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The emergence of SARS-CoV-2 in December 2019 resulted in the COVID-19 pandemic. Recurring disease outbreaks repeatedly overloaded the public health sector and severely affected the global economy. We developed a candidate COVID-19 vaccine based on a recombinant Newcastle disease virus (NDV) vaccine vector, encoding a pre-fusion stabilized full-length Spike protein obtained from the original SARS-CoV-2 Wuhan isolate. Vaccination of hamsters by intra-muscular injection or intra-nasal instillation induced high neutralizing antibody responses. Intranasal challenge infection with SARS-CoV-2 strain Lelystad demonstrated that both vaccination routes provided partial protection in the upper respiratory tract, and almost complete protection in the lower respiratory tract, as measured by suppressed viral loads and absence of histological lung lesions. Activity wheel measurements demonstrated that animals vaccinated by intranasal inoculation rapidly recovered to normal activity. NDV constructs encoding the spike of SARS-CoV-2 may be attractive candidates for development of intra-nasal COVID-19 booster vaccines.

Coronavirus (CoV) spikes mediate receptor binding and membrane fusion, making them prime targets for neutralising antibodies. In the cases of SARS-CoV, SARS-CoV-2, and MERS-CoV, spikes transition freely between open and closed conformations to balance host cell attachment and immune evasion. The open conformation exposes domain S1B, allowing it to bind to proteinaceous cell surface receptors. It also facilitates protein refolding during spike-mediated membrane fusion. However, with a single exception, the pre-fusion spikes of all other CoVs studied so far have been observed exclusively in the closed state. This raises the possibility of regulation, where spikes more commonly transition to open states in response to specific cues, rather than spontaneously. In our study, using cryo-EM and molecular dynamics simulations, we show that the spike protein of the common cold human coronavirus HKU1 undergoes local and long-range conformational changes upon binding a sialoglycan-based primary receptor to domain S1A. This binding triggers the transition of S1Bdomains to the open state via allosteric inter-domain cross-talk. Our findings paint a more elaborate picture of CoV attachment, with possibilities of dual receptor usage and priming of entry as a means of immune escape.

STING induces transcription of pro-inflammatory genes upon activation at the Golgi apparatus. Many of the regulators involved in STING activation are unknown. We found that ACBD3 and other phosphatidylinositol 4-phosohate (PI4P) regulating proteins play a critical role in STING activation. We show that proper STING localization and activation at the Golgi depended on ACBD3 and PI4KB expression. Furthermore, depleting PI4P by inactivating PI4KB or overexpressing Sac1 diminished STING activation. STING signalling was also regulated by the lipid-shuttling protein OSBP, which removes PI4P from the Golgi. OSBP inhibition by the FDA-approved antifungal itraconazole and other OSBP inhibitors greatly enhanced STING activation by increasing the levels of STING-activating phospholipids. Itraconazole-enhanced STING activation resulted in a hundred to thousand-fold increased expression of interferon-beta and other cytokines. In conclusion, the phospholipid PI4P is critical for STING activation and manipulating PI4P levels is a promising therapeutic strategy to alter the STING immune response.

The nucleocapsid protein N of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enwraps and condenses the viral genome for packaging but is also an antagonist of the innate antiviral defense. It suppresses the integrated stress response (ISR), purportedly by interacting with stress granule (SG) assembly factors G3BP1 and 2, and inhibits type I interferon responses. To elucidate its mode of action, we systematically deleted and over-expressed distinct regions and domains. We show that N via domain N2b blocks PKR-mediated ISR activation, as measured by suppression of ISR-induced translational arrest and SG formation. N2b mutations that prevent dsRNA binding abrogate these activities also when introduced in the intact N protein. Substitutions reported to block post-translation modifications of N or its interaction with G3BP1/2 did not have a detectable additive effect. In an encephalomyocarditis virus-based infection model, N2b - but not a derivative defective in RNA binding - prevented PKR activation, inhibited β-interferon expression and promoted virus replication. Apparently, SARS-CoV-2 N inhibits innate immunity by sequestering dsRNA to prevent activation of PKR and RIG-I-like receptors. Similar observations were made for the N protein of human coronavirus 229E, suggesting that this may be a general trait conserved among members of other orthocoronavirus (sub)genera.SIGNIFICANCE STATEMENTSARS-CoV-2 nucleocapsid protein N is an antagonist of innate immunity but how it averts virus detection by intracellular sensors remains subject to debate. We provide evidence that SARS-CoV-2 N, by sequestering dsRNA through domain N2b, prevents PKR-mediated activation of the integrated stress response as well as detection by RIG-I-like receptors and ensuing type I interferon expression. This function, conserved in human coronavirus 229E, is not affected by mutations that prevent posttranslational modifications, previously implicated in immune evasion, or that target its binding to stress granule scaffold proteins. Our findings further our understanding of how SARS-CoV-2 evades innate immunity, how this may drive viral evolution and why increased N expression may have been a selective advantage to SARS-CoV-2 variants of concern.

