C60-based Multivalent Glycoporphyrins Inhibit SARS-CoV-2 Specific Interaction with the DC-SIGN Transmembrane Receptor.

Since WHO has declared the COVID-19 outbreak a global pandemic, nearly seven million deaths have been reported. This efficient spread of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is facilitated by the ability of the spike glycoprotein to bind multiple cell membrane receptors. Although ACE2 is identified as the main receptor for SARS-CoV-2, other receptors could play a role in viral entry. Among others, C-type lectins such as DC-SIGN are identified as efficient trans-receptor for SARS-CoV-2 infection, so the use of glycomimetics to inhibit the infection through the DC-SIGN blockade is an encouraging approach. In this regard, multivalent nanostructures based on glycosylated [60]fullerenes linked to a central porphyrin scaffold have been designed and tested against DC-SIGN-mediated SARS-CoV-2 infection. First results show an outstanding inhibition of the trans-infection up to 90%. In addition, a deeper understanding of nanostructure-receptor binding is achieved through microscopy techniques, high-resolution NMR experiments, Quartz Crystal Microbalance experiments, and molecular dynamic simulations.

Superior neutralizing response after first versus second SARS-CoV-2 infection in fully vaccinated individuals.

Currently, the majority of the population has been vaccinated against COVID-19 and/or has experienced SARS-CoV-2 infection either before or after vaccination. The immunological response to repeated episodes of infections is not completely clear. We measured SARS-CoV-2 specific neutralization titers by a pseudovirus assay after BA.1 infection and RBD-specific immunoglobulin G (IgG), immunoglobulin A (IgA), and immunoglobulin M (IgM) in a cohort of COVID-19 uninfected and triple vaccinated individuals (breakthrough infection group, BTI) as compared with those previously infected by SARS-CoV-2 (reinfection group, REI) who underwent identical vaccination schedule. SARS-CoV-2 specific neutralizing response after BA.1 infection was significantly higher in the BTI group as compared with the REI. Furthermore, neutralization titers in REI were not significant different from convalescent non reinfected controls. RBD-specific IgG and IgA, but not IgM, were also significantly higher in BTI as compared with REI. Our results show that the first episode of SARS-CoV-2 infection induces a significant increase in neutralizing titers in triple vaccinated individuals and that previous SARS-CoV-2 infection compromise significantly the neutralization response induced by reinfection, even by divergent SARS-CoV-2 variants and at least up to 2 years postinfection, suggesting a fundamental limitation in inducing effective booster through the intranasal route in previously infected individuals.

MAFB shapes human monocyte-derived macrophage response to SARS-CoV-2 and controls severe COVID-19 biomarker expression.

Monocyte-derived macrophages, the major source of pathogenic macrophages in COVID-19, are oppositely instructed by macrophage CSF (M-CSF) or granulocyte macrophage CSF (GM-CSF), which promote the generation of antiinflammatory/immunosuppressive MAFB+ (M-MØ) or proinflammatory macrophages (GM-MØ), respectively. The transcriptional profile of prevailing macrophage subsets in severe COVID-19 led us to hypothesize that MAFB shapes the transcriptome of pulmonary macrophages driving severe COVID-19 pathogenesis. We have now assessed the role of MAFB in the response of monocyte-derived macrophages to SARS-CoV-2 through genetic and pharmacological approaches, and we demonstrate that MAFB regulated the expression of the genes that define pulmonary pathogenic macrophages in severe COVID-19. Indeed, SARS-CoV-2 potentiated the expression of MAFB and MAFB-regulated genes in M-MØ and GM-MØ, where MAFB upregulated the expression of profibrotic and neutrophil-attracting factors. Thus, MAFB determines the transcriptome and functions of the monocyte-derived macrophage subsets that underlie pulmonary pathogenesis in severe COVID-19 and controls the expression of potentially useful biomarkers for COVID-19 severity.

The role of DC-SIGN as a trans-receptor in infection by MERS-CoV.

DC-SIGN is a C-type lectin expressed in myeloid cells such as immature dendritic cells and macrophages. Through glycan recognition in viral envelope glycoproteins, DC-SIGN has been shown to act as a receptor for a number of viral agents such as HIV, Ebola virus, SARS-CoV, and SARS-CoV-2. Using a system of Vesicular Stomatitis Virus pseudotyped with MERS-CoV spike protein, here, we show that DC-SIGN is partially responsible for MERS-CoV infection of dendritic cells and that DC-SIGN efficiently mediates trans-infection of MERS-CoV from dendritic cells to susceptible cells, indicating a potential role of DC-SIGN in MERS-CoV dissemination and pathogenesis.

