ABSTRACT
The SARS-CoV-2 pandemic made evident that we count with few coronavirus-fighting drugs. Here we aimed to identify a cost-effective antiviral with broad spectrum activity and high safety and tolerability profiles. We began elaborating a list of 116 drugs previously used to treat other pathologies or characterized in pre-clinical studies with potential to treat coronavirus infections. We next employed molecular modelling tools to rank the 44 most promising inhibitors and tested their efficacy as antivirals against a panel of and {beta} coronavirus, e.g., the HCoV-229E and SARS-CoV-2 viruses. Four drugs, OSW-1, U18666A, hydroxypropyl-{beta}-cyclodextrin (H{beta}CD) and phytol, showed antiviral activity against both HCoV-229E (in MRC5 cells) and SARS-CoV-2 (in Vero E6 cells). The mechanism of action of these compounds was studied by transmission electron microscopy (TEM) and by testing their capacity to inhibit the entry of SARS-CoV-2 pseudoviruses in ACE2-expressing HEK-293T cells. The entry was inhibited by H{beta}CD and U18666A, yet only H{beta}CD could inhibit SARS-CoV-2 replication in the pulmonary cells Calu-3. With these results and given that cyclodextrins are widely used for drug encapsulation and can be safely administered to humans, we further tested 6 native and modified cyclodextrins, which confirmed {beta}-cyclodextrins as the most potent inhibitors of SARS-CoV-2 replication in Calu-3 cells. All accumulated data points to {beta}-cyclodextrins as promising candidates to be used in the therapeutic treatments for SARS-CoV-2 and possibly other respiratory viruses.
ABSTRACT
The pandemic caused by the new coronavirus SARS-CoV-2 has made evident the need for broad-spectrum, efficient antiviral treatments to combat emerging and re-emerging viruses. Plitidepsin is an antitumor agent of marine origin that has also shown a potent pre-clinical efficacy against SARS-CoV-2. Plitidepsin targets the host protein eEF1A (eukaryotic translation factor 1 alpha 1) and affects viral infection at an early, post-entry step. Because electron microscopy is a valuable tool to study virus-cell interactions and the mechanism of action of antiviral drugs, in this work we have used transmission electron microscopy (TEM) to evaluate the effects of plitidepsin in SARS-CoV-2 infection in cultured Vero E6 cells 24 and 48h post-infection. In the absence of plitidepsin, TEM morphological analysis showed double-membrane vesicles (DMVs), organelles that support coronavirus genome replication, single-membrane vesicles with viral particles, large vacuoles with groups of viruses and numerous extracellular virions attached to the plasma membrane. When treated with plitidepsin, no viral structures were found in SARS-CoV-2-infected Vero E6 cells. Immunogold detection of SARS-CoV-2 nucleocapsid (N) protein and double-stranded RNA (dsRNA) provided clear signals in cells infected in the absence of plitidepsin, but complete absence in cells infected and treated with plitidepsin. The present study shows that plitidepsin completely blocks the biogenesis of viral replication organelles and the morphogenesis of virus progeny. Electron microscopy morphological analysis coupled to immunogold labeling of SARS-CoV-2 products offers a unique approach to understand how antivirals such as plitidepsin work.
ABSTRACT
The current COVID-19 vaccines have been associated with a decline in infection rates, prevention of severe disease and decrease in mortality rates. However, new variants of concern (VoCs) are continuously evolving, making the development of new accessible COVID-19 vaccines essential in order to mitigate the pandemic. Here we present data on preclinical studies in mice of a receptor-binding domain (RBD)-based recombinant protein vaccine candidate (PHH-1V) consisting of an RBD fusion heterodimer containing the B.1.351 and B.1.1.7 SARS-CoV-2 VoCs and formulated with the SQBA adjuvant, an oil-in-water emulsion produced by HIPRA. BALB/c and K18-hACE2 mice were immunized with different recombinant RBD fusion heterodimer doses, following a two-dose prime-and-boost schedule. Vaccination induced a CD4+ and CD8+ T cell response and RBD-binding antibodies with neutralising activity against various VoCs with a good tolerability profile. Significantly, a 10-{micro}g or 20-{micro}g RBD fusion heterodimer/dose vaccination conferred 100% efficacy, preventing mortality in SARS-CoV-2 infected K18-hACE2 mice. These findings demonstrate the feasibility of this recombinant vaccine strategy.
