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Introduction: The ongoing emergence of SARS-CoV-2 variants poses significant challenges to existing therapeutics. The spike (S) glycoprotein is central to both viral entry and cell-to-cell transmission via syncytia formation, a process that confers resistance to neutralizing antibodies. The mechanisms underlying this resistance, particularly in relation to spike-mediated fusion, remain poorly understood., Methods: We analyzed two clinical SARS-CoV-2 isolates differing by a single amino acid substitution in the S protein. Using biochemical and cell-based assays, we evaluated entry kinetics, syncytia formation, and the neutralizing efficacy of convalescent sera. These parameters were further correlated with S-mediated cell-cell fusion activity., Results: The single amino acid substitution significantly altered entry kinetics and enhanced syncytia formation. This modification did not diminished the neutralizing capacity of convalescent sera, but it increased the efficiency of S-induced cell-cell fusion. These findings highlight the mutation's impact on viral transmissibility and immune evasion., Discussion: Our study demonstrates that even minor changes in the S protein can profoundly influence SARS-CoV-2 transmissibility and resistance to antibody-mediated neutralization. Understanding the molecular basis of S-mediated cell-cell fusion is crucial for anticipating the impact of emerging variants and developing next-generation therapeutic strategies. These insights provide a framework for predicting variant fitness and optimizing treatment approaches against future SARS-CoV-2 variants., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Criscuolo, Giuliani, Castelli, Cavallaro, Sisti, Burioni, Ferrari, Mancini, Locatelli and Clementi.)

(1) Background: Our aim is the evaluation of the neutralizing activity of BNT162b2 mRNA vaccine-induced antibodies in different in vitro cellular models, as this still represents one of the surrogates of protection against SARS-CoV-2 viral variants. (2) Methods: The entry mechanisms of SARS-CoV-2 in three cell lines (Vero E6, Vero E6/TMPRSS2 and Calu-3) were evaluated with both pseudoviruses and whole virus particles. The neutralizing capability of sera collected from vaccinated subjects was characterized through cytopathic effects and Real-Time RT PCR. (3) Results: In contrast to Vero E6 and Vero E6/TMPRSS2, Calu-3 allowed the evaluation of both viral entry mechanisms, resembling what occurs during natural infection. The choice of an appropriate cellular model can decisively influence the determination of the neutralizing activity of antibodies against SARS-CoV-2 variants. Indeed, the lack of correlation between neutralizing data in Calu-3 and Vero E6 demonstrated that testing the antibody inhibitory activity by using a single cell model possibly results in an inaccurate characterization. (4) Conclusions: Cellular systems allowing only one of the two viral entry pathways may not fully reflect the neutralizing activity of vaccine-induced antibodies moving increasingly further away from possible correlates of protection from SARS-CoV-2 infection.

Background: Platelet activation and thrombotic events characterizes COVID-19., Objectives: To characterize platelet activation and determine if SARS-CoV-2 induces platelet activation., Patients/methods: We investigated platelet activation in 119 COVID-19 patients at admission in a university hospital in Milan, Italy, between March 18 and May 5, 2020. Sixty-nine subjects (36 healthy donors, 26 patients with coronary artery disease, coronary artery disease, and seven patients with sepsis) served as controls., Results: COVID-19 patients had activated platelets, as assessed by the expression and distribution of HMGB1 and von Willebrand factor, and by the accumulation of platelet-derived (plt) extracellular vesicles (EVs) and HMGB1 + plt-EVs in the plasma. P-selectin upregulation was not detectable on the platelet surface in a fraction of patients (55%) and the concentration of soluble P-selectin in the plasma was conversely increased. The plasma concentration of HMGB1 + plt-EVs of patients at hospital admission remained in a multivariate analysis an independent predictor of the clinical outcome, as assessed using a 6-point ordinal scale (from 1 = discharged to 6 = death). Platelets interacting in vitro with SARS-CoV-2 underwent activation, which was replicated using SARS-CoV-2 pseudo-viral particles and purified recombinant SARS-CoV-2 spike protein S1 subunits. Human platelets express CD147, a putative coreceptor for SARS-CoV-2, and Spike-dependent platelet activation, aggregation and granule release, release of soluble P-selectin and HMGB1 + plt-EVs abated in the presence of anti-CD147 antibodies., Conclusions: Hence, an early and intense platelet activation, which is reproduced by stimulating platelets in vitro with SARS-CoV-2, characterizes COVID-19 and could contribute to the inflammatory and hemostatic manifestations of the disease., (© 2021 International Society on Thrombosis and Haemostasis.)

