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.

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.

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.

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.

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.

Template-primer binding affinity and RNase H cleavage specificity contribute to the strand transfer efficiency of HIV-1 reverse transcriptase.

During reverse transcription of the HIV-1 genome, two strand-transfer events occur. Both events rely on the RNase H cleavage activity of reverse transcriptases (RTs) and template homology. Using a panel of mutants of HIV-1BH10 (group M/subtype B) and HIV-1ESP49 (group O) RTs and in vitro assays, we demonstrate that there is a strong correlation between RT minus-strand transfer efficiency and template-primer binding affinity. The highest strand transfer efficiencies were obtained with HIV-1ESP49 RT mutants containing the substitutions K358R/A359G/S360A, alone or in combination with V148I or T355A/Q357M. These HIV-1ESP49 RT mutants had been previously engineered to increase their DNA polymerase activity at high temperatures. Now, we found that RTs containing RNase H-inactivating mutations (D443N or E478Q) were devoid of strand transfer activity, whereas enzymes containing F61A or L92P had very low strand transfer activity. The strand transfer defect produced by L92P was attributed to a loss of template-primer binding affinity and, more specifically, to the higher dissociation rate constants (koff) shown by RTs bearing this substitution. Although L92P also deleteriously affected the RT's nontemplated nucleotide addition activity, neither nontemplated nucleotide addition activity nor the RT's clamp activities contributed to increased template switching when all tested mutant and WT RTs were considered. Interestingly, our results also revealed an association between efficient strand transfer and the generation of secondary cleavages in the donor RNA, consistent with the creation of invasion sites. Exposure of the elongated DNA at these sites facilitate acceptor (RNA or DNA) binding and promote template switching.

Unprecedented Thiacalixarene Fucoclusters as Strong Inhibitors of Ebola cis-Cell Infection and HCMV-gB Glycoprotein/DC-SIGN C-type Lectin Interaction.

Glycan-protein interactions control numerous biological events from cell-cell recognition and signaling to pathogen host cell attachment for infections. To infect cells, some viruses bind to immune cells with the help of DC-SIGN (dendritic cell [DC]-specific ICAM3-grabbing nonintegrin) C-type lectin expressed on dendritic and macrophage cell membranes, via their envelope protein. Prevention of this infectious interaction is a serious therapeutic option. Here, we describe the synthesis of the first water-soluble tetravalent fucocluster pseudopeptide-based 1,3-alternate thiacalixarenes as viral antigen mimics designed for the inhibition of DC-SIGN, to prevent viral particle uptake. Their preparation exploits straightforward convergent strategies involving one-pot Ugi four-component (Ugi-4CR) and azido-alkyne click chemistry reactions as key steps. Surface plasmon resonance showed strong inhibition of DC-SIGN interaction properties by tetravalent ligands designed with high relative potencies and ß avidity factors. All ligands block DC-SIGN active sites at nanomolar IC50 preventing cis-cell infection by Ebola viral particles pseudotyped with EBOV glycoprotein (Zaire species of Ebola virus) on Jurkat cells that express DC-SIGN. In addition, we observed strong inhibition of DC-SIGN/human cytomegalovirus (HCMV)-gB recombinant glycoprotein interaction. This finding opens the way to the simple development of new models of water-soluble glycocluster-based thia-calixarenes with wide-ranging antimicrobial activities.

Design, synthesis and biological evaluation of 3-hydroxyquinazoline-2,4(1H,3H)-diones as dual inhibitors of HIV-1 reverse transcriptase-associated RNase H and integrase.

A novel series of 3-hydroxyquinazoline-2,4(1H,3H)-diones derivatives has been designed and synthesized. Their biochemical characterization revealed that most of the compounds were effective inhibitors of HIV-1 RNase H activity at sub to low micromolar concentrations. Among them, II-4 was the most potent in enzymatic assays, showing an IC50 value of 0.41 ±â€¯0.13 µM, almost five times lower than the IC50 obtained with ß-thujaplicinol. In addition, II-4 was also effective in inhibiting HIV-1 IN strand transfer activity (IC50 = 0.85 ±â€¯0.18 µM) but less potent than raltegravir (IC50 = 71 ±â€¯14 nM). Despite its relatively low cytotoxicity, the efficiency of II-4 in cell culture was limited by its poor membrane permeability. Nevertheless, structure-activity relationships and molecular modeling studies confirmed the importance of tested 3-hydroxyquinazoline-2,4(1H,3H)-diones as useful leads for further optimization.

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.

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.

Glycofullerenes inhibit viral infection.

Water-soluble glycofullerenes based on a hexakis-adduct of [60]fullerene with an octahedral addition pattern are very attractive compounds providing a spherical presentation of carbohydrates. These tools have been recently described and they have been used to interact with lectins in a multivalent manner. Here, we present the use of these glycofullerenes, including new members with 36 mannoses, as compounds able to inhibit a DC-SIGN-dependent cell infection by pseudotyped viral particles. The results obtained in these experiments demonstrate for the first time that these glycoconjugates are adequate to inhibit efficiently an infection process, and therefore, they can be considered as very promising and interesting tools to interfere in biological events where lectins such as DC-SIGN are involved.