TLR7 Activation in M-CSF-Dependent Monocyte-Derived Human Macrophages Potentiates Inflammatory Responses and Prompts Neutrophil Recruitment.

Toll-like receptor 7 (TLR7) is an endosomal pathogen-associated molecular pattern (PAMP) receptor that senses single-stranded RNA (ssRNA) and whose engagement results in the production of type I IFN and pro-inflammatory cytokines upon viral exposure. Recent genetic studies have established that a dysfunctional TLR7-initiated signaling is directly linked to the development of inflammatory responses. We present evidence that TLR7 is preferentially expressed by monocyte-derived macrophages generated in the presence of M-CSF (M-MØ). We now show that TLR7 activation in M-MØ triggers a weak MAPK, NFκB, and STAT1 activation and results in low production of type I IFN. Of note, TLR7 engagement reprograms MAFB+ M-MØ towards a pro-inflammatory transcriptional profile characterized by the expression of neutrophil-attracting chemokines (CXCL1-3, CXCL5, CXCL8), whose expression is dependent on the transcription factors MAFB and AhR. Moreover, TLR7-activated M-MØ display enhanced pro-inflammatory responses and a stronger production of neutrophil-attracting chemokines upon secondary stimulation. As aberrant TLR7 signaling and enhanced pulmonary neutrophil/lymphocyte ratio associate with impaired resolution of virus-induced inflammatory responses, these results suggest that targeting macrophage TLR7 might be a therapeutic strategy for viral infections where monocyte-derived macrophages exhibit a pathogenic role.

N'-Phenylacetohydrazide Derivatives as Potent Ebola Virus Entry Inhibitors with an Improved Pharmacokinetic Profile.

Ebola virus (EBOV) is a single-strand RNA virus belonging to the Filoviridae family, which has been associated to most Ebola virus disease outbreaks to date, including the West African and the North Kivu epidemics between 2013 and 2022. This unprecedented health emergency prompted the search for effective medical countermeasures. Following up on the carbazole hit identified in our previous studies, we synthetized a new series of compounds, which demonstrated to prevent EBOV infection in cells by acting as virus entry inhibitors. The in vitro inhibitory activity was evaluated through the screening against surrogate models based on viral pseudotypes and further confirmed using replicative EBOV. Docking and molecular dynamics simulations joined to saturation transfer difference-nuclear magnetic resonance (STD-NMR) and mutagenesis experiments to elucidate the biological target of the most potent compounds. Finally, in vitro metabolic stability and in vivo pharmacokinetic studies were performed to confirm their therapeutic potential.

Cross neutralization of SARS-CoV-2 omicron subvariants after repeated doses of COVID-19 mRNA vaccines.

We have measured the humoral response to messenger RNA (mRNA) vaccines in COVID-19 naïve and convalescent individuals. Third doses of mRNA COVID-19 vaccines induced a significant increase in potency and breadth of neutralization against SARS-CoV-2 variants of concern (VoC) including Omicron subvariants BA.1, BA.2, and BA.2.12.1, that were cross-neutralized at comparable levels and less for BA.4/5. This booster effect was especially important in naïve individuals that only after the third dose achieved a level that was comparable with that of vaccinated COVID-19 convalescents except for BA.4/5. Avidity of RBD-binding antibodies was also significantly increased in naïve individuals after the third dose, indicating an association between affinity maturation and cross neutralization of VoC. These results suggest that at least three antigenic stimuli by infection or vaccination with ancestral SARS-CoV-2 sequences are required to induce high avidity cross-neutralizing antibodies. Nevertheless, the circulation of new subvariants such as BA.4/5 with partial resistance to neutralization will have to be closely monitored and eventually consider for future vaccine developments.

Potent Neutralizing Activity of Polyclonal Equine Antibodies Against Severe Acute Respiratory Syndrome Coronavirus 2 Variants of Concern.