The ongoing evolution of SARS-CoV-2 has resulted in the emergence of Omicron, which displays striking immune escape potential. Many of its mutations localize to the spike protein ACE2 receptor-binding domain, annulling the neutralizing activity of most therapeutic monoclonal antibodies. Here we describe a receptor-blocking human monoclonal antibody, 87G7, that retains ultrapotent neutralization against SARS-CoV-2 variants including the Alpha, Beta, Gamma, Delta and Omicron (BA.1/BA.2) Variants-of-Concern (VOCs). Structural analysis reveals that 87G7 targets a patch of hydrophobic residues in the ACE2-binding site that are highly conserved in SARS-CoV-2 variants, explaining its broad neutralization capacity. 87G7 protects mice and/or hamsters against challenge with all current SARS-CoV-2 VOCs. Our findings may aid the development of sustainable antibody-based strategies against COVID-19 that are more resilient to SARS-CoV-2 antigenic diversity., The MANCO project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 101003651). This work made use of the Dutch national e infrastructure with the support of the SURF Cooperative using grant no. EINF-2453. This research was funded by the Deutsche Forschungsgemeinschaft (DFG; German Research Foundation) - 398066876/GRK 2485/1; BMBF (Federal Ministry of Education and Research) project entitled RAPID (Risk assessment in re-pandemic respiratory infectious diseases), 01KI1723G, Ministry of Science and Culture of Lower Saxony in Germany (14 - 76103-184 CORONA-15/20)

The SARS-CoV-2 virus responsible for the COVID-19 pandemic has so far infected more than 160 million people globally, and continues to undergo genomic evolution. Emerging SARS-CoV-2 variants show increased infectivity and may lead to resistance against immune responses of previously immunized individuals or existing therapeutics, especially antibody-based therapies. Several monoclonal antibody therapeutics authorized for emergency use or in development have been shown to lose potency against some SARS-CoV-2 variants. Cocktails of two different monoclonal antibodies constitute a promising approach to protect against novel emerging variants as long as both antibodies are potent, but come with increased development complexity and therefore cost. As an alternative, we developed two multispecific DARPin® therapeutics, each combining three independent DARPin® domains binding the SARS-CoV-2 spike protein in one molecule, to potently neutralize the virus and overcome virus escape. Here, we show in a panel of in vitro studies that the multispecific DARPin® therapeutic design incorporated in our clinical candidate ensovibep (MP0420), achieved high neutralization potencies against the circulating SARS-CoV-2 variants B.1.1.7 (UK variant), B.1.351 (South African variant), P.1 (Brazilian variant), B.1.429 (South Californian variant), B.1.526 (New York variant), R.1 (Japanese variant), A.23.1 (Ugandan variant), and B.1.617 (Indian variant), and there is strong evidence that ensovibep also potently neutralizes the Indian variant B.1.618 based on the analysis of the key point mutations in the spike protein of this variant. Additionally, viral passaging experiments show potent protection by ensovibep and MP0423 against development of escape mutations. Finally, we demonstrate that the cooperative binding of the individual modules in a multispecific DARPin® antiviral is key for potent virus inhibition and protection from escape variants. These results, combined with the relatively small size and high production yields of DARPin® molecules, suggest that ensovibep is a highly valuable alternative to monoclonal antibody cocktails for global supply and demonstrate the strength of the DARPin® platform for achieving potent and lasting virus inhibition for SARS-CoV-2 and possibly other viruses.