The type I Interferon (IFN-I)-induced ubiquitin-like modifier Interferon-Stimulated Gene 15 (ISG15) plays a crucial role in the innate immune response against viral infections. ISG15 is conjugated to target proteins by an enzymatic cascade, called ISGylation. While ubiquitin-specific protease 18 (USP18) serves as the major deISGylase counteracting ISG15 conjugation, ISG15 cross-reactive deubiquitylating enzymes (DUBs) have also been reported. Here, we identify USP24 as an ISG15 cross-reactive DUB through activity-based protein profiling. USP24 processes pro-ISG15 and ISG15-linked substrates in vitro, and its depletion increases ISG15 conjugates following interferon stimulation in cells without altering canonical IFN-I signaling. USP24 knockout cells show increased IFN-β and ISG expression upon activation of cytosolic RNA sensing via viral mimicry. Proteomic analysis identifies RNA helicase Moloney leukemia virus 10 (MOV10) as a specific target of USP24 for deISGylation. Our data revealed that ISGylation of MOV10 promotes MOV10 interaction with IFIT3 and enhances IFN-β production/secretion in response to viral stimuli. This process is negatively regulated by USP24, which directly deISGylates MOV10. Our data highlight USP24's role in modulating ISGylation and IFN-I production, suggesting USP24 as a potential therapeutic agent for infectious and inflammatory diseases.

Abstract The expanding geographical spread of Usutu virus (USUV) poses an increasing threat to bird populations and to human health. Here we assessed a chimeric USUV vaccine candidate that was constructed using the live attenuated yellow fever virus (YFV) YF-17D platform. In our chimeric virus, the pre-membrane (PrM) and envelope (E) proteins of YFV were replaced with those of USUV. Similar chimeras of YF-17D with otherflaviviruses have been reported to be attenuated in vivo, making them promising modified live virus vaccine candidates. We used either wild type USUV PrME or USUV PrME containing rationally designed mutations in the E protein that were expected to (further) attenuate the virus, based on their effect in the context of other orthoflaviviruses. In cell culture the YF-17D/USUV chimeric viruses displayed reduced fitness compared to both YF-17D and USUV, with the chimera containing the mutated USUV E exhibiting the slowest growth kinetics. However, in our interferon α/β receptor deficient (Ifnar−/−) mouse model the chimeric YF-17D/USUV viruses caused rapid lethality in all animals, with survival times that were only marginally better compared to wild type USUV. It was surprising that introduction of the PrME region from the naturally low pathogenic USUV into the YF-17D platform yielded viruses that caused lethal infections in our model, while in contrast other neuroinvasive orthoflaviviruses are attenuated in this same chimeric platform. All control mice that were infected with a 1000-fold higher dose of YF-17D survived. We also discovered that the mutations in E that are attenuating in related viruses did not result in attenuation in the Ifnar−/− mouse model, neither in the context of USUV itself or the YF-17D chimera. This may be due to the immunocompromised nature of the mice or because these mutations are not attenuating in the context of USUV. This work highlights some complications and unpredictability related to the use of the chimeric live attenuated YF-17D vaccine platform and introduction of rationally designed mutations with a predicted attenuating effect. Furthermore, our work illustrates that infection models that are able to better predict the phenotype and safety of (chimeric) USUV/orthoflavivirus vaccine candidates are needed.

In order to detect and respond to invading pathogens, mammals have evolved a battery of pattern recognition receptors. Among these, RIG-I-like receptors (RLR) are cytosolic RNA sensors that play an essential role in the innate immune response against RNA viruses, including coronaviruses. In return, coronaviruses have acquired diverse strategies to impair RLR-mediated immune responses to enable productive infection. Viral innate immune evasion mechanisms have been well studied for highly pathogenic human coronaviruses (HCoVs), and often, these activities are thought to be linked to the severe symptoms these viruses can cause. Whether other coronaviruses, including human common cold coronaviruses, display similar activities has remained understudied. Here, we present evidence that the main protease (Mpro) of common cold HCoV-229E acts as an interferon (IFN) and NF-κB antagonist by disrupting RLR-mediated antiviral signalling. Furthermore, we show that HCoV-229E, HCoV-OC43 and MERS-CoV Mpros are able to directly cleave NEMO. We also show that HCoV-229E Mpro induces the cleavage and/or degradation of multiple other RLR pathway components, including MDA5, TBK1 and IKKε. Finally, we show that HCoV-229E infection leads to a delayed innate immune response that is accompanied by a decrease in NEMO protein levels. Our results suggest that NEMO degradation during HCoV-229E infection could be mediated, in part, by cellular degradation pathways, in addition to viral Mpro-mediated cleavage. Altogether, our research unveils innate immune evasion activities of the Mpros of low-pathogenic coronaviruses, which, despite their low pathogenicity, appear to share functionalities previously described for highly pathogenic HCoVs.

