Targeting membrane-bound viral RNA synthesis reveals potent inhibition of diverse coronaviruses including the middle East respiratory syndrome virus.

Coronaviruses raise serious concerns as emerging zoonotic viruses without specific antiviral drugs available. Here we screened a collection of 16671 diverse compounds for anti-human coronavirus 229E activity and identified an inhibitor, designated K22, that specifically targets membrane-bound coronaviral RNA synthesis. K22 exerts most potent antiviral activity after virus entry during an early step of the viral life cycle. Specifically, the formation of double membrane vesicles (DMVs), a hallmark of coronavirus replication, was greatly impaired upon K22 treatment accompanied by near-complete inhibition of viral RNA synthesis. K22-resistant viruses contained substitutions in non-structural protein 6 (nsp6), a membrane-spanning integral component of the viral replication complex implicated in DMV formation, corroborating that K22 targets membrane bound viral RNA synthesis. Besides K22 resistance, the nsp6 mutants induced a reduced number of DMVs, displayed decreased specific infectivity, while RNA synthesis was not affected. Importantly, K22 inhibits a broad range of coronaviruses, including Middle East respiratory syndrome coronavirus (MERS-CoV), and efficient inhibition was achieved in primary human epithelia cultures representing the entry port of human coronavirus infection. Collectively, this study proposes an evolutionary conserved step in the life cycle of positive-stranded RNA viruses, the recruitment of cellular membranes for viral replication, as vulnerable and, most importantly, druggable target for antiviral intervention. We expect this mode of action to serve as a paradigm for the development of potent antiviral drugs to combat many animal and human virus infections

Quantification of interferon signaling in avian cells

Activation of the type I interferon (IFN) response is an essential defense mechanism against invading pathogens such as viruses. This chapter describes two protocols to quantify activation of the chicken IFN response through analysis of gene expression by real-time quantitative PCR and by quantification of bioactive IFN protein using a bioassay.</p

Quantification of infectious bronchitis coronavirus by titration in vitro and in ovo

Quantification of the number of infectious viruses in a sample is a basic virological technique. In this chapter we provide a detailed description of three techniques to estimate the number of viable infectious avian coronaviruses in a sample. All three techniques are serial dilution assays, better known as titrations.</p

Infectious bronchitis coronavirus limits interferon production by inducing a host shutoff that requires accessory protein 5b

During infection of their host cell, viruses often inhibit production of host proteins, a process which is referred to as host shutoff. By doing this, viruses limit production of antiviral proteins and increase production capacity for viral proteins. Coronaviruses from the Alpha- and Betacoronavirus genera, such as severe acute respiratory syndrome coronavirus (SARS-CoV) establish host shutoff via their non-structural protein 1 (nsp1). The genomes of Gamma- and Deltacoronaviruses however do not encode nsp1, and it has been suggested that these viruses do not induce host shutoff. Here we show that infectious bronchitis Gammacoronavirus (IBV) does induce host shutoff and we find that its accessory protein 5b is indispensable for this function. Importantly, we found that 5b-null viruses, unlike wild type viruses, induce production of high concentrations of type I interferon protein in vitro, indicating that host shutoff by IBV plays an important role in antagonizing the host's innate immune response. Altogether we demonstrate that 5b is a functional equivalent of nsp1 thereby answering the long-standing question whether lack of nsp1 in Gammacoronaviruses is compensated for by another viral protein. As such, our study is a significant step forward in the understanding of coronavirus biology and closes a gap in the understanding of some IBV virulence strategies. IMPORTANCE: Many viruses inhibit protein synthesis of their host cell to enhance virus replication and antagonize anti-viral defense mechanisms. This process is referred to as 'host-shutoff'. We have studied gene expression and protein synthesis in chicken cells infected with the important poultry pathogen, infectious bronchitis virus (IBV). We show that IBV inhibits synthesis of host proteins, including that of type I interferon, a key component of the antiviral response. The IBV-induced host shutoff however, does not require degradation of host RNA. Furthermore, we demonstrate that accessory protein 5b of IBV plays a crucial role in the onset of the host shutoff. Our findings suggest that inhibition of host protein synthesis is a common feature of coronaviruses and primarily serves to inhibit the antiviral response of the host

