SARS-CoV-2 B.1.351 (beta) variant shows enhanced infectivity in K18-hACE2 transgenic mice and expanded tropism to wildtype mice compared to B.1 variant

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.

Clinical course impacts early kinetics and long-term magnitude and amplitude of SARS-CoV-2 neutralizing antibodies beyond one year after infection

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.

Elucidation of ligand binding and dimerization of NADPH:protochlorophyllide (Pchlide) oxidoreductase from pea (Pisum sativum L.) by structural analysis and simulations.

NADPH:protochlorophyllide (Pchlide) oxidoreductase (POR) is a key enzyme of chlorophyll biosynthesis in angiosperms. It is one of few known photoenzymes, which catalyzes the light-activated trans-reduction of the C17-C18 double bond of Pchlide's porphyrin ring. Due to the light requirement, dark-grown angiosperms cannot synthesize chlorophyll. No crystal structure of POR is available, so to improve understanding of the protein's three-dimensional structure, its dimerization, and binding of ligands (both the cofactor NADPH and substrate Pchlide), we computationally investigated the sequence and structural relationships among homologous proteins identified through database searches. The results indicate that α4 and α7 helices of monomers form the interface of POR dimers. On the basis of conserved residues, we predicted 11 functionally important amino acids that play important roles in POR binding to NADPH. Structural comparison of available crystal structures revealed that they participate in formation of binding pockets that accommodate the Pchlide ligand, and that five atoms of the closed tetrapyrrole are involved in non-bonding interactions. However, we detected no clear pattern in the physico-chemical characteristics of the amino acids they interact with. Thus, we hypothesize that interactions of these atoms in the Pchlide porphyrin ring are important to hold the ligand within the POR binding site. Analysis of Pchlide binding in POR by molecular docking and PELE simulations revealed that the orientation of the nicotinamide group is important for Pchlide binding. These findings highlight the complexity of interactions of porphyrin-containing ligands with proteins, and we suggest that fit-inducing processes play important roles in POR-Pchlide interactions.

Siglec-1 on dendritic cells mediates SARS-CoV-2 trans-infection of target cells while on macrophages triggers proinflammatory responses

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.

Previous SARS-CoV-2 infection increases B.1.1.7 cross-neutralization by vaccinated individuals.

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.

Protection against reinfection with D614- or G614-SARS-CoV-2 isolates in hamsters

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.

Stable neutralizing antibody levels six months after mild and severe COVID-19 episode

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.

Search for SARS-CoV-2 inhibitors in currently approved drugs to tackle COVID-19 pandemia

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.