The COVID-19 pandemic caused by SARS-CoV-2 has made the development of safe and effective vaccines a critical priority. To date, four vaccines have been approved by European and American authorities for preventing COVID-19, but the development of additional vaccine platforms with improved supply and logistics profiles remains a pressing need. Here we report the preclinical evaluation of a novel COVID-19 vaccine candidate based on the electroporation of engineered, synthetic cDNA encoding a viral antigen in the skeletal muscle. We constructed a set of prototype DNA vaccines expressing various forms of the SARS-CoV-2 spike (S) protein and assessed their immunogenicity in animal models. Among them, COVID-eVax-a DNA plasmid encoding a secreted monomeric form of SARS-CoV-2 S protein receptor-binding domain (RBD)-induced the most potent anti-SARS-CoV-2 neutralizing antibody responses (including against the current most common variants of concern) and a robust T cell response. Upon challenge with SARS-CoV-2, immunized K18-hACE2 transgenic mice showed reduced weight loss, improved pulmonary function, and lower viral replication in the lungs and brain. COVID-eVax conferred significant protection to ferrets upon SARS-CoV-2 challenge. In summary, this study identifies COVID-eVax as an ideal COVID-19 vaccine candidate suitable for clinical development. Accordingly, a combined phase I-II trial has recently started., Competing Interests: Declaration of interests A.C. and M.M. are Evvivax employees. E.M., F.P., G.R., A.M., L.L., L.L., E. Salvatori, M. Cappalletti, F.F.F., E.D., V.C., and L.A. are Takis employees. G. Caselli and L.R. are Rottapharm Biotech employees. Takis and Rottapharm Biotech are jointly developing COVID-eVax. M.I. participates in advisory boards/consultancies for or receives funding from Gilead Sciences, Roche, Third Rock Ventures, Amgen, Allovir, Asher Bio. L.G.G is a member of the board of directors at Genenta Science and Epsilon Bio and participates in advisory boards/consultancies for Gilead Sciences, Roche, and Arbutus Biopharma., (Copyright © 2021 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Background and Aims: Vaccines, to limit SARS-CoV-2 infection, were produced and reliable assays are needed for their evaluation. The WHO produced an International-Standard (WHO-IS) to facilitate the standardization/comparison of serological methods. The WHO-IS, produced in limited amount, was never tested for reproducibility. This study aims at developing a reproducible and accessible working standard (WS) to complement the WHO-IS., Materials and Methods: Sera from vaccinated individuals were used to produce the WSs. The WHO-IS, the WSs and single serum samples (n = 48) were tested on 6 quantitative serological devices. Neutralization assays were performed for the 48 samples and compared with their antibody titers., Results: The WS carry an antibody titer 20-fold higher than the WHO-IS. It was reproducible, showed both good linearity and insignificant intra- and inter-laboratory variability. However, the WSs behave differently from the WHO-IS. Analysis of the 48 samples showed that single correlation factors are not sufficient to harmonize results from different assays. Yet, all the devices predict neutralization activity based on the antibody titer., Conclusions: A reproducible and highly concentrated WS, specific for IgG against SARS-CoV-2 Spike-glycoprotein was produced. Such characteristics make it particularly suited for the harmonization of commercially available assays and the consequent evaluation of post-vaccinated individuals., (Copyright © 2021 Elsevier B.V. All rights reserved.)