Infectious Bronchitis Coronavirus Inhibits STAT1 Signaling and Requires Accessory Proteins for Resistance to Type I Interferon Activity

The innate immune response is the first line of defense against viruses, and type I interferon (IFN) is a critical component of this response. Similar to other viruses, the gammacoronavirus infectious bronchitis virus (IBV) has evolved under evolutionary pressure to evade and counteract the IFN response to enable its survival. Previously, we reported that IBV induces a delayed activation of the IFN response. In the present work, we describe the resistance of IBV to IFN and the potential role of accessory proteins herein. We show that IBV is fairly resistant to the antiviral state induced by IFN and identify that viral accessory protein 3a is involved in resistance to IFN, as its absence renders IBV less resistant to IFN treatment. In addition to this, we found that independently of its accessory proteins, IBV inhibits IFN-mediated phosphorylation and translocation of STAT1. In summary, we show that IBV uses multiple strategies to counteract the IFN response.</p

Trimeric SARS-CoV-2 Spike Proteins Produced from CHO Cells in Bioreactors Are High-Quality Antigens

The spike protein of the pandemic human corona virus is essential for its entry into human cells. In fact, most neutralizing antibodies against Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) are directed against the Virus-surface exposed spike protein, making it the antigen of choice for use in vaccines and diagnostic tests. In the current pandemic context, global demand for spike proteins has rapidly increased and could exceed hundreds of grams to kilograms annually. Coronavirus spikes are large heavily glycosylated homo-trimeric complexes, with inherent instability. The poor manufacturability now threatens the availability of these proteins for vaccines and diagnostic tests. Here, we outline scalable, Good Manufacturing Practice (GMP) compliant, and chemically defined processes for the production of two cell-secreted stabilized forms of the trimeric spike proteins (Wuhan and D614G variant). The processes are chemically defined and based on clonal suspension-CHO cell populations and on protein purification via a two-step scalable downstream process. The trimeric conformation was confirmed using electron microscopy and HPLC analysis. Binding to susceptible cells was shown using a virus-inhibition assay. The diagnostic sensitivity and specificity for detection of serum SARS-CoV-2-specific-immunoglobulin molecules was found to exceed that of spike fragments (Spike subunit-1, S1 and Receptor Binding Domain, RBD). The process described here will enable production of sufficient high-quality trimeric spike protein to meet the global demand for SARS-CoV-2 diagnostic tests and potentially vaccines

Neutralising antibodies against the SARS-CoV-2 Delta variant induced by Alhydroxyquim-II-adjuvanted trimeric spike antigens

ABSTRACT Global control of COVID-19 will require the deployment of vaccines capable of inducing long-term protective immunity against SARS-CoV-2 variants. In this report, we describe an adjuvanted subunit candidate vaccine that affords elevated, sustained and cross-variant SARS-CoV-2 neutralising antibodies (NAbs) in multiple animal models. Alhydroxiquim-II is a TLR7/8 small-molecule agonist chemisorbed on aluminium hydroxide. Vaccination with Alhydroxiquim-II combined with a stabilized, trimeric form of the SARS-CoV-2 spike protein (termed CoVac-II) resulted in high-titre NAbs in mice, with no decay in responses over an 8-month period. NAbs from sera of CoVac-II-immunized mice, horses and rabbits were broadly neutralising against SARS-CoV-2 variants. Boosting long-term CoVac-II-immunized mice with adjuvanted spike protein from the Beta variant markedly increased levels of NAb titres against multiple SARS-CoV-2 variants; notably high titres against the Delta variant were observed. These data strongly support the clinical assessment of Alhydroxiquim-II-adjuvanted spike proteins to protect against SARS-CoV-2 variants of concern

High-Titer Neutralizing Antibodies against the SARS-CoV-2 Delta Variant Induced by Alhydroxyquim-II-Adjuvanted Trimeric Spike Antigens

