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

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.

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

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

Neutralizing antibodies (nAbs) that target the SARS-CoV-2 spike protein are approved for treatment of COVID-19. However, with the emergence of variants of concern, there is a need for new treatment options. We report a novel format that enables modular assembly of bi-paratopic, tetravalent nAbs with antigen-binding sites from two distinct nAbs. The tetravalent nAb was purified in high yield, and it exhibited biophysical characteristics that were comparable to those of approved therapeutic antibodies. The tetravalent nAb bound to the spike protein trimer at least 100-fold more tightly than bivalent IgGs (apparent KD < 1 pM), and it exhibited extremely high potencies against a broad array of pseudoviruses, chimeric viruses, and authentic virus variants. Together, these results establish the tetravalent diabody-Fc-Fab as a robust, modular platform for rapid production of drug-grade nAbs with potencies and breadth of coverage that greatly exceed those of conventional bivalent IgGs.

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

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

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 mild to moderate COVID-19.

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

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 SummaryThe 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.

SARS-CoV-2 B.1.617 Indian variants: are electrostatic potential changes responsible for a higher transmission rate?

Lineage B.1.617+, also known as G/452R.V3, is a recently described SARS-CoV-2 variant under investigation (VUI) firstly identified in October 2020 in India. As of May 2021, three sublineages labelled as B.1.617.1, B.1.617.2 and B.1.617.3 have been already identified, and their potential impact on the current pandemic is being studied. This variant has 13 amino acid changes, three in its spike protein, which are currently of particular concern: E484Q, L452R and P681R. Here we report a major effect of the mutations characterizing this lineage, represented by a marked alteration of the surface electrostatic potential (EP) of the Receptor Binding Domain (RBD) of the spike protein. Enhanced RBD-EP is particularly noticeable in the B.1.617.2 sublineage, which shows multiple replacements of neutral or negatively-charged amino acids with positively-charged amino acids. We here hypothesize that this EP change can favor the interaction between the B.1.617+RBD and the negatively-charged ACE2 thus conferring a potential increase in the virus transmission.

SARS-CoV-2 shifting transmission dynamics and hidden reservoirs limited the efficacy of public health interventions in Italy

We investigated SARS-CoV-2 transmission dynamics in Italy, one of the countries hit hardest by the pandemic, using phylodynamic analysis of viral genetic and epidemiological data. We observed the co-circulation of at least 13 different SARS-CoV-2 lineages over time, which were linked to multiple importations and characterized by large transmission clusters concomitant with a high number of infections. Subsequent implementation of a three-phase nationwide lockdown strategy greatly reduced infection numbers and hospitalizations. Yet we present evidence of sustained viral spread among sporadic clusters acting as "hidden reservoirs" during summer 2020. Mathematical modelling shows that increased mobility among residents eventually catalyzed the coalescence of such clusters, thus driving up the number of infections and initiating a new epidemic wave. Our results suggest that the efficacy of public health interventions is, ultimately, limited by the size and structure of epidemic reservoirs, which may warrant prioritization during vaccine deployment.

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

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.

Steroid-responsive severe encephalopathy in SARS-CoV-2 infection

Covid-19 infection has the potential for targeting the central nervous system and several neurological symptoms have been described in patients with severe respiratory distress. Here we described the case of a 60-year old subject with SARS-CoV-2 infection but only mild respiratory abnormalities who developed an akinetic mutism due to encephalitis. MRI was negative whereas EEG showed generalized theta slowing. CSF analyses during the acute stage were negative for SARS-CoV-2, positive for pleocytosis and hyperproteinorrachia, and showed increased IL-8 and TNF- concentrations while other infectious or autoimmune disorders were excluded. A progressive clinical improvement along with a reduction of CSF parameters was observed after high-dose steroid treatment, thus arguing for an inflammatory-mediated brain involvement related to Covid-19.

Mesenchymal stromal cells primed with paclitaxel provide a new approach for cancer therapy.

BACKGROUND: Mesenchymal stromal cells may represent an ideal candidate to deliver anti-cancer drugs. In a previous study, we demonstrated that exposure of mouse bone marrow derived stromal cells to Doxorubicin led them to acquire anti-proliferative potential towards co-cultured haematopoietic stem cells (HSCs). We thus hypothesized whether freshly isolated human bone marrow Mesenchymal stem cells (hMSCs) and mature murine stromal cells (SR4987 line) primed in vitro with anti-cancer drugs and then localized near cancer cells, could inhibit proliferation. METHODS AND PRINCIPAL FINDINGS: Paclitaxel (PTX) was used to prime culture of hMSCs and SR4987. Incorporation of PTX into hMSCs was studied by using FICT-labelled-PTX and analyzed by FACS and confocal microscopy. Release of PTX in culture medium by PTX primed hMSCs (hMSCsPTX) was investigated by HPLC. Culture of Endothelial cells (ECs) and aorta ring assay were used to test the anti-angiogenic activity of hMSCsPTX and PTX primed SR4987(SR4987PTX), while anti-tumor activity was tested in vitro on the proliferation of different tumor cell lines and in vivo by co-transplanting hMSCsPTX and SR4987PTX with cancer cells in mice. Nevertheless, despite a loss of cells due to chemo-induced apoptosis, both hMSCs and SR4987 were able to rapidly incorporate PTX and could slowly release PTX in the culture medium in a time dependent manner. PTX primed cells acquired a potent anti-tumor and anti-angiogenic activity in vitro that was dose dependent, and demonstrable by using their conditioned medium or by co-culture assay. Finally, hMSCsPTX and SR4987PTX co-injected with human cancer cells (DU145 and U87MG) and mouse melanoma cells (B16) in immunodeficient and in syngenic mice significantly delayed tumor takes and reduced tumor growth. CONCLUSIONS: These data demonstrate, for the first time, that without any genetic manipulation, mesenchymal stromal cells can uptake and subsequently slowly release PTX. This may lead to potential new tools to increase efficacy of cancer therapy.