SARS-CoV-2 fine-tunes the interferon (IFN)-induced antiviral responses, which play a key role in preventing coronavirus disease 2019 (COVID-19) progression. Indeed, critically ill patients show an impaired type I IFN response accompanied by elevated inflammatory cytokine and chemokine levels, responsible for cell and tissue damage and associated multi-organ failure. Here, the early interaction between SARS-CoV-2 and immune cells was investigated by interrogating an in vitro human peripheral blood mononuclear cell (PBMC)-based experimental model. We found that, even in absence of a productive viral replication, the virus mediates a vigorous TLR7/8-dependent production of both type I and III IFNs and inflammatory cytokines and chemokines, known to contribute to the cytokine storm observed in COVID-19. Interestingly, we observed how virus-induced type I IFN secreted by PBMC enhances anti-viral response in infected lung epithelial cells, thus, inhibiting viral replication. This type I IFN was released by plasmacytoid dendritic cells (pDC) via an ACE-2-indipendent but Neuropilin-1-dependent mechanism. Viral sensing regulates pDC phenotype by inducing cell surface expression of PD-L1 marker, a feature of type I IFN producing cells. Coherently to what observed in vitro, asymptomatic SARS-CoV-2 infected subjects displayed a similar pDC phenotype associated to a very high serum type I IFN level and induction of anti-viral IFN-stimulated genes in PBMC. Conversely, hospitalized patients with severe COVID-19 display very low frequency of circulating pDC with an inflammatory phenotype and high levels of chemokines and pro-inflammatory cytokines in serum. This study further shed light on the early events resulting from the interaction between SARS-CoV-2 and immune cells occurring in vitro and confirmed ex vivo. These observations can improve our understanding on the contribution of pDC/type I IFN axis in the regulation of the anti-viral state in asymptomatic and severe COVID-19 patients., Competing Interests: The authors have declared that no competing interests exist.

SARS-CoV-2 spike is evolving to maximize transmissibility and evade the humoral response. The massive genomic sequencing of SARS-CoV-2 isolates has led to the identification of single-point mutations and deletions, often having the recurrence of hotspots, associated with advantageous phenotypes. We report the isolation and molecular characterization of a SARS-CoV-2 strain, belonging to a lineage (C.36) not previously associated with concerning traits, which shows decreased susceptibility to vaccine sera neutralization.

Plenty of serologic tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been developed so far, thus documenting the importance of evaluating the relevant features of the immune response to this viral agent. The performance of these assays is currently under investigation. Amongst them, LIAISON® SARS-CoV-2 S1/S2 IgG by DiaSorin and Elecsys Anti-SARS-CoV-2 cobas® by Roche are currently used by laboratory medicine hospital departments in Italy and many other countries. In the present study, we firstly compared two serologic tests on serum samples collected at two different time points from 46 laboratory-confirmed coronavirus disease-2019 (COVID-19) subjects. Secondly, 85 negative serum samples collected before the SARS-CoV-2 pandemic were analyzed. Thirdly, possible correlations between antibody levels and the resulting neutralizing activity against a clinical isolate of SARS-CoV-2 were evaluated. Results revealed that both tests are endowed with low sensitivity on the day of hospital admission, which increased to 97.8% and 100% for samples collected after 15 days for DiaSorin and Roche tests, respectively. The specificity evaluated for the two tests ranges from 96.5% to 100%, respectively. Importantly, a poor direct correlation between antibody titers and neutralizing activity levels was evidenced in the present study. These data further shed light on both potentials and possible limitations related to SARS-CoV-2 serology. In this context, great efforts are still necessary for investigating antibody kinetics to develop novel diagnostic algorithms. Moreover, further investigations on the role of neutralizing antibodies and their correlate of protection will be of paramount importance for the development of effective vaccines., (© 2020 Wiley Periodicals LLC.)

The SARS-CoV-2 protease (3CLpro) is one of the key targets for the development of efficacious drugs for COVID-19 treatment due to its essential role in the life cycle of the virus and exhibits high conservation among coronaviruses. Recent studies have shown that flavonoids, which are small natural molecules, have antiviral activity against coronaviruses (CoVs), including SARS-CoV-2. In this study, we identified the docking sites and binding affinity of several natural compounds, similar to flavonoids, and investigated their inhibitory activity towards 3CLpro enzymatic activity. The selected compounds were then tested in vitro for their cytotoxicity, for antiviral activity against SARS-CoV-2, and the replication of other coronaviruses in different cell lines. Our results showed that Baicalein (100 μg/mL) exerted strong 3CLpro activity inhibition (>90%), whereas Hispidulin and Morin displayed partial inhibition. Moreover, Baicalein, up to 25 μg/mL, hindered >50% of SARS-CoV-2 replication in Vero E6 cultures. Lastly, Baicalein displayed antiviral activity against alphacoronavirus (Feline-CoV) and betacoronavirus (Bovine-CoV and HCoV-OC43) in the cell lines. Our study confirmed the antiviral activity of Baicalein against SARS-CoV-2 and demonstrated clear evidence of its pan-coronaviral activity. [ABSTRACT FROM AUTHOR]