Global control of COVID-19 will require the deployment of vaccines capable of inducing long-term protective immunity against SARS-CoV-2 variants. In this report, we describe an adjuvanted subunit candidate vaccine that affords elevated, sustained, and cross-variant SARS-CoV-2 neutralizing antibodies (NAbs) in multiple animal models. Alhydroxiquim-II is a Toll-Like Receptor (TLR) 7/8 small-molecule agonist chemisorbed on aluminum hydroxide (Alhydrogel). Vaccination with Alhydroxiquim-II combined with a stabilized, trimeric form of the SARS-CoV-2 spike protein (termed CoVac-II) resulted in high-titer NAbs in mice, with no decay in responses over an 8-month period. NAbs from sera of CoVac-II-immunized mice, horses and rabbits were broadly neutralizing against SARS-CoV-2 variants. Boosting long-term CoVac-II-immunized mice with adjuvanted spike protein from the Beta variant markedly increased levels of NAb titers against multiple SARS-CoV-2 variants; notably, high titers against the Delta variant were observed. These data strongly support the clinical assessment of Alhydroxiquim-II-adjuvanted spike proteins to protect against SARS-CoV-2 variants of concern. IMPORTANCE There is an urgent need for next-generation COVID-19 vaccines that are safe, demonstrate high protective efficacy against SARS-CoV-2 variants and can be manufactured at scale. We describe a vaccine candidate (CoVac-II) that is based on stabilized, trimeric spike antigen produced in an optimized, scalable and chemically defined production process. CoVac-II demonstrates strong and persistent immunity after vaccination of mice, and is highly immunogenic in multiple animal models, including rabbits and horses. We further show that prior immunity can be boosted using a recombinant spike antigen from the Beta variant; importantly, plasma from boosted mice effectively neutralize multiple SARS-CoV-2 variants in vitro, including Delta. The strong humoral and Th1-biased immunogenicity of CoVac-II is driven by use of Alhydroxiquim-II (AHQ-II), the first adjuvant in an authorized vaccine that acts through the dual Toll-like receptor (TLR)7 and TLR8 pathways, as part of the Covaxin vaccine. Our data suggest AHQ-II/spike protein combinations could constitute safe, affordable, and mass-manufacturable COVID-19 vaccines for global distribution

Analysis of recombinant HCoV-229E nsp6 mutants.

<p>(<b>A</b>) Predicted topological structure of HCoV-229E nsp6 indicating the location of K22 resistance mutations. Concerning transmembrane domains VI and VII two proposed topologies are shown. (<b>B-C</b>) Recombinant nsp6 mutant viruses are resistant to K22. MRC-5 cells were inoculated with nsp6 recombinant HCoV-229E^H121L, HCoV-229E^M159V, HCoV-229E^H121L/M159V or wild-type HCoV-229E at a moi of 0.05 for 45 min at 4°C, and K22 (10 µM) was added at specific time points relative to the end of inoculation period. The infectious cell culture medium and cells were harvested after 24 h of incubation at 37°C, and copy numbers of cell-associated (CA) or extracellular (EX) viral RNA was determined. Data shown are means (±SD) of duplicate determinations from two independent experiments. (<b>D-F</b>) Replication kinetics of recombinant nsp6 mutant viruses. MRC-5 cells were inoculated with nsp6 recombinant HCoV-229E^H121L, HCoV-229E^M159V, HCoV-229E^H121L/M159V or wild-type HCoV-229E at an moi of 0.05 for 1 h at 4°C. The infectious cell culture medium and cells were harvested at specific time points relative to the end of inoculation period, and copy numbers of cell-associated (CA; <b>D</b>) or extracellular (EX; <b>E</b>) viral RNA and infectivity (<b>F</b>) was determined. Data shown are means (±SD) of duplicate determinations from two independent experiments.</p

Alignment of coronavirus nsp6 sequences.

<p>Alignment of nsp6 sequences derived from coronaviruses used in this study was performed with Geneious Software (Biomatters Ltd, New Zealand). Coronavirus species and corresponding GenBank accession numbers are indicated. Membrane domains predicted by TMHMM Server v. 2.0 (<a href="http://www.cbs.dtu.dk/services/TMHMM/" target="_blank">http://www.cbs.dtu.dk/services/TMHMM/</a>) are indicated by cyan shading while conserved amino acid residues are highlighted by black/grey shading. K22 resistance-conferring mutations in HCoV-229E nsp6, identified in this study, are depicted.</p