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1.
Int J Mol Sci ; 23(10)2022 May 16.
Article in English | MEDLINE | ID: covidwho-1855647

ABSTRACT

The dramatic experience with SARS-CoV-2 has alerted the scientific community to be ready to face new epidemics/pandemics caused by new variants. Among the therapies against the pandemic SARS-CoV-2 virus, monoclonal Antibodies (mAbs) targeting the Spike glycoprotein have represented good drugs to interfere in the Spike/ Angiotensin Converting Enzyme-2 (ACE-2) interaction, preventing virus cell entry and subsequent infection, especially in patients with a defective immune system. We obtained, by an innovative phage display selection strategy, specific binders recognizing different epitopes of Spike. The novel human antibodies specifically bind to Spike-Receptor Binding Domain (RBD) in a nanomolar range and interfere in the interaction of Spike with the ACE-2 receptor. We report here that one of these mAbs, named D3, shows neutralizing activity for virus infection in cell cultures by different SARS-CoV-2 variants and retains the ability to recognize the Omicron-derived recombinant RBD differently from the antibodies Casirivimab or Imdevimab. Since anti-Spike mAbs, used individually, might be unable to block the virus cell entry especially in the case of resistant variants, we investigated the possibility to combine D3 with the antibody in clinical use Sotrovimab, and we found that they recognize distinct epitopes and show additive inhibitory effects on the interaction of Omicron-RBD with ACE-2 receptor. Thus, we propose to exploit these mAbs in combinatorial treatments to enhance their potential for both diagnostic and therapeutic applications in the current and future pandemic waves of coronavirus.


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Monoclonal , Antibodies, Monoclonal, Humanized , Antibodies, Neutralizing , Antibodies, Viral , COVID-19/drug therapy , Epitopes , Humans , Spike Glycoprotein, Coronavirus/chemistry , Viral Envelope Proteins/chemistry
2.
Genet Med ; 2022 May 05.
Article in English | MEDLINE | ID: covidwho-1819495

ABSTRACT

PURPOSE: Emerging evidence suggest that infection-dependent hyperactivation of complement system (CS) may worsen COVID-19 outcome. We investigated the role of predicted high impact rare variants - referred as qualifying variants (QVs) - of CS genes in predisposing asymptomatic COVID-19 in elderly individuals, known to be more susceptible to severe disease. METHODS: Exploiting exome sequencing data and 56 CS genes, we performed a gene-based collapsing test between 164 asymptomatic subjects (aged ≥60 years) and 56,885 European individuals from the Genome Aggregation Database. We replicated this test comparing the same asymptomatic individuals with 147 hospitalized patients with COVID-19. RESULTS: We found an enrichment of QVs in 3 genes (MASP1, COLEC11, and COLEC10), which belong to the lectin pathway, in the asymptomatic cohort. Analyses of complement activity in serum showed decreased activity of lectin pathway in asymptomatic individuals with QVs. Finally, we found allelic variants associated with asymptomatic COVID-19 phenotype and with a decreased expression of MASP1, COLEC11, and COLEC10 in lung tissue. CONCLUSION: This study suggests that genetic rare variants can protect from severe COVID-19 by mitigating the activity of lectin pathway and prothrombin. The genetic data obtained through ES of 786 asymptomatic and 147 hospitalized individuals are publicly available at http://espocovid.ceinge.unina.it/.