Several anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) monoclonal antibodies (mAbs) have received emergency authorization for coronavirus disease 2019 (COVID-19) treatment. However, most of these mAbs are not active against the highly mutated Omicron SARS-CoV-2 subvariants. We have tested a polyclonal approach of equine anti-SARS-CoV-2 F(ab')2 antibodies that achieved a high level of neutralizing potency against all SARS-CoV-2 variants of concern tested including Omicron BA.1, BA.2, BA.2.12 and BA.4/5. A repertoire of antibodies targeting conserved epitopes in different regions of the spike protein could plausibly account for this remarkable breadth of neutralization. These results warrant the clinical investigation of equine polyclonal F(ab')2 antibodies as a novel therapeutic strategy against COVID-19.

Topological and Multivalent Effects in Glycofullerene Oligomers as EBOLA Virus Inhibitors.

The synthesis of new biocompatible antiviral materials to fight against the development of multidrug resistance is being widely explored. Due to their unique globular structure and excellent properties, [60]fullerene-based antivirals are very promising bioconjugates. In this work, fullerene derivatives with different topologies and number of glycofullerene units were synthesized by using a SPAAC copper free strategy. This procedure allowed the synthesis of compounds 1-3, containing from 20 to 40 mannose units, in a very efficient manner and in short reaction times under MW irradiation. The glycoderivatives were studied in an infection assay by a pseudotyped viral particle with Ebola virus GP1. The results obtained show that these glycofullerene oligomers are efficient inhibitors of EBOV infection with IC50s in the nanomolar range. In particular, compound 3, with four glycofullerene moieties, presents an outstanding relative inhibitory potency (RIP). We propose that this high RIP value stems from the appropriate topological features that efficiently interact with DC-SIGN.

New insights into the role of endosomal proteins for African swine fever virus infection.

African swine fever virus (ASFV) infectious cycle starts with the viral adsorption and entry into the host cell. Then, the virus is internalized via clathrin/dynamin mediated endocytosis and macropinocytosis. Similar to other viruses, ASF virion is then internalized and incorporated into the endocytic pathway. While the endosomal maturation entails luminal acidification, the decrease in pH acts on the multilayer structure of the virion dissolving the outer capsid. Upon decapsidation, the inner viral membrane is exposed to interact with the limiting membrane of the late endosome for fusion. Viral fusion is then necessary for the egress of incoming virions from endosomes into the cytoplasm, however this remains an intriguing and yet essential process for infection, specifically for the egress of viral nucleic acid into the cytoplasm for replication. ASFV proteins E248R and E199L, located at the exposed inner viral membrane, might be implicated in the fusion step. An interaction between these viral proteins and cellular endosomal proteins such as the Niemann-Pick C type 1 (NPC1) and lysosomal membrane proteins (Lamp-1 and -2) was shown. Furthermore, the silencing of these proteins impaired ASFV infection. It was also observed that NPC1 knock-out cells using CRISPR jeopardized ASFV infection and that the progression and endosomal exit of viral cores was arrested within endosomes at viral entry. These results suggest that the interactions of ASFV proteins with some endosomal proteins might be important for the membrane fusion step. In addition to this, reductions on ASFV infectivity and replication in NPC1 KO cells were accompanied by fewer and smaller viral factories. Our findings pave the way to understanding the role of proteins of the endosomal membrane in ASFV infection.

Neutralizing Response Against SARS-CoV-2 Variants 8 Months After BNT162b2 Vaccination in Naive and COVID-19-Convalescent Individuals.

We have investigated the evolution of the neutralizing response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants at 8 months after Pfizer-BNT162b2 vaccination in coronavirus disease 2019 (COVID-19)-naive (n = 21) and COVID-19-convalescent (n = 21) individuals. Neutralizing levels declined for all variants (range 2- to 3.7-fold). Eight months after vaccination, a significant proportion (4/21) of naive individuals lacked detectable neutralizing activity against the highly transmissible SARS-CoV-2 delta variant. In the convalescent group, the impressive high initial humoral response resulted in detectable neutralizing antibody levels against all variants throughout this period.

Prime-Boost Vaccination With BNT162b2 Induces High Neutralizing Activity Against SARS-CoV-2 Variants in Naïve and COVID-19-Convalescent Individuals.

