ABSTRACT
A new SARS-CoV-2 clade (GV) characterized by S substitution A222V, first reported from Spain in March, is rapidly spreading across Europe. To establish the A222V variant involvement in the infection rise in Italy, all GISAID sequences from Italy and those from our Laboratory (Lazio) in the period June-October were analysed. A222V, first recognized in August, represents 11.2% of sequences in this period, reaching 100% of autochthonous sequences in October, supporting increased GV circulation in Italy.
ABSTRACT
BackgroundEpidemiological, virological and pathogenetic characteristics of SARS-CoV-2 infection are under evaluation. A better understanding of the pathophysiology associated with COVID-19 is crucial to improve treatment modalities and to develop effective prevention strategies. Transcriptomic and proteomic data on the host response against SARS-CoV-2 still have anecdotic character; currently available data from other coronavirus infections are therefore a key source of information. MethodsWe investigated selected molecular aspects of three human coronavirus (HCoV) infections, namely SARS-CoV, MERS-CoV and HCoV-229E, through a network based-approach. A functional analysis of HCoV-host interactome was carried out in order to provide a theoretic host-pathogen interaction model for HCoV infections and in order to translate the results in prediction for SARS-CoV-2 pathogenesis. The 3D model of S-glycoprotein of SARS-CoV-2 was compared to the structure of the corresponding SARS-CoV, HCoV-229E and MERS-CoV S-glycoprotein. SARS-CoV, MERS-CoV, HCoV-229E and the host interactome were inferred through published protein-protein interactions (PPI) as well as gene co-expression, triggered by HCoV S-glycoprotein in host cells. ResultsAlthough the amino acid sequences of the S-glycoprotein were found to be different between the various HCoV, the structures showed high similarity, but the best 3D structural overlap shared by SARS-CoV and SARS-CoV-2, consistent with the shared ACE2 predicted receptor. The host interactome, linked to the S-glycoprotein of SARS-CoV and MERS-CoV, mainly highlighted innate immunity pathway components, such as Toll Like receptors, cytokines and chemokines. ConclusionsIn this paper, we developed a network-based model with the aim to define molecular aspects of pathogenic phenotypes in HCoV infections. The resulting pattern may facilitate the process of structure-guided pharmaceutical and diagnostic research with the prospect to identify potential new biological targets.
ABSTRACT
COVID-19 pandemic is becoming one of the most dramatic health, social and economic global challenges in recent history. Testing is one of the main components of the public health response to contain the virus spreading. There is an urgent need to expand testing capacity and antigen rapid tests (Ag RDT) represent good candidates for point-of-care and mass surveillance testing to rapidly identify people with SARS-CoV-2 infection, counterbalancing lower sensitivity as compared to the gold standard molecular tests with timeliness of results and possibility of recurred testing. Here, we report preliminary data of the testing algorithm implemented at the points-of-entry (airports and port) in Lazio Region (Central Italy) on travelers arriving between 17th of August to 15th of October, 2020, using the STANDARD F COVID-19 Antigen Fluorescence ImmunoAssay. Our findings show that the probability of molecular confirmation of Ag RDT positive results is directly dependent from the semi-quantitative results of this Ag RDT, and that the molecularly confirmed samples actually harbor infectious virus. These results support the public health strategies based on early screening campaigns in settings where molecular testing is not feasible or easily accessible, using rapid and simple point of care tests, able to rapidly identify those subjects who are at highest risk of spreading SARS-CoV-2 infection.