3.
Int J Mol Sci ; 23(8)2022 Apr 09.
Article in English | MEDLINE | ID: covidwho-1785747

ABSTRACT

Tracing the appearance and evolution of virus variants is essential in the management of the COVID-19 pandemic. Here, we focus on SARS-CoV-2 spread in Italian patients by using viral sequences deposited in public databases and a tracing procedure which is used to monitor the evolution of the pandemic and detect the spreading, within the infected population of emergent sub-clades with a potential positive selection. Analyses of a collection of monthly samples focused on Italy highlighted the appearance and evolution of all the main viral sub-trees emerging at the end of the first year of the pandemic. It also identified additional expanding subpopulations which spread during the second year (i.e., 2021). Three-dimensional (3D) modelling of the main amino acid changes in mutated viral proteins, including ORF1ab (nsp3, nsp4, 2'-o-ribose methyltransferase, nsp6, helicase, nsp12 [RdRp]), N, ORF3a, ORF8, and spike proteins, shows the potential of the analysed structural variations to result in epistatic modulation and positive/negative selection pressure. These analyzes will be of importance to the early identification of emerging clades, which can develop into new "variants of concern" (i.e., VOC). These analyses and settings will also help SARS-CoV-2 coronet genomic centers in other countries to trace emerging worldwide variants.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/epidemiology , Humans , Mutation , Pandemics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/metabolism
4.
Int J Mol Sci ; 23(4)2022 Feb 09.
Article in English | MEDLINE | ID: covidwho-1690219

ABSTRACT

The development of prophylactic agents against the SARS-CoV-2 virus is a public health priority in the search for new surrogate markers of active virus replication. Early detection markers are needed to follow disease progression and foresee patient negativization. Subgenomic RNA transcripts (with a focus on sgN) were evaluated in oro/nasopharyngeal swabs from COVID-19-affected patients with an analysis of 315 positive samples using qPCR technology. Cut-off Cq values for sgN (Cq < 33.15) and sgE (Cq < 34.06) showed correlations to high viral loads. The specific loss of sgN in home-isolated and hospitalized COVID-19-positive patients indicated negativization of patient condition, 3-7 days from the first swab, respectively. A new detection kit for sgN, gene E, gene ORF1ab, and gene RNAse P was developed recently. In addition, in vitro studies have shown that 2'-O-methyl antisense RNA (related to the sgN sequence) can impair SARS-CoV-2 N protein synthesis, viral replication, and syncytia formation in human cells (i.e., HEK-293T cells overexpressing ACE2) upon infection with VOC Alpha (B.1.1.7)-SARS-CoV-2 variant, defining the use that this procedure might have for future therapeutic actions against SARS-CoV-2.


Subject(s)
COVID-19/virology , Coronavirus Nucleocapsid Proteins/genetics , SARS-CoV-2/physiology , Virus Replication/physiology , Coronavirus Nucleocapsid Proteins/analysis , Giant Cells/drug effects , Giant Cells/virology , HEK293 Cells , Humans , Limit of Detection , Nasopharynx/virology , Phosphoproteins/analysis , Phosphoproteins/genetics , RNA, Antisense/pharmacology , RNA, Viral , Ribonuclease P/genetics , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , Sensitivity and Specificity , Social Isolation , Viral Load , Viroporin Proteins/genetics , Virus Replication/drug effects
5.
Molecules ; 27(3)2022 Jan 27.
Article in English | MEDLINE | ID: covidwho-1674736

ABSTRACT

Butyrate is a major gut microbiome metabolite that regulates several defense mechanisms against infectious diseases. Alterations in the gut microbiome, leading to reduced butyrate production, have been reported in patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. A new butyrate releaser, useful for all the known applications of butyrate, presenting physiochemical characteristics suitable for easy oral administration, (N-(1-carbamoyl-2-phenyl-ethyl) butyramide (FBA), has been recently developed. We investigated the protective action of FBA against SARS-CoV-2 infection in the human small intestine and enterocytes. Relevant aspects of SARS-CoV-2 infection were assessed: infectivity, host functional receptor angiotensin-converting enzyme-2 (ACE2), transmembrane protease serine 2 (TMPRSS2), neuropilin-1 (NRP1), pro-inflammatory cytokines expression, genes involved in the antiviral response and the activation of Nf-kB nuclear factor (erythroid-derived 2-like) 2 (Nfr2) pathways. We found that FBA positively modulates the crucial aspects of the infection in small intestinal biopsies and human enterocytes, reducing the expression of ACE2, TMPRSS2 and NRP1, pro-inflammatory cytokines interleukin (IL)-15, monocyte chemoattractant protein-1 (MCP-1) and TNF-α, and regulating several genes involved in antiviral pathways. FBA was also able to reduce the number of SARS-CoV-2-infected cells, and ACE2, TMPRSS2 and NRP1 expression. Lastly, through the inhibition of Nf-kB and the up-regulation of Nfr2, it was also able to reduce the expression of pro-inflammatory cytokines IL-15, MCP-1 and TNF-α in human enterocytes. The new butyrate releaser, FBA, exerts a preventive action against SARS-CoV-2 infection. It could be considered as an innovative strategy to limit COVID-19.