The ongoing coronavirus disease 2019 pandemic has forced the clinical and scientific community to try drug repurposing of existing antiviral agents as a quick option against severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2). Under this scenario, interferon (IFN) β-1a, whose antiviral potential is already known, and which is a drug currently used in the clinical management of multiple sclerosis, may represent as a potential candidate. In this report, we demonstrate that IFN-β-1a was highly effective in inhibiting in vitro SARS-CoV-2 replication at clinically achievable concentration when administered after virus infection. [ABSTRACT FROM AUTHOR]

While the SARS-CoV-2 pandemic is heavily hitting the world, it is of extreme importance that significant in vitro observations guide the quick set up of clinical trials. In this study, we evidence that the anti-SARS-CoV2 activity of a clinically achievable hydroxychloroquine concentration is maximized only when administered before and after the infection of Vero E6 and Caco-2 cells. This suggests that only a combined prophylactic and therapeutic use of hydroxychloroquine may be effective in limiting viral replication in patients. [ABSTRACT FROM AUTHOR]

Background: Studies reporting the long-term humoral response after receiving the BNT162b2 COVID-19 vaccine are important to drive future vaccination strategies. Yet, available literature is scarce. Covidiagnostix is a multicenter study designed to assess the antibody response in >1000 healthcare professionals (HCPs) who received the BNT162b2 vaccine. Methods: Serum was tested at time-0 (T0), before the first dose, T1, T2, and T3, respectively, 21, 42, and 180 days after T0. Antibodies against the SARS-CoV-2 nucleocapsid-protein were measured to assess SARS-CoV-2 infections, whereas antibodies against the receptor-binding domain of the spike protein were measured to assess the vaccine response. Neutralization activity against the D614G, B.1.1.7, and B.1.351 variants were also analyzed. Results: Six months post-vaccination HCPs showed an antibody titer decrease of approximately 70%, yet, the titer was still one order of magnitude higher than that of seropositive individuals before vaccination. We identified 12 post-vaccination infected HCPs. None showed severe symptoms. Interestingly, most of them showed titers at T2 above the neutralization thresholds obtained from the neutralization activity experiments. Conclusion: Vaccination induces a humoral response which is well detectable even six months post-vaccination. Vaccination prevents severe COVID-19 cases, yet post-vaccination infection is possible even in the presence of a high anti-S serum antibody titer. [ABSTRACT FROM AUTHOR]

The plaque reduction neutralization test (PRNT) is a preferred method for the detection of functional, SARS-CoV-2 specific neutralizing antibodies from serum samples. Alternatively, surrogate enzyme-linked immunosorbent assays (ELISAs) using ACE2 as the target structure for the detection of neutralization-competent antibodies have been developed. They are capable of high throughput, have a short turnaround time, and can be performed under standard laboratory safety conditions. However, there are very limited data on their clinical performance and how they compare to the PRNT. We evaluated three surrogate immunoassays (GenScript SARS-CoV-2 Surrogate Virus Neutralization Test Kit (GenScript Biotech, Piscataway Township, NJ, USA), the TECO® SARS-CoV-2 Neutralization Antibody Assay (TECOmedical AG, Sissach, Switzerland), and the Leinco COVID-19 ImmunoRank™ Neutralization MICRO-ELISA (Leinco Technologies, Fenton, MO, USA)) and one automated quantitative SARS-CoV-2 Spike protein-based IgG antibody assay (Abbott GmbH, Wiesbaden, Germany) by testing 78 clinical samples, including several follow-up samples of six BNT162b2 (BioNTech/Pfizer, Mainz, Germany/New York, NY, USA) vaccinated individuals. Using the PRNT as a reference method, the overall sensitivity of the examined assays ranged from 93.8 to 100% and specificity ranged from 73.9 to 91.3%. Weighted kappa demonstrated a substantial to almost perfect agreement. The findings of our study allow these assays to be considered when a PRNT is not available. However, the latter still should be the preferred choice. For optimal clinical performance, the cut-off value of the TECO assay should be individually adapted. [ABSTRACT FROM AUTHOR]