ABSTRACT
In the last months, many studies have clearly described several mechanisms of SARS-CoV-2 infection at cell and tissue level. Host conditions and comorbidities were identified as risk factors for severe and fatal disease courses, but the mechanisms of interaction between host and SARS-CoV-2 determining the grade of COVID- 19 severity, are still unknown. We provide a network analysis on protein-protein interactions (PPI) between viral and host proteins to better identify host biological responses, induced by both whole proteome of SARS-CoV-2 and specific viral proteins. A host-virus interactome was inferred on published PPI, using an explorative algorithm (Random Walk with Restart) triggered by all the 28 proteins of SARS-CoV-2, or each single viral protein one-by-one. The functional analysis for all proteins, linked to many aspects of COVID-19 pathogenesis, allows to identify the subcellular districts, where SARS-CoV-2 proteins seem to be distributed, while in each interactome built around one single viral protein, a different response was described, underlining as ORF8 and ORF3a modulated cardiovascular diseases and pro-inflammatory pathways, respectively. Finally, an explorative network-based approach was applied to Bradykinin Storm, highlighting a possible direct action of ORF3a and NS7b to enhancing this condition. This network-based model for SARS-CoV-2 infection could be a framework for pathogenic evaluation of specific clinical outcomes. We identified possible host responses induced by specific proteins of SARS-CoV-2, underlining the important role of specific viral accessory proteins in pathogenic phenotypes of severe COVID-19 patients.
ABSTRACT
SARS-CoV-2 is a novel coronavirus, not encountered before by humans. The wide spectrum of clinical expression of SARS-CoV-2 illness suggests that individual immune responses to SARS-CoV-2 play a crucial role in determining the clinical course after first infection. Immunological studies have focussed on patients with moderate to severe disease, demonstrating excessive inflammation in tissues and organ damage. In order to understand the basis of the protective immune response in COVID-19, we performed a longitudinal follow-up, flow-cytometric and serological analysis of innate and adaptive immunity in 64 adults with a spectrum of clinical presentations: 28 healthy SARS-CoV-2-negative contacts of COVID-19 cases; 20 asymptomatic SARS-CoV-2-infected cases; 8 patients with Mild COVID-19 disease and 8 cases of Severe COVID-19 disease. Our data show that high frequency of NK cells and early and transient increase of specific IgA, IgM and, to a lower extent, IgG are associated to asymptomatic SARS-CoV-2 infection. By contrast, monocyte expansion and high and persistent levels of IgA and IgG, produced relatively late in the course of the infection, characterize severe disease. Modest increase of monocytes and different kinetics of antibodies are detected in mild COVID-19. The importance of innate NK cells and the short-lived antibody response of asymptomatic individuals and patients with mild disease suggest that only severe COVID-19 may result in protective memory established by the adaptive immune response.
ABSTRACT
Coronavirus disease 2019 (COVID-19) vaccines are proving to be very effective in preventing severe illness; however, although rare, post-vaccine infections have been reported. The present study describes 94 infections (47.9% symptomatic, 52.1% asymptomatic), occurred in Lazio Region (Central Italy) in the first trimester 2021, after first or second dose of mRNA BNT162b2 vaccine. Median viral load at diagnosis was independent from number and time of vaccine dose administration, despite the higher proportion of samples with low viral load observed in fully vaccinated individuals. More importantly, infectious virus was cultured from NPS collected from both asymptomatic and symptomatic vaccinated individuals, suggesting that, at least in principle, they can transmit the infection to susceptible people. The majority of the post-vaccine infections here reported, showed pauci/asymptomatic clinical course, confirming the impact of vaccination on COVID-19 disease. Most cases (78%) showed infection in presence of neutralizing antibodies at the time of infection diagnosis, presumably attributable to vaccination, due to the concomitant absence of anti-N IgG in most cases. The proportion of post-vaccine infections attributed either to Alpha and Gamma VOCs was similar to the proportion observed in the contemporary unvaccinated population in Lazio region. In addition, mutational analysis did not suggest enrichment of a defined set of Spike protein substitutions depending on the vaccination status. Characterization of host and virus factors associated with vaccine breakthrough, coupled with intensive and continuous monitoring of involved viral strains, is crucial to adopt informed vaccination strategies.