Subject(s)
Butyrates/pharmacology , COVID-19/drug therapy , SARS-CoV-2/metabolism , Antiviral Agents/pharmacology , Butyrates/metabolism , COVID-19/metabolism , Caco-2 Cells , Enterocytes/drug effects , Enterocytes/metabolism , Gene Expression/genetics , Gene Expression Regulation/genetics , Humans , Intestines/drug effects , Intestines/metabolism , Male , SARS-CoV-2/drug effects , SARS-CoV-2/pathogenicity
6.
Front Public Health ; 9: 801609, 2021.
Article in English | MEDLINE | ID: covidwho-1604379

ABSTRACT

As of November 17, 2021, SARS-CoV-2 (Severe Acute Respiratory Syndrome CoronaVirus 2), the causative agent of COVID-19 (COronaVIrus Disease 19), has infected ~250 million people worldwide, causing around five million deaths. Titers of anti-SARS-CoV-2 neutralizing antibodies were relatively stable for at least 9 months in a population-based study conducted in Wuhan, China, both in symptomatic and in asymptomatic individuals. In the mass screening campaign conducted in the town of Ariano Irpino (Avellino, Italy) in May, 2020, 5.7% (95% CI: 5.3-6-1) of the 13,444 asymptomatic citizens screened were positive for anti-nucleocapsid antibodies against SARS-CoV-2. Among these, 422 citizens were re-tested for anti SARS-CoV-2 antibodies in January, 2021 and/or in April, 2021 and enrolled in this longitudinal observational study. Median (interquartile range) age of the study cohort was 46 years (29-59), with 47 (11.1%) participants of minor age, while 217 (51.4%) participants were females. There was no evidence of re-infection in any of the subjects included. Presence of anti-nuclear antibodies antibodies (Elecysis, Roche) was reported in 95.7 and 93.7% of evaluable participants in January and April, 2021. Multiple logistic regression analysis used to explore associations between age, sex and seroprevalence showed that adults vs. minors had significantly lower odds of having anti-S1 antibodies (Biorad) both in January, 2021 and in April, 2021. Our findings showed that antibodies remained detectable at least 11.5 months after infection in >90% of never symptomatic cases. Further investigation is required to establish duration of immunity against SARS-CoV-2.


Subject(s)
COVID-19 , SARS-CoV-2 , Adult , Female , Follow-Up Studies , Humans , Immunity, Humoral , Middle Aged , Seroepidemiologic Studies
7.
J Funct Foods ; 87: 104787, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1607061

ABSTRACT

Fermented foods have been proposed in limiting SARS-CoV-2 infection. Emerging evidence suggest the efficacy of cow's milk fermented with the probiotic L. paracasei CBAL74 (FM-CBAL74) in preventing infectious diseases. We evaluated the protective action of FM-CBAL74 against SARS-CoV-2 infection in human enterocytes. Relevant aspects of SARS-CoV-2 infection were assessed: infectivity, host functional receptor angiotensin-converting enzyme-2 (ACE2), transmembrane protease serine 2 (TMPRSS2), and pro-inflammatory cytokines expression (IL-6, IL-15, IL-1ß, VEGFß, TNF-α, MCP-1, CXCL1). Pre-incubation with FM-CBA L74 reduced the number of infected cells. The expression of ACE2 and the pro-inflammatory cytokines IL-6, VEGFß, IL-15, IL-1ß was downregulated by the pre-treatment with this fermented food. No effect on TMPRSS2, MCP-1, TNF-α and CXCL1 expression was observed. Modulating the crucial aspects of the infection, the fermented food FM-CBAL74 exerts a preventive action against SARS-CoV-2. These evidence could pave the way to innovative nutritional strategy to mitigate the COVID-19.

