike protein as well as clinically isolated SARS-CoV-2 strains. We focused our attention on doxycycline which has been suggested as potential therapeutic candidate for treating COVID-19 and is currently employed in about twenty clinical trials. Doxycycline resulted effective in inhibiting the transduction of pseudovirus but it did not affect the entry and replication of SARS-CoV-2. The results obtained underline the need to define more stringent and controlled pharmacological approaches before wasting precious time and resources with clinical trials.

Molecular Tracing of SARS-CoV-2 in Italy in the First Three Months of the Epidemic (preprint)

The aim of this study is the characterization and genomic tracing by phylogenetic analyses of 59 new SARS-CoV-2 Italian isolates obtained from patients attending clinical centres in North and Central Italy until the end of April 2020. All but one of the newly characterized genomes belonged to the lineage B.1, the most frequently identified in European countries, including Italy. Only a single sequence was found to belong to lineage B. A mean of 6 nucleotide substitutions per viral genome was observed, without significant differences between synonymous and non-synonymous mutations, indicating genetic drift as a major source for virus evolution. tMRCA estimation confirmed the probable origin of the epidemic between the end of January and the beginning of February with a rapid increase in the number of infections between the end of February and mid-March. Since early February, an effective reproduction number (Re) greater than 1 was estimated, which then increased reaching the peak of 2.3 in early March, confirming the circulation of the virus before the first COVID-19 cases were documented. Continuous use of state-of-the-art methods for molecular surveillance is warranted to trace virus circulation and evolution and inform effective prevention and containment of future SARS-CoV-2 outbreaks.

Molecular tracing of SARS-CoV-2 in Italy in the first three months of the epidemic (preprint)

The aim of this study is the characterization and genomic tracing by phylogenetic analyses of 59 new SARS-CoV-2 Italian isolates obtained from patients attending clinical centres in North and Central Italy until the end of April 2020. All but one of the newly characterized genomes belonged to the lineage B.1, the most frequently identified in European countries, including Italy. Only a single sequence was found to belong to lineage B. A mean of 6 nucleotide substitutions per viral genome was observed, without significant differences between synonymous and non-synonymous mutations, indicating genetic drift as a major source for virus evolution. tMRCA estimation confirmed the probable origin of the epidemic between the end of January and the beginning of February with a rapid increase in the number of infections between the end of February and mid-March. Since early February, an effective reproduction number (Re) greater than 1 was estimated, which then increased reaching the peak of 2.3 in early March, confirming the circulation of the virus before the first COVID-19 cases were documented. Continuous use of state-of-the-art methods for molecular surveillance is warranted to trace virus circulation and evolution and inform effective prevention and containment of future SARS-CoV-2 outbreaks.

The clinical spectrum of encephalitis in COVID-19 disease: the ENCOVID multicentre study (preprint)

Background: Several preclinical and clinical investigations have argued for nervous system involvement in SARS-CoV-2 infection. Some sparse case reports have described various forms of encephalitis in COVID-19 disease, but very few data have focused on clinical presentations, clinical course, response to treatment and outcomes yet. Objective: to describe the clinical phenotype, laboratory and neuroimaging findings of encephalitis associated with SARS-CoV-2 infection, their relationship with respiratory function and inflammatory parameters and their clinical course and response to treatment. Design: The ENCOVID multicentre study was carried out in 13 centres in northern Italy between February 20th and May 31st, 2020. Only patients with altered mental status and at least two supportive criteria for encephalitis with full infectious screening, CSF, EEG, MRI data and a confirmed diagnosis of SARS-CoV-2 infection were included. Clinical presentation and laboratory markers, severity of COVID-19 disease, response to treatment and outcomes were recorded. Results: Out of 45 cases screened, twenty-five cases of encephalitis positive for SARS-CoV-2 infection with full available data were included. The most common symptoms at onset were delirium (68%), aphasia/dysarthria (24%) and seizures (24%). CSF showed hyperproteinorrachia and/or pleocytosis in 68% of cases whereas SARS-CoV-2 RNA by RT-PCR resulted negative. Based on MRI, cases were classified as ADEM (n=3), limbic encephalitis (LE, n=2), encephalitis with normal imaging (n=13) and encephalitis with MRI alterations (n=7). ADEM and LE cases showed a delayed onset compared to the other encephalitis (p=0.001) and were associated with previous more severe COVID-19 respiratory involvement. Patients with MRI alterations exhibited worse response to treatment and final outcomes compared to other encephalitis. Conclusions and relevance: We found a wide clinical spectrum of encephalitis associated with COVID19 infection, underlying different pathophysiological mechanisms. Response to treatment and final outcome strongly depended on specific CNS-manifestations.