ABSTRACT
BackgroundThe emerging threat represented by SARS-CoV-2 variants, demands the development of therapies for better clinical management of COVID-19. MAD0004J08 is an extremely potent Fc-engineered monoclonal antibody (mAb) able to neutralise in vitro all current SARS-CoV-2 variants of concern (VoCs). This ongoing study, evaluates safety, pharmacokinetics and SARS-CoV-2 sera neutralization effect of MAD0004J08 when administered as single dose intramuscularly in healthy adults. MethodWe conducted a dose escalation study with sequential enrolment of three cohorts, each with an increasing dose level of MAD0004J08 (48mg, 100mg and 400mg). Within each cohort, 10 young healthy adults were randomized with 4:1 ratio to a single intramuscular (i.m.) injection of MAD0004J08 or placebo. The primary endpoint is the proportion of subjects with severe and/or serious treatment emergent adverse events (TEAEs) within 7 days post-treatment. Secondary endpoints reported in this paper are the proportion of subjects with solicited TEAEs up 7 days post dosing, MAD0004J08 serum concentrations and neutralising activity versus the original SARS-COV-2 Wuhan virus at different timepoints post-dosing. As post-hoc analyses, we compared the sera neutralising titres of subjects who received MAD0004J08 with those of people that had received the COVID-19 BNT162b2 mRNA vaccine in the previous sixty days (n=10) and COVID-19 convalescent patients (n=20), and assessed serum neutralisation activity against the B.1.1.7 (alpha), B.1.351 (beta) and B.1.1.248 (gamma) SARS-CoV-2 variants of concern. FindingsA total of 30 subjects, 10 per cohort, were enrolled and randomized. Data up to 30 days were available and analysed in this report. No severe TEAEs were reported in any of the cohorts in the 7 days post-treatment. MAD0004J08 was detected in the sera of treated subjects within few hours post-administration and reached almost maximal levels on day 8. The geometric mean neutralising titres (GMT) assessed against the original Wuhan virus peaked on day 8 and ranged 226 - 905, 905 - 2,560, and 1,280 - 5,120 for cohort 1, 2 and 3 respectively. The sera neutralising GMT in MAD0004J08 treated subjects in all the three cohorts were found to be 1{middle dot}5-54{middle dot}5-fold higher compared to sera from convalescent patients and 1{middle dot}83- 76{middle dot}4-fold higher compared to sera from COVID-19 vaccinees. Finally, GMT in MAD0004J08 treated subjects showed high neutralising titres versus the B.1.1.7 (alpha), B.1.351 (beta) and B.1.1.248 (gamma) SARS-CoV-2 VoCs. InterpretationA single dose administration of MAD0004J08 via i.m. route is safe and well tolerated and results in a rapid systemic distribution of the MAD0004J08 and sera neutralising titres higher than COVID-19 convalescent and vaccinated subjects. A single dose administration of MAD0004J08 is also sufficient to effectively neutralise major SARS-CoV-2 variants of concern. Based on these results, a Phase 2-3 trial is ongoing to further assess the safety, dosage, and efficacy of MAD0004J08 in asymptomatic or mild-moderate symptomatic COVID-19 patients. FundingEU Malaria Fund, Ministero dello Sviluppo Economico, Ministero della Salute, Regione Toscana, Toscana Life Sciences Sviluppo and European Research Council. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSWe searched PUBMED, MEDLINE and MedRxiv for clinical trials, meta-analyses and randomized controlled trials evaluating the antibody neutralization titres vs. different SARS-CoV-2 variants of concern obtained from subjects who received monoclonal antibodies for the treatment of COVID-19 using the following search terms: ("COVID-19" OR "SARS-CoV-2") AND ("monoclonal antibody" OR "neutralising antibody") AND ("variants" OR "variants of concern"). No relevant studies were identified. Added value of this studyThis is the first human study assessing safety, PK and neutralising potential of MAD0004J08, a monoclonal antibody against SARS-CoV-2 wild type Wuhan virus and variants of concern, administered intramuscularly at low dosages (48, 100 and 400 mg). MAD0004J08 showed to be safe and well tolerated in the tested dose range. Anti-spike antibodies were detected in the sera of tested SARS-CoV-2 negative healthy adults few hours post-injection. In addition, the sera obtained from MAD0004J08treated subjects, showed to have high neutralisation titres against the Wuhan virus, the B.1.1.7 (alpha), B.1.351 (beta) and B.1.1.248 (gamma) variants of concern. Implications of all the available evidenceA potent monoclonal antibody such as MAD0004J08, capable of neutralising multiple variants of concern of SARS-CoV-2 rapidly and long lastingly when given as a single intramuscular injection. The antibody, presently tested in a phase 2-3 efficacy trial, can be a major advancement in the prophylaxis and clinical management of COVID-19, because of its broad spectrum, ease of use in non-hospital settings and economic sustainability.