8.
ACS Omega ; 6(50): 34945-34953, 2021 Dec 21.
Article in English | MEDLINE | ID: covidwho-1586043

ABSTRACT

Numerous reverse transcription polymerase chain reaction (RT-PCR) tests have emerged over the past year as the gold standard for detecting millions of cases of SARS-CoV-2 reported daily worldwide. However, problems with critical shortages of key reagents such as PCR primers and RNA extraction kits and unpredictable test reliability related to high viral replication cycles have triggered the need for alternative methodologies to PCR to detect specific COVID-19 proteins. Several authors have developed methods based on liquid chromatography with tandem mass spectrometry (LC-MS/MS) to confirm the potential of the technique to detect two major proteins, the spike and the nucleoprotein, of COVID-19. In the present work, an S-Trap mini spin column digestion protocol was used for sample preparation prodromal to LC-MS/MS analysis in multiple reactions monitoring ion mode (MRM) to obtain a comprehensive method capable of detecting different viral proteins. The developed method was applied to n. 81 oro/nasopharyngeal swabs submitted in parallel to quantitative reverse transcription PCR (RT-qPCR) assays to detect RdRP, the S and N genes specific for COVID-19, and the E gene for all Sarbecoviruses, including SARS-CoV-2 (with cycle negativity threshold set to 40). A total of 23 peptides representative of the six specific viral proteins were detected in the monitoring of 128 transitions found to have good ionic currents extracted in clinical samples that reacted differently to the PCR assay. The best instrumental response came from the FLPFQFGR sequence of spike [558-566] peptide used to test the analytical performance of the method that has good sensitivity with a low false-negative rate. Transition monitoring using a targeted MS approach has the great potential to detect the fragmentation reactions of any peptide molecularly defined by a specific amino acid sequence, offering the extensibility of the approach to any viral sequence including derived variants and thus providing insights into the development of new types of clinical diagnostics.

9.
Front Oncol ; 11: 758146, 2021.
Article in English | MEDLINE | ID: covidwho-1526778

ABSTRACT

We analyze the fundamental functions of Prune_1 in brain pathophysiology. We discuss the importance and maintenance of the function of Prune_1 and how its perturbation influences both brain pathological conditions, neurodevelopmental disorder with microcephaly, hypotonia, and variable brain anomalies (NMIHBA; OMIM: 617481), and tumorigenesis of medulloblastoma (MB) with functional correlations to other tumors. A therapeutic view underlying recent discoveries identified small molecules and cell penetrating peptides to impair the interaction of Prune_1 with protein partners (e.g., Nm23-H1), thus further impairing intracellular and extracellular signaling (i.e., canonical Wnt and TGF-ß pathways). Identifying the mechanism of action of Prune_1 as responsible for neurodevelopmental disorders (NDDs), we have recognized other genes which are found overexpressed in brain tumors (e.g., MB) with functional implications in neurodevelopmental processes, as mainly linked to changes in mitotic cell cycle processes. Thus, with Prune_1 being a significant target in NDDs, we discuss how its network of action can be dysregulated during brain development, thus generating cancer and metastatic dissemination.

