Particulate multivalent presentation of the receptor binding domain induces protective immune responses against MERS-CoV

Middle East respiratory syndrome coronavirus (MERS-CoV) is a WHO priority pathogen for which vaccines are urgently needed. Using an immune-focusing approach, we created self-assembling particles multivalently displaying critical regions of the MERS-CoV spike protein ─fusion peptide, heptad repeat 2, and receptor binding domain (RBD) ─ and tested their immunogenicity and protective capacity in rabbits. Using a "plug-and-display" SpyTag/SpyCatcher system, we coupled RBD to lumazine synthase (LS) particles producing multimeric RBD-presenting particles (RBD-LS). RBD-LS vaccination induced antibody responses of high magnitude and quality (avidity, MERS-CoV neutralizing capacity, and mucosal immunity) with cross-clade neutralization. The antibody responses were associated with blocking viral replication and upper and lower respiratory tract protection against MERS-CoV infection in rabbits. This arrayed multivalent presentation of the viral RBD using the antigen-SpyTag/LS-SpyCatcher is a promising MERS-CoV vaccine candidate and this platform may be applied for the rapid development of vaccines against other emerging vi

Chimeric camel/human heavy-chain antibodies protect against MERS-CoV infection

Middle East respiratory syndrome coronavirus (MERS-CoV) continues to cause outbreaks in humans as a result of spillover events from dromedaries. In contrast to humans, MERS-CoV–exposed dromedaries develop only very mild infections and exceptionally potent virus-neutralizing antibody responses. These strong antibody responses may be caused by affinity maturation as a result of repeated exposure to the virus or by the fact that dromedaries—apart from conventional antibodies—have relatively unique, heavy chain–only antibodies (HCAbs). These HCAbs are devoid of light chains and have long complementarity-determining regions with unique epitope binding properties, allowing them to recognize and bind with high affinity to epitopes not recognized by conventional antibodies. Through direct cloning and expression of the variable heavy chains (VHHs) of HCAbs from the bone marrow of MERS-CoV–infected dromedaries, we identified several MERS-CoV–specific VHHs or nanobodies. In vitro, these VHHs efficiently blocked virus entry at picomolar concentrations. The selected VHHs bind with exceptionally high affinity to the receptor binding domain of the viral spike protein. Furthermore, camel/human chimeric HCAbs—composed of the camel VHH linked to a human Fc domain lacking the CH1 exon—had an extended half-life in the serum and protected mice against a lethal MERS-CoV challenge. HCAbs represent a promising alternative strategy to develop novel interventions not only for MERS-CoV but also for other emerging pathogens.info:eu-repo/semantics/publishedVersio

Isolation of cross-reactive monoclonal antibodies against divergent human coronaviruses that delineate a conserved and vulnerable site on the spike protein

The coronavirus spike glycoprotein, located on the virion surface, is the key mediator of cell entry. As such, it is an attractive target for the development of protective antibodies and vaccines. Here we describe two human monoclonal antibodies, 1.6C7 and 28D9, that display a remarkable cross-reactivity against distinct species from three Betacoronavirus subgenera, capable of binding the spike proteins of SARS-CoV and SARS-CoV-2, MERS-CoV and the endemic human coronavirus HCoV-OC43. Both antibodies, derived from immunized transgenic mice carrying a human immunoglobulin repertoire, blocked MERS-CoV infection in cells, whereas 28D9 also showed weak cross-neutralizing potential against HCoV-OC43, SARS-CoV and SARS-CoV-2 in a neutralization-sensitive virus pseudotyping system, but not against authentic virus. Both cross-reactive monoclonal antibodies were found to target the stem helix in the spike protein S2 fusion subunit which, in the prefusion conformation of trimeric spike, forms a surface exposed membrane-proximal helical bundle, that is antibody-accessible. We demonstrate that administration of these antibodies in mice protects from a lethal MERS-CoV challenge in both prophylactic and/or therapeutic models. Collectively, these antibodies delineate a conserved, immunogenic and vulnerabe site on the spike protein which spurs the development of broad-range diagnostic, preventive and therapeutic measures against coronaviruses.The project was co-financed by a grant from the Zoonotic Anticipation and Preparedness Initiative [ZAPI project; Innovative Medicines Initiative (IMI) grant agreement no. 115760], with the assistance and financial support of IMI and the European Commission, and in-kind contributions from European Federation of Pharmaceutical Industries and Associations partners. The collaboration project is cofunded by the PPP Allowance made available by Health~Holland, Top Sector Life Sciences & Health, to stimulate public-private partnerships. This study was also partially financed by grants from the Ministry of Science and Innovation of Spain (BIO2016-75549-R AEI/FEDER, UE) and NIH (2PO1AIO6O699). The mice used to generate the mAbs produced in this study were provided by Harbour Antibodies BV, a daughter company of Harbour Biomed (http://www.harbourbiomed.com). Chunyan Wang was supported by a grant from the China Scholarship Council.Peer reviewe

In silico structure-based design and synthesis of novel anti-RSV compounds

Respiratory syncytial virus (RSV) is the major cause for respiratory tract disease in infants and young children. Currently, no licensed vaccine or a selective antiviral drug against RSV infections are available. Here, we describe a structure-based drug design approach that led to the synthesis of a novel series of zinc-ejecting compounds active against RSV replication. 30 compounds, sharing a common dithiocarbamate moiety, were designed and prepared to target the zinc finger motif of the M2-1 protein. A library of ∼12,000 small fragments was docked to explore the area surrounding the zinc ion. Among these, seven ligands were selected and used for the preparation of the new derivatives. The results reported here may help the development of a lead compound for the treatment of RSV infections

Schematic representations of the dual luciferase reporter constructs, and of transfection and infection assays.