BACKGROUND: The objective of this study was to investigate the neutralizing response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VoC) during coronavirus disease 2019 (COVID-19) convalescence and after vaccination. METHODS: COVID-19-convalescent and -naïve individuals were tested for neutralizing activity against SARS-CoV-2 VoC Alpha, Beta, Gamma, and Delta at 1 and 7 months postinfection and 4-6 weeks after BNT162b2 vaccination. RESULTS: Vaccination induced a high neutralizing response in naïve individuals. Interestingly, vaccination of convalescent patients induced a boosted response that was able to neutralize all VoC at high titers. CONCLUSIONS: Vaccination with BNT162b2 induced high levels of neutralization against SARS-CoV-2 VoC in most patients; this is especially beneficial in COVID-19-convalescent individuals.

DC/L-SIGN recognition of spike glycoprotein promotes SARS-CoV-2 trans-infection and can be inhibited by a glycomimetic antagonist.

The efficient spread of SARS-CoV-2 resulted in a unique pandemic in modern history. Despite early identification of ACE2 as the receptor for viral spike protein, much remains to be understood about the molecular events behind viral dissemination. We evaluated the contribution of C-type lectin receptors (CLRS) of antigen-presenting cells, widely present in respiratory mucosa and lung tissue. DC-SIGN, L-SIGN, Langerin and MGL bind to diverse glycans of the spike using multiple interaction areas. Using pseudovirus and cells derived from monocytes or T-lymphocytes, we demonstrate that while virus capture by the CLRs examined does not allow direct cell infection, DC/L-SIGN, among these receptors, promote virus transfer to permissive ACE2+ Vero E6 cells. A glycomimetic compound designed against DC-SIGN, enable inhibition of this process. These data have been then confirmed using authentic SARS-CoV-2 virus and human respiratory cell lines. Thus, we described a mechanism potentiating viral spreading of infection.

Prolonged SARS-CoV-2 cell culture replication in respiratory samples from patients with severe COVID-19.

OBJECTIVES: This study compares the infectivity of SARS-CoV-2 in respiratory samples from patients with mild COVID-19 with those from hospitalized patients with severe bilateral pneumonia. In severe COVID-19, we also analysed the presence of neutralizing activity in paired sera. METHODS: We performed cell cultures on 193 real-time reverse transcription polymerase chain reaction respiratory samples, positive for SARS-CoV-2, obtained from 189 patients at various times, from clinical diagnosis to follow-up. Eleven samples were obtained from asymptomatic individuals, 91 samples from 91 outpatients with mild forms of COVID-19 and 91 samples from 87 inpatients with severe pneumonia. In these patients, neutralizing activity was analysed in 30 paired sera collected after symptom onset >10 days. RESULTS: We detected a cytopathic effect (CPE) in 91/193 (47%) samples. Viral viability was maintained for up to 10 days in patients with mild COVID-19. In patients with severe COVID-19, the virus remained viable for up to 32 days after the onset of symptoms. Patients with severe COVID-19 presented infectious virus at a significantly higher rate in the samples with moderate to low viral load (cycle threshold value ≥ 26): 32/75 (43%) versus 14/63 (22%) for mild cases (p < 0.01). We observed a positive CPE despite the presence of clear neutralizing activity (NT50 > 1:1024 in 10% (3/30) of samples. DISCUSSION: Patients with severe COVID-19 might shed viable virus during prolonged periods of up to 4 weeks after symptom onset, even when presenting high cycle threshold values in their respiratory samples and despite having developed high neutralizing antibody titres.

Identification of potential inhibitors of protein-protein interaction useful to fight against Ebola and other highly pathogenic viruses.

Despite the efforts to develop new treatments against Ebola virus (EBOV) there is currently no antiviral drug licensed to treat patients with Ebola virus disease (EVD). Therefore, there is still an urgent need to find new drugs to fight against EBOV. In order to do this, a virtual screening was done on the druggable interaction between the EBOV glycoprotein (GP) and the host receptor NPC1 with a subsequent selection of compounds for further validation. This screening led to the identification of new small organic molecules with potent inhibitory action against EBOV infection using lentiviral EBOV-GP-pseudotype viruses. Moreover, some of these compounds have shown their ability to interfere with the intracellular cholesterol transport receptor NPC1 using an ELISA-based assay. These preliminary results pave the way to hit to lead optimization programs that lead to successful candidates.

Synthesis of Highly Efficient Multivalent Disaccharide/[60]Fullerene Nanoballs for Emergent Viruses.