10.
Sci Signal ; 14(690)2021 07 06.
Article in English | MEDLINE | ID: covidwho-1299215

ABSTRACT

Inorganic polyphosphates (polyPs) are linear polymers composed of repeated phosphate (PO4 3-) units linked together by multiple high-energy phosphoanhydride bonds. In addition to being a source of energy, polyPs have cytoprotective and antiviral activities. Here, we investigated the antiviral activities of long-chain polyPs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In molecular docking analyses, polyPs interacted with several conserved amino acid residues in angiotensin-converting enzyme 2 (ACE2), the host receptor that facilitates virus entry, and in viral RNA-dependent RNA polymerase (RdRp). ELISA and limited proteolysis assays using nano- LC-MS/MS mapped polyP120 binding to ACE2, and site-directed mutagenesis confirmed interactions between ACE2 and SARS-CoV-2 RdRp and identified the specific amino acid residues involved. PolyP120 enhanced the proteasomal degradation of both ACE2 and RdRp, thus impairing replication of the British B.1.1.7 SARS-CoV-2 variant. We thus tested polyPs for functional interactions with the virus in SARS-CoV-2-infected Vero E6 and Caco2 cells and in primary human nasal epithelial cells. Delivery of a nebulized form of polyP120 reduced the amounts of viral positive-sense genomic and subgenomic RNAs, of RNA transcripts encoding proinflammatory cytokines, and of viral structural proteins, thereby presenting SARS-CoV-2 infection in cells in vitro.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Polyphosphates/pharmacology , SARS-CoV-2/drug effects , Administration, Inhalation , Amino Acid Sequence , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antiviral Agents/administration & dosage , Antiviral Agents/chemistry , COVID-19/metabolism , COVID-19/virology , Caco-2 Cells , Chlorocebus aethiops , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Coronavirus RNA-Dependent RNA Polymerase/genetics , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Cytokines/metabolism , HEK293 Cells , Host Microbial Interactions/drug effects , Host Microbial Interactions/genetics , Host Microbial Interactions/physiology , Humans , In Vitro Techniques , Models, Biological , Molecular Docking Simulation , Nebulizers and Vaporizers , Polyphosphates/administration & dosage , Polyphosphates/chemistry , Proteasome Endopeptidase Complex/metabolism , Protein Interaction Domains and Motifs , Proteolysis/drug effects , RNA, Viral/genetics , RNA, Viral/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Sequence Homology, Amino Acid , Signal Transduction/drug effects , Vero Cells , Virus Replication/drug effects
11.
Sci Rep ; 11(1): 11046, 2021 05 26.
Article in English | MEDLINE | ID: covidwho-1246388

ABSTRACT

Among the therapies against the pandemic SARS-CoV-2 virus, monoclonal Antibodies (mAbs) targeting the Spike glycoprotein represent good candidates to interfere in the Spike/ACE2 interaction, preventing virus cell entry. Since anti-spike mAbs, used individually, might be unable to block the virus entry in the case of resistant mutations, we designed an innovative strategy for the isolation of multiple novel human scFvs specific for the binding domain (RBD) of Spike. By panning a large phage display antibody library on immobilized RBD, we obtained specific binders by eluting with ACE2 in order to identify those scFvs recognizing the epitope of Spike interacting with its receptor. We converted the novel scFvs into full size IgG4, differently from the previously isolated IgG1 mAbs, to avoid unwanted potential side effects of IgG1 potent effector functions on immune system. The novel antibodies specifically bind to RBD in a nanomolar range and interfere in the interaction of Spike with ACE2 receptor, either used as purified protein or when expressed on cells in its native conformation. Furthermore, some of them have neutralizing activity for virus infection in cell cultures by using two different SARS-CoV-2 isolates including the highly contagious VOC 202012/01 variant and could become useful therapeutic tools to fight against the SARS-CoV-2 virus.