ABSTRACT
BackgroundUnderstanding the determinants of long-term immune responses to SARS-CoV-2 and the concurrent impact of vaccination and emerging variants of concern will guide optimal strategies to achieve global protection against the COVID-19 pandemic. MethodsA prospective cohort of 332 COVID-19 patients was followed beyond one year. Plasma neutralizing activity was evaluated using HIV-based reporter pseudoviruses expressing different SARS-CoV-2 spikes and was longitudinally analyzed using mixed-effects models. FindingsLong-term neutralizing activity was stable beyond one year after infection in mild/asymptomatic and hospitalized participants. However, longitudinal models suggest that hospitalized individuals generate both short- and long-lived memory B cells, while outpatient responses were dominated by long-lived B cells. In both groups, vaccination boosted responses to natural infection, although viral variants, mainly B.1.351, reduced the efficacy of neutralization. Importantly, despite showing higher neutralization titers, hospitalized patients showed lower cross-neutralization of B.1.351 variant compared to outpatients. Multivariate analysis identified severity of primary infection as the factor that independently determines both the magnitude and the inferior cross-neutralization activity of long-term neutralizing responses. ConclusionsNeutralizing response induced by SARS-CoV-2 is heterogeneous in magnitude but stable beyond one year after infection. Vaccination boosts these long-lasting natural neutralizing responses, counteracting the significant resistance to neutralization of new viral variants. Severity of primary infection determines higher magnitude but poorer quality of long-term neutralizing responses.
ABSTRACT
SARS-CoV-2 variants display enhanced transmissibility and/or immune evasion and can be generated in humans or animals, like minks, thus generating new reservoirs. The continuous surveillance of animal susceptibility to new variants is necessary to predict pandemic evolution. In this study we demonstrate that, compared to the B.1 SARS-CoV-2 variant, K18-hACE2 transgenic mice challenged with the B.1.351 variant displayed a faster progression of infection. Furthermore, we also report that B.1.351 can establish infection in wildtype mice, while B.1 cannot. B.1.351-challenged wildtype mice showed a milder infection than transgenic mice, confirmed by detectable viral loads in oropharyngeal swabs and tissues, lung pathology, immunohistochemistry and serology. In silico models supported these findings by demonstrating that the Spike mutations in B.1.351 resulted in increased affinity for both human and murine ACE2 receptors. Overall, this study highlights the plasticity of SARS-CoV-2 animal susceptibility landscape, which may contribute to viral persistence and expansion.
ABSTRACT
COVID-19 pandemic is not yet under control by vaccination, and effective antivirals are critical for preparedness. Here we report that macrophages and dendritic cells, key antigen presenting myeloid cells (APCs), are largely resistant to SARS-CoV-2 infection. APCs effectively captured viruses within cellular compartments that lead to antigen degradation. Macrophages sense SARS-CoV-2 and released higher levels of cytokines, including those related to cytokine storm in severe COVID-19. The sialic acid-binding Ig-like lectin 1 (Siglec-1/CD169) present on APCs, which interacts with sialylated gangliosides on membranes of retroviruses or filoviruses, also binds SARS-CoV-2 via GM1. Blockage of Siglec-1 receptors by monoclonal antibodies reduces SARS-CoV-2 uptake and transfer to susceptible target cells. APCs expressing Siglec-1 and carrying SARS-CoV-2 are found in pulmonary tissues of non-human primates. Single cell analysis reveals the in vivo induction of cytokines in those macrophages. Targeting Siglec-1 could offer cross-protection against SARS-CoV-2 and other enveloped viruses that exploit APCs for viral dissemination, including those yet to come in future outbreaks.
ABSTRACT
To assess the potential impact of predominant circulating SARS-CoV-2 variants on neutralizing activity of infected and/or vaccinated individuals, we analyzed neutralization of pseudoviruses expressing the spike of the original Wuhan strain, the D614G and B.1.1.7 variants. Our data show that parameters of natural infection (time from infection and infecting variant) determined cross-neutralization. Importantly, upon vaccination, previously infected individuals developed equivalent B.1.1.7 and Wuhan neutralizing responses. In contrast, uninfected vaccinees showed reduced neutralization against B.1.1.7. FundingThis study was funded by Grifols, the Departament de Salut of the Generalitat de Catalunya, the Spanish Health Institute Carlos III, CERCA Programme/Generalitat de Catalunya, and the crowdfunding initiatives #joemcorono, BonPreu/Esclat and Correos.
ABSTRACT
Reinfections with SARS-CoV-2 have already been documented in humans, although its real incidence is currently unknown. Besides having great impact on public health, this phenomenon raises the question if immunity generated by a single infection is sufficient to provide sterilizing/protective immunity to a subsequent SARS-CoV-2 re-exposure. The Golden Syrian hamster is a manageable animal model to explore immunological mechanisms able to counteract COVID-19, as it recapitulates pathological aspects of mild to moderately affected patients. Here, we report that SARS-CoV-2-inoculated hamsters resolve infection in the upper and lower respiratory tracts within seven days upon inoculation with the Cat01 (G614) SARS-CoV-2 isolate. Three weeks after primary challenge, and despite high titers of neutralizing antibodies, half of the animals were susceptible to reinfection by both identical (Cat01, G614) and variant (WA/1, D614) SARS-CoV-2 isolates. However, upon re-inoculation, only nasal tissues were transiently infected with much lower viral replication than those observed after the first inoculation. These data indicate that a primary SARS-CoV-2 infection is not sufficient to elicit a sterilizing immunity in hamster models but protects against lung disease.