ABSTRACT
Human monoclonal antibodies are safe, preventive and therapeutic tools, that can be rapidly developed to help restore the massive health and economic disruption caused by the Covid-19 pandemic. By single cell sorting 4277 SARS-CoV-2 spike protein specific memory B cells from 14 Covid-19 survivors, 453 neutralizing antibodies were identified and 220 of them were expressed as IgG. Up to 65,9% of monoclonals neutralized the wild type virus at a concentration of >500 ng/mL, 23,6% neutralized the virus in the range of 100 - 500 ng/mL and 9,1% had a neutralization potency in the range of 10 - 100 ng/mL. Only 1,4% neutralized the authentic virus with a potency of 1-10 ng/mL. We found that the most potent neutralizing antibodies are extremely rare and recognize the RBD, followed in potency by antibodies that recognize the S1 domain, the S-protein trimeric structure and the S2 subunit. The three most potent monoclonal antibodies identified were able to neutralize the wild type and D614G mutant viruses with less than 10 ng/mL and are good candidates for the development of prophylactic and therapeutic tools against SARS-CoV-2. One Sentence SummaryExtremely potent neutralizing human monoclonal antibodies isolated from Covid-19 convalescent patients for prophylactic and therapeutic interventions.
ABSTRACT
In the absence of approved drugs or vaccines, there is a pressing need to develop tools for therapy and prevention of Covid-19. Human monoclonal antibodies have very good probability of being safe and effective tools for therapy and prevention of SARS-CoV-2 infection and disease. Here we describe the screening of PBMCs from seven people who survived Covid-19 infection to isolate human monoclonal antibodies against SARS-CoV-2. Over 1,100 memory B cells were single-cell sorted using the stabilized prefusion form of the spike protein and incubated for two weeks to allow natural production of antibodies. Supernatants from each cell were tested by ELISA for spike protein binding, and positive antibodies were further tested for neutralization of spike binding to receptor(s) on Vero E6 cells and for virus neutralization in vitro. From the 1,167 memory B specific for SARS-CoV-2, we recovered 318 B lymphocytes expressing human monoclonals recognizing the spike protein and 74 of these were able to inhibit the binding of the spike protein to the receptor. Finally, 17 mAbs were able to neutralize the virus when assessed for neutralization in vitro. Lead candidates to progress into the drug development pipeline will be selected from the panel of neutralizing antibodies identified with the procedure described in this study. One Sentence SummaryNeutralizing human monoclonal antibodies isolated from Covid-19 convalescent patients for therapeutic and prophylactic interventions.