Subject(s)
Antibodies, Neutralizing/metabolism , Antibodies, Viral/metabolism , COVID-19/therapy , Immunoglobulin G/metabolism , Immunotherapy/methods , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Monoclonal , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/immunology , Antibodies, Viral/genetics , Antibodies, Viral/immunology , COVID-19/immunology , Cells, Cultured , Epitopes , Humans , Immunoglobulin G/immunology , Pandemics , Protein Binding , Protein Domains/genetics , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
12.
Front Public Health ; 9: 649781, 2021.
Article in English | MEDLINE | ID: covidwho-1231426

ABSTRACT

The onset of the new SARS-CoV-2 coronavirus encouraged the development of new serologic tests that could be additional and complementary to real-time RT-PCR-based assays. In such a context, the study of performances of available tests is urgently needed, as their use has just been initiated for seroprevalence assessment. The aim of this study was to compare four chemiluminescence immunoassays and one immunochromatography test for SARS-Cov-2 antibodies for the evaluation of the degree of diffusion of SARS-CoV-2 infection in Salerno Province (Campania Region, Italy). A total of 3,185 specimens from citizens were tested for anti-SARS-CoV-2 antibodies as part of a screening program. Four automated immunoassays (Abbott and Liaison SARS-CoV-2 CLIA IgG and Roche and Siemens SARS-CoV-2 CLIA IgM/IgG/IgA assays) and one lateral flow immunoassay (LFIA Technogenetics IgG-IgM COVID-19) were used. Seroprevalence in the entire cohort was 2.41, 2.10, 1.82, and 1.85% according to the Liaison IgG, Abbott IgG, Siemens, and Roche total Ig tests, respectively. When we explored the agreement among the rapid tests and the serologic assays, we reported good agreement for Abbott, Siemens, and Roche (Cohen's Kappa coefficient 0.69, 0.67, and 0.67, respectively), whereas we found moderate agreement for Liaison (Cohen's kappa coefficient 0.58). Our study showed that Abbott and Liaison SARS-CoV-2 CLIA IgG, Roche and Siemens SARS-CoV-2 CLIA IgM/IgG/IgA assays, and LFIA Technogenetics IgG-IgM COVID-19 have good agreement in seroprevalence assessment. In addition, our findings indicate that the prevalence of IgG and total Ig antibodies against SARS-CoV-2 at the time of the study was as low as around 3%, likely explaining the amplitude of the current second wave.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Immunoassay , Immunoglobulin M , Italy , Luminescence , Sensitivity and Specificity , Seroepidemiologic Studies
13.
Diagnostics (Basel) ; 11(2)2021 Feb 12.
Article in English | MEDLINE | ID: covidwho-1085113

ABSTRACT

The COVID-19 pandemic has forced diagnostic laboratories to focus on the early diagnostics of SARS-CoV-2. The positivity of a molecular test cannot respond to the question regarding the viral capability to replicate, spread, and give different clinical effects. Despite the fact that some targets are covered by commercially-available assays, the identification of new biomarkers is desired in order to improve the quality of the information given by these assays. Therefore, since the subgenomic transcripts (sgN and sgE) are considered markers of viral activity, we evaluated these subgenomic transcripts in relation to the genomic amplification obtained using five different commercial CE-IVD tools. Methods: Five CE-IVD kits were compared in terms of their capability to detect both synthetic SARS-CoV-2 viral constructs (spiked in TMB or PBS medium) and targets (N, E, RdRp and Orf1ab genes) in twenty COVID-19-positive patients' swabs. The sgN and sgE were assayed by real-time RT-qPCR and digital PCR. Results: None of the diagnostic kits missed the viral target genes when they were applied to targets spiked in TMB or PBS (at dilutions ranging from 100 pg to 0.1 pg). Nevertheless, once they were applied to RNA extracted from the patients' swabs, the superimposability ranged from 50% to 100%, regardless of the extraction procedure. The sgN RNA transcript was detected only in samples with a higher viral load (Ct ≤ 22.5), while sgE was within all of the Ct ranges. Conclusions: The five kits show variable performances depending on the assay layout. It is worthy of note that the detection of the sgN transcript is associated with a higher viral load, thus representing a new marker of early and more severe infection.

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