ABSTRACT
Understanding mid-term kinetics of immunity to SARS-CoV-2 is the cornerstone for public health control of the pandemic and vaccine development. However, current evidence is rather based on limited measurements, thus losing sight of the temporal pattern of these changes1-6. In this longitudinal analysis, conducted on a prospective cohort of COVID-19 patients followed up to 242 days, we found that individuals with mild or asymptomatic infection experienced an insignificant decay in neutralizing activity that persisted six months after symptom onset or diagnosis. Hospitalized individuals showed higher neutralizing titers, which decreased following a two-phase pattern, with an initial rapid decline that significantly slowed after day 80. Despite this initial decay, neutralizing activity at six months remained higher among hospitalized individuals. The slow decline in neutralizing activity at mid-term contrasted with the steep slope of antibody titers change, reinforcing the hypothesis that the quality of immune response evolves over the post-convalescent stage4,5.
ABSTRACT
BackgroundThere is a crucial need for effective therapies that are immediately available to counteract COVID-19 disease. Recently, ELISA binding cross-reactivity against components of human epidemic coronaviruses with currently available intravenous immunoglobulins (IVIG) Gamunex-C and Flebogamma DIF (5% and 10%) have been reported. In this study, the same products were tested for neutralization activity against SARS-CoV-2, SARS-CoV and MERS-CoV and their potential as an antiviral therapy. MethodsThe neutralization capacity of six selected lots of IVIG was assessed against SARS-CoV-2 (two different isolates), SARS-CoV and MERS-CoV in cell cultures. Infectivity neutralization was measured by determining the percent reduction in plaque-forming units (PFU) and by cytopathic effects for two IVIG lots in one of the SARS-CoV-2 isolates. Neutralization was quantified using the plaque reduction neutralization test 50 (PRNT50) in the PFU assay and the half maximal inhibitory concentration (IC50) in the cytopathic/cytotoxic method (calculated as the minus log10 dilution which reduced the viral titer by 50%). ResultsAll IVIG preparations showed neutralization of both SARS-CoV-2 isolates, ranging from 79 to 89.5% with PRNT50 titers from 4.5 to >5 for the PFU method and ranging from 47.0%-64.7% with an IC50 ~1 for the cytopathic method. All IVIG lots produced neutralization of SARS-CoV ranging from 39.5 to 55.1 % and PRNT50 values ranging from 2.0 to 3.3. No IVIG preparation showed significant neutralizing activity against MERS-CoV. ConclusionIn cell culture neutralization assays, the tested IVIG products contain antibodies with significant cross-neutralization capacity against SARS-CoV-2 and SARS-CoV. However, no neutralization capacity was demonstrated against MERS-CoV. These preparations are currently available and may be immediately useful for COVID-19 management.
ABSTRACT
There is an urgent need to identify therapeutics for the treatment of Coronavirus diseases 2019 (COVID-19). Although different antivirals are given for the clinical management of SARS-CoV-2 infection, their efficacy is still under evaluation. Here, we have screened existing drugs approved for human use in a variety of diseases, to compare how they counteract SARS-CoV-2-induced cytopathic effect and viral replication in vitro. Among the potential 72 antivirals tested herein that were previously proposed to inhibit SARS-CoV-2 infection, only 18% had an IC50 below 25 M or 102 IU/mL. These included plitidepsin, novel cathepsin inhibitors, nelfinavir mesylate hydrate, interferon 2-alpha, interferon-gamma, fenofibrate, camostat along the well-known remdesivir and chloroquine derivatives. Plitidepsin was the only clinically approved drug displaying nanomolar efficacy. Four of these families, including novel cathepsin inhibitors, blocked viral entry in a cell-type specific manner. Since the most effective antivirals usually combine therapies that tackle the virus at different steps of infection, we also assessed several drug combinations. Although no particular synergy was found, inhibitory combinations did not reduce their antiviral activity. Thus, these combinations could decrease the potential emergence of resistant viruses. Antivirals prioritized herein identify novel compounds and their mode of action, while independently replicating the activity of a reduced proportion of drugs which are mostly approved for clinical use. Combinations of these drugs should be tested in animal models to inform the design of fast track clinical trials.