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.

Preclinical immune efficacy against SARS-CoV-2 beta B.1.351 variant by MVA-based vaccine candidates.

The constant appearance of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VoCs) has jeopardized the protective capacity of approved vaccines against coronavirus disease-19 (COVID-19). For this reason, the generation of new vaccine candidates adapted to the emerging VoCs is of special importance. Here, we developed an optimized COVID-19 vaccine candidate using the modified vaccinia virus Ankara (MVA) vector to express a full-length prefusion-stabilized SARS-CoV-2 spike (S) protein, containing 3 proline (3P) substitutions in the S protein derived from the beta (B.1.351) variant, termed MVA-S(3Pbeta). Preclinical evaluation of MVA-S(3Pbeta) in head-to-head comparison to the previously generated MVA-S(3P) vaccine candidate, expressing a full-length prefusion-stabilized Wuhan S protein (with also 3P substitutions), demonstrated that two intramuscular doses of both vaccine candidates fully protected transgenic K18-hACE2 mice from a lethal challenge with SARS-CoV-2 beta variant, reducing mRNA and infectious viral loads in the lungs and in bronchoalveolar lavages, decreasing lung histopathological lesions and levels of proinflammatory cytokines in the lungs. Vaccination also elicited high titers of anti-S Th1-biased IgGs and neutralizing antibodies against ancestral SARS-CoV-2 Wuhan strain and VoCs alpha, beta, gamma, delta, and omicron. In addition, similar systemic and local SARS-CoV-2 S-specific CD4+ and CD8+ T-cell immune responses were elicited by both vaccine candidates after a single intranasal immunization in C57BL/6 mice. These preclinical data support clinical evaluation of MVA-S(3Pbeta) and MVA-S(3P), to explore whether they can diversify and potentially increase recognition and protection of SARS-CoV-2 VoCs.

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.

Dendritic Cell-Mediated Cross-Priming by a Bispecific Neutralizing Antibody Boosts Cytotoxic T Cell Responses and Protects Mice against SARS-CoV-2.

Administration of neutralizing antibodies (nAbs) has proved to be effective by providing immediate protection against SARS-CoV-2. However, dual strategies combining virus neutralization and immune response stimulation to enhance specific cytotoxic T cell responses, such as dendritic cell (DC) cross-priming, represent a promising field but have not yet been explored. Here, a broadly nAb, TNT , are first generated by grafting an anti-RBD biparatopic tandem nanobody onto a trimerbody scaffold. Cryo-EM data show that the TNT structure allows simultaneous binding to all six RBD epitopes, demonstrating a high-avidity neutralizing interaction. Then, by C-terminal fusion of an anti-DNGR-1 scFv to TNT , the bispecific trimerbody TNT DNGR-1 is generated to target neutralized virions to type 1 conventional DCs (cDC1s) and promote T cell cross-priming. Therapeutic administration of TNT DNGR-1, but not TNT , protects K18-hACE2 mice from a lethal SARS-CoV-2 infection, boosting virus-specific humoral responses and CD8+ T cell responses. These results further strengthen the central role of interactions with immune cells in the virus-neutralizing antibody activity and demonstrate the therapeutic potential of the Fc-free strategy that can be used advantageously to provide both immediate and long-term protection against SARS-CoV-2 and other viral infections.

Novel RNase H Inhibitors Blocking RNA-directed Strand Displacement DNA Synthesis by HIV-1 Reverse Transcriptase.

In retroviruses, strand displacement DNA-dependent DNA polymerization catalyzed by the viral reverse transcriptase (RT) is required to synthesize double-stranded proviral DNA. In addition, strand displacement during RNA-dependent DNA synthesis is critical to generate high-quality cDNA for use in molecular biology and biotechnology. In this work, we show that the loss of RNase H activity due to inactivating mutations in HIV-1 RT (e.g. D443N or E478Q) has no significant effect on strand displacement while copying DNA templates, but has a large impact on DNA polymerization in reactions carried out with RNA templates. Similar effects were observed with ß-thujaplicinol and other RNase H active site inhibitors, including compounds with dual activity (i.e., characterized also as inhibitors of HIV-1 integrase and/or the RT DNA polymerase). Among them, dual inhibitors of HIV-1 RT DNA polymerase/RNase H activities, containing a 7-hydroxy-6-nitro-2H-chromen-2-one pharmacophore were found to be very potent and effective strand displacement inhibitors in RNA-dependent DNA polymerization reactions. These findings might be helpful in the development of transcriptomics technologies to obtain more uniform read coverages when copying long RNAs and for the construction of more representative libraries avoiding biases towards 5' and 3' ends, while providing valuable information for the development of novel antiretroviral agents.