ABSTRACT
Safe and effective vaccines against coronavirus disease 2019 (COVID-19) are urgently needed to control the ongoing pandemic. Although impressive progress has been made with several COVID-19 vaccines already approved, it is clear that those developed so far cannot meet the global vaccine demand. We have developed a COVID-19 vaccine based on a replication-defective gorilla adenovirus expressing the stabilized pre-fusion SARS-CoV-2 Spike protein, named GRAd-COV2. We aimed to assess the safety and immunogenicity of a single-dose regimen of this vaccine in healthy younger and older adults to select the appropriate dose for each age group. To this purpose, a phase 1, dose-escalation, open-label trial was conducted including 90 healthy subjects, (45 aged 18-55 years and 45 aged 65-85 years), who received a single intramuscular administration of GRAd-CoV2 at three escalating doses. Local and systemic adverse reactions were mostly mild or moderate and of short duration, and no serious AE was reported. Four weeks after vaccination, seroconversion to Spike/RBD was achieved in 43/44 young volunteers and in 45/45 older subjects. Consistently, neutralizing antibodies were detected in 42/44 younger age and 45/45 older age volunteers. In addition, GRAd-COV2 induced a robust and Th1-skewed T cell response against the S antigen in 89/90 subjects from both age groups. Overall, the safety and immunogenicity data from the phase 1 trial support further development of this vaccine. One Sentence SummaryGRAd-COV2, a candidate vaccine for COVID-19 based on a novel gorilla adenovirus, is safe and immunogenic in younger and older adults
ABSTRACT
Patients with cancer are at higher risk of severe coronavirus infectious disease 2019 (COVID-19), but the mechanisms underlying virus-host interactions during cancer therapies remain elusive. When comparing nasopharyngeal swabs from cancer and non-cancer patients for RT-qPCR cycle thresholds measuring acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in 1063 patients (58% with cancer, 89% COVID-19+), we found that malignant disease favors the magnitude and duration of viral RNA shedding concomitant with prolonged serum elevations of type 1 IFN that anticorrelated with anti-RBD IgG antibodies. Chronic viral RNA carriers exhibited the typical immunopathology of severe COVID-19 at the early phase of infection including circulation of immature neutrophils, depletion of non-conventional monocytes and a general lymphopenia that, however, was accompanied by a rise in plasmablasts, activated follicular T helper cells, and non-naive Granzyme B+ FasL+, EomehighTCF-1high, PD-1+CD8+ Tc1 cells. Virus-induced lymphopenia worsened cancer-associated lymphocyte loss, and low lymphocyte counts correlated with chronic SARS-CoV-2 RNA shedding, COVID-19 severity and a higher risk of cancer-related death in the first and second surge of the pandemic. Lymphocyte loss correlated with significant changes in metabolites from the polyamine and biliary salt pathways as well as increased blood DNA from Enterobacteriaceae and Micrococcaceae gut family members in long term viral carriers. We surmise that cancer therapies may exacerbate the paradoxical association between lymphopenia and COVID-19-related immunopathology, and that the prevention of COVID-19-induced lymphocyte loss may reduce cancer-associated death.
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BackgroundIn a previous open-label trial, early anakinra treatment guided by elevated soluble urokinase plasminogen activator receptor (suPAR) prevented progression of COVID-19 pneumonia into respiratory failure. MethodsIn the SAVE-MORE multicenter trial, 594 hospitalized patients with moderate and severe COVID-19 pneumonia and plasma suPAR 6 ng/ml or more and receiving standard-of-care were 1:2 randomized to subcutaneous treatment with placebo or 100 mg anakinra once daily for 10 days. The primary endpoint was the overall clinical status of the 11-point World Health Organization ordinal Clinical Progression Scale (WHO-CPS) at day 28. The changes of the WHO-CPS and of the sequential organ failure assessment (SOFA) score were the main secondary endpoints. ResultsAnakinra-treated patients were distributed to lower strata of WHO-CPS by day 28 (adjusted odds ratio-OR 0.36; 95%CI 0.26-0.50; P<0.001); anakinra protected from severe disease or death (6 or more points of WHO-CPS) (OR: 0.46; P: 0.010). The median absolute decrease of WHO-CPS in the placebo and anakinra groups from baseline was 3 and 4 points respectively at day 28 (OR 0.40; P<0.0001); and 2 and 3 points at day 14 (OR 0.63; P: 0.003); the absolute decrease of SOFA score was 0 and 1 points (OR 0.63; P: 0.004). 28-day mortality decreased (hazard ratio: 0.45; P: 0.045). Hospital stay was shorter. ConclusionsEarly start of anakinra treatment guided by suPAR provides 2.78 times better improvement of overall clinical status in moderate and severe COVID-19 pneumonia. (Sponsored by the Hellenic Institute for the Study of Sepsis ClinicalTrials.gov identifier, NCT04680949)
ABSTRACT
The COVID-19 pandemic caused by the {beta}-coronavirus SARS-CoV-2 has made the development of safe and effective vaccines a critical global priority. To date, four vaccines have already 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, a technology previously utilized for cancer vaccines. 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 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 significantly 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 in Italy.