Background: The coronavirus disease 2019 (COVID-19) outbreak, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been declared a global pandemic. It is well-established that SARS-CoV-2 infection can lead to dysregulated immune responses. Arginase-1 (Arg1), which has a pivotal role in immune cells, can be expressed in most of the myeloid cells, e.g., neutrophils and macrophages. Arg1 has been associated with the suppression of antiviral immune responses. Methods: Whole blood was taken from 21 COVID-19 patients and 21 healthy individuals, and after RNA extraction and complementary DNA (cDNA) synthesis, gene expression of Arg1 was measured by real-time PCR. Results: The qPCR results showed that the expression of Arg1 was significantly increased in COVID-19 patients compared to healthy individuals (p < 0.01). The relative expression analysis demonstrated there were approximately 2.3 times increased Arg1 expression in the whole blood of COVID-19 patients. Furthermore, the receiver operating characteristic (ROC) analysis showed a considerable diagnostic value for Arg1 expression in COVID-19 (p = 0.0002 and AUC = 0.8401). Conclusion: Arg1 might be a promising marker in the pathogenesis of the disease, and it could be a valuable diagnostic tool. [ABSTRACT FROM AUTHOR]

The outbreak of coronavirus disease 2019 (COVID-19) is posing a threat to global health. This disease has different clinical manifestations and different outcomes. The immune response to the novel 2019 coronavirus is complex and involves both innate and adaptive immunity. In this context, cell-mediated immunity plays a vital role in effective immunity against SARS-CoV-2. Significant differences have been observed when comparing severe and non-severe patients. Since these immunological characteristics have not been fully elucidated, we aimed to use cluster analysis to investigate the immune cell patterns in patients with COVID-19 who required hospitalization but not intensive care. We identified four clusters of different immunological patterns, the worst being characterized by total lymphocytes, T helper lymphocytes CD4+ (CD4+), T cytotoxic lymphocytes CD8+ (CD8+) and natural killer (NK) cells below the normal range, together with natural killer lymphocyte granzyme < 50% (NK granzyme+) and antibody-secreting plasma cells (ASCs) equal to 0 with fatal outcomes. In the worst group, 50% of patients died in the intensive care unit. Moreover, a negative trend was found among four groups regarding total lymphocytes, CD4+, CD8+ and B lymphocytes (p < 0.001, p < 0.005, p < 0.000, p < 0.044, respectively). This detailed analysis of immune changes may have prognostic value. It may provide a new perspective for identifying subsets of COVID-19 patients and selecting novel prospective treatment strategies. Notwithstanding these results, this is a preliminary report with a small sample size, and our data may not be generalizable. Further cohort studies with larger samples are necessary to quantify the prognostic value's weight, according to immunological changes in COVID-19 patients, for predicting prognoses and realizing improvements in clinical conditions. [ABSTRACT FROM AUTHOR]

Severe coronavirus disease 2019 (COVID-19) is characterized by overproduction of immune mediators, but the role of interferons (IFNs) of the type I (IFN-I) or type III (IFN-III) families remains debated. We scrutinized the production of IFNs along the respiratory tract of COVID-19 patients and found that high levels of IFN-III, and to a lesser extent IFN-I, characterize the upper airways of patients with high viral burden but reduced disease risk or severity. Production of specific IFN-III, but not IFN-I, members denotes patients with a mild pathology and efficiently drives the transcription of genes that protect against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In contrast, compared to subjects with other infectious or noninfectious lung pathologies, IFNs are overrepresented in the lower airways of patients with severe COVID-19 that exhibit gene pathways associated with increased apoptosis and decreased proliferation. Our data demonstrate a dynamic production of IFNs in SARS-CoV-2-infected patients and show IFNs play opposing roles at distinct anatomical sites. [Display omitted] • IFN expression in COVID-19 varies based on location, viral load, age, and severity • IFN-λ1 and IFN-λ3 drive protective ISGs in the upper airways of mildly ill patients • Critical patients express IFN-αβ and IFN-λ2 and have low ISGs and high p53 expression • Epithelial cells efficiently produce protective IFN-λ1, while cDCs express IFN-λ2,3 An in-depth analysis of IFNs in COVID-19 reveals differences in their roles based on anatomical location, viral load, age, and disease severity. In the upper respiratory tract, high levels of IFN-III are protective and result in mild disease in spite of higher SARS-CoV-2 viral burden, while the lower airways of patients with severe COVID-19 demonstrate elevated IFN-I and IFN-III, cell death, and a reduction in IFN-stimulated genes. [ABSTRACT FROM AUTHOR]