<p>A) Schematic outline of the firefly and <i>Gaussia</i> luciferase reporter constructs. The firefly and <i>Gaussia</i> luciferase genes, flanked by 3′ and 5′ UTR of the NP segment, were inserted in antisense orientation between a PolI promoter and a ribozyme sequence, resulting in FNP and GNP, respectively. The extended <i>Gaussia</i> luciferase reporter construct (GFsNP) additionally contains the 3′ terminal half of the firefly luciferase gene (indicated as Fs) behind the stop codon of the <i>Gaussia</i> gene. B) HEK 293T cells were transfected with one or both reporter constructs (single or co-transfection). Luciferase expression is induced by expression of viral RNA polymerases (PB1, PB2, PA) and NP either by simultaneous co-transfection of expression plasmids (transfection assay) or by infection with IAV at an MOI 1 at 24 h post-transfection (infection assay). The firefly and <i>Gaussia</i> luciferase expression levels can be measured consecutively using a dual luciferase assay system (Promega) 24 h post-transfection or post-infection.</p

vRNA segment lengths and UTR sequences of IAV-WSN used in the reporter constructs.

a<p>The conserved regions in the 3′ and 5′ UTRs are underlined. The start and stop codons are italicized. The bold characters indicate the mutated nucleotides.</p

The effect of gene length and segment UTR.

<p>A) Plasmids encoding firefly (FNP) or <i>Gaussia</i> luciferase reporter constructs were transfected alone (Single; s) or in combination (Co; c). Luciferase expression was induced by simultaneous co-transfection of polymerase and NP expression plasmids (transfection assay). A) Normalized ratio of firefly to <i>Gaussia</i> luciferase activity (Fluc/Gluc) after single or co-transfection of FNP and GNP or GFsNP (extended version). B) Normalized ratio of firefly to <i>Gaussia</i> luciferase activity (Fluc/Gluc) after single or co-transfection of FNP and different versions of the extended <i>Gaussia</i> reporter construct carrying UTRs derived from the eight IAV-WSN genome segments. C) Correlation between fold inhibition of firefly luciferase expression upon co-transfection of a <i>Gaussia</i> luciferase reporter construct and the corresponding <i>Gaussia</i> luciferase expression levels after single transfection is shown.</p

The effect of panhandle-stabilizing mutations in the UTR.

<p>A) Schematic representation of the proposed conformational structure of IAV-WSN wild type NP UTR in corkscrew or panhandle conformation (left panel; refs 10 and 30) and the improved base-pairing by panhandle-stabilizing mutations in the 3′ (NPph) or 5′ (NPphR) UTR (right panel). B) Normalized ratio of firefly to <i>Gaussia</i> luciferase activity (Fluc/Gluc) after single or co-transfection of FNP and different versions of the extended <i>Gaussia</i> reporter construct carrying either NP, NPph or NPphR UTRs (GFsNP, GFsNPph, and GFsNPphR, respectively). C) Normalized ratio of firefly to <i>Gaussia</i> luciferase activity (Fluc/Gluc) after single or co-transfection of firefly luciferase constructs with NP or NPphs UTR (FNP and FNPph, respectively) and the short or extended <i>Gaussia</i> reporter construct carrying either NP (GNP and GFsNP) or NPph (GNPph and GFsNPph) UTRs.</p

Comparison of transfection and infection assay.

<p>Luciferase activity of firefly (FNP or FNPph) or <i>Gaussia</i> (GNP or GNPph) luciferase reporter constructs using the transfection (A) or infection assay (B). B) Cells were infected with IAV-WSN at an MOI of 1 TCID50 units per cells, which resulted in approximately 50% infected cells as determined by immunocytochemical analysis using NP-specific antibodies. C) Fold difference in luciferase expression levels between FNPph and FNP and between GNPph and GNP in either the transfection or infection assay. D) Quantitative RT-PCR analysis of mRNA levels derived from GNP or GNPph using the transfection (trans) or infection (inf) assay. For the transfection assay, cells were co-transfected with expression plasmids encoding PB1, PB2, PA and NP. For the infection assay, cells transfected with reporter plasmids were infected with IAV-WSN. The comparative Ct method was used to determine the relative mRNA levels using the housekeeping gene GAPDH as a reference. The mRNA levels were normalized relative to the mRNA expression level of the GNP reporter construct in the transfection assay.</p