After the last epidemic of the Zika virus (ZIKV) in Brazil that peaked in 2016, growing evidence has been demonstrated of the link between this teratogenic flavivirus and microcephaly cases. However, no vaccine or antiviral drug has been approved yet. ZIKV and Dengue viruses (DENV) entry to the host cell takes place through several receptors, including dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), so that the blockade of this receptor through multivalent glycoconjugates supposes a promising biological target to inhibit the infection process. In order to get enhanced multivalency in biocompatible systems, tridecafullerenes appended with up to 360 1,2-mannobiosides have been synthesized using a strain-promoted cycloaddition of azides to alkynes (SPAAC) strategy. These systems have been tested against ZIKV and DENV infection, showing an outstanding activity in the picomolar range.

Survey of Crimean-Congo Hemorrhagic Fever Enzootic Focus, Spain, 2011-2015.

During 2011-2015, we conducted a Crimean-Congo hemorrhagic fever virus (CCHFV) survey in captured ticks that were feeding mainly on wild and domestic ungulates in Spain, where presence of this virus had been reported previously. We detected CCHFV RNA in Hyalomma lusitanicum and H. marginatum ticks for 3 of the 5 years. The rate of infected ticks was 2.78% (44/1,579), which was similar to those for other countries in Europe with endemic foci for CCHFV (Kosovo, Bulgaria, and Albania). These data confirm the established spread of CCHFV into western Europe. Phylogenetic study of the small RNA segment showed Africa-3 clade as the only genotype identified, although we observed cocirculation of genetic variants during 2011 and 2015. We could not rule out genetic reassortments because of lack of sequence data for the medium and large RNA segments of the virus genome.

Nanocarbon-Based Glycoconjugates as Multivalent Inhibitors of Ebola Virus Infection.

SWCNTs, MWCNTs, and SWCNHs have been employed as virus-mimicking nanocarbon platforms for the multivalent presentation of carbohydrates in an artificial Ebola virus infection model assay. These carbon nanoforms have been chemically modified by the covalent attachment of glycodendrons and glycofullerenes using the CuAAC "click chemistry" approach. This modification dramatically increases the water solubility of these structurally different nanocarbons. Their efficiency in blocking DC-SIGN-mediated viral infection by an artificial Ebola virus has been tested in a cellular experimental assay, finding that glycoconjugates based on MWCNTs functionalized with glycofullerenes are potent inhibitors of viral infection.

Broad Neutralizing Activity Against Ebolaviruses Lacking the Mucin-Like Domain in Convalescent Plasma Specimens From Patients With Ebola Virus Disease.

Background: In Ebola virus (EBOV) infection, the specific neutralizing activity of convalescent plasma against other members of the Ebolavirus genus has not been extensively analyzed. Methods: We measured the neutralizing activity in plasma from 3 survivors of the recent outbreak due to the Makona variant of EBOV and tested its neutralizing potency against other variants of EBOV (ie, Mayinga and Kikwit) and against Sudan virus (SUDV), Bundibugyo virus (BDBV), and Reston virus (RESTV), using a glycoprotein (GP)-pseudotyped lentiviral system both with full-length GP and in vitro-cleaved GP (GPCL). Results: Convalescent plasma specimens from survivors of EBOV infection showed low neutralizing activity against full-length GPs of SUDV, BDBV, RESTV, and EBOV variants Mayinga and Kikwit. However, broad and potent neutralizing activity was observed against the GPCL forms of SUDV, BDBV, and RESTV. Discussion: Removal of the mucin-like domain and glycan cap from the GP of members of the Ebolavirus genus presumably exposes conserved epitopes in or in the vicinity of the receptor binding site and internal fusion loop that are readily amenable to neutralization. These types of broad neutralizing antibodies could be induced by using immunogens mimicking GPCL.

Autochthonous Crimean-Congo Hemorrhagic Fever in Spain.

Crimean-Congo hemorrhagic fever (CCHF) is a widely distributed, viral, tickborne disease. In Europe, cases have been reported only in the southeastern part of the continent. We report two autochthonous cases in Spain. The index patient acquired the disease through a tick bite in the province of Ávila - 300 km away from the province of Cáceres, where viral RNA from ticks was amplified in 2010. The second patient was a nurse who became infected while caring for the index patient. Both were infected with the African 3 lineage of this virus. (Funded by Red de Investigación Cooperativa en Enfermedades Tropicales [RICET] and Efficient Response to Highly Dangerous and Emerging Pathogens at EU [European Union] Level [EMERGE].).