Vault RNAs (vtRNAs) are a family of four small non-coding RNAs that regulate multiple cellular pathways in human cells. VtRNA expression is increased upon infection with certain RNA and DNA viruses, suggesting that they either promote or restrict viral replication. However, their precise molecular function remains unclear. We show that replication of picornaviruses, alphaviruses, and beta-coronaviruses broadly enhances vtRNA expression. Genetic loss of vtRNAs inhibits replication of Sindbis virus (SINV) and encephalomyocarditis virus (EMCV), independent of the antiviral type I interferon response. A proteomic screen demonstrates that vtRNAs associate with RNA-binding proteins ELAVL1 and hnRNP C in uninfected and infected cells. During infection, vtRNAs facilitate cytoplasmic localization of ELAVL1 and hnRNP C, where these proteins associate with viral RNA and support SINV replication. Together, our findings reveal a proviral function of vtRNAs during SINV and EMCV infection, which presents therapeutic opportunities to fight infectious diseases.

Background:Adult solid organ transplant recipients (SOTRs) have decreased responsiveness to severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) vaccination and higher incidence of infection, but there are few data on the serological response in pediatric SOTR. The aim of this study was to determine serological response to SARS-CoV-2 vaccination in pediatric liver (LT) and kidney transplant (KT) recipients and compare it with adult SOTR. Methods:A European, prospective, multicenter study was performed. Samples were taken at 7 and 32 wk following COVID-19 vaccination and serological endpoints were measured by ELISA. Results:A total of 42 pediatric (16 post-LT and 26 post-KT) and 117 adult (all post-LT) were included. All pediatric participants and 94% adult participants received mRNA vaccines. Paediatric SOTR patients had significantly higher anti-Spike IgG levels than adult participants at week 7 (114 220.7 [59 285.92-220 058.55] versus 8756.7 [5643.69-13 586.71], P < 0.0001) and week 32 (46 113.2 [10 992.91-193 436.14] versus 8207.0 [3561.20-18 913.43], P = 0.0032). No significant difference in week 7 anti-Spike IgG response was found between pediatric LT and KT (129 434.4 [51 888.64-322 869.69] versus 105 304.5 [39 910.20-277 849.50], P = 0.9854). No differences were seen between children and adults in the rate of decline of anti-Spike IgG between weeks 7 and 32 (P = 0.8000). Male sex and hemolytic-uremic syndrome or postischemic kidney disease were associated with lower anti-Spike IgG levels at week 7 in pediatric SOTR. Conclusions:Paediatric SOTR demonstrate greater SARS-CoV-2 vaccine responses than comparable adult SOTR patients. These data support efficacy and safety of SARS-CoV-2 vaccination in child SOTR and may alleviate vaccine hesitancy in this patient group.

Usutu virus (USUV) is a mosquito-borne zoonotic flavivirus with a geographic range that has expanded over recent years. Maintained in a transmission cycle between mosquito vectors and avian reservoirs the virus can cause large seasonal outbreaks in bird populations, but spillover into mammalian hosts has also been reported. While usually mild or asymptomatic in humans, neurological disorders are increasingly observed, which has boosted interest and the need for better understanding of the pathogenesis of various USUV lineages. In this study we inoculated interferon α/β receptor knockout (Ifnar−/−) mice with decreasing doses of USUV, monitoring symptoms and survival to determine a less lethal dose, and we directly compared isolates from three different viral lineages. We found that a Dutch isolate of USUV Africa-3 lineage is lethal at a dose of 20 pfu per mouse, which is considerably lower than what was anticipated based upon the literature. A Europe-2 strain showed an even higher virulence in this mouse model, compared to strains from Africa-3 and Europe-3 lineages—though this was not reflected in in vitro studies. These results enhance our understanding of the pathogenicity of different USUV strains and provide guidance for the use of low doses for inoculation in an Ifnar−/− animal model.

Usutu virus (USUV) is an emerging orthoflavivirus, which mainly affects birds but in rare cases can cause severe neuroinvasive disease in humans. The virus relies on a multitude of host cell proteins, molecules and cellular processes for its replication, and must subvert host antiviral responses to establish a successful infection. Studying the complex network of virus-host protein interactions by proteomics approaches can therefore provide new insights in the replication cycle of USUV and its pathogenesis. We have previously shown that the USUV protein NS4A acts as an antagonist of the antiviral interferon response, and here we further map the host interaction partners of USUV NS4A using proximity labeling coupled to mass spectrometry. The resulting NS4A interactome revealed many host proteins involved in the autophagy pathway. We showed that both USUV infection and overexpression of USUV NS4A can indeed induce the autophagy pathway. However, stimulation or inhibition of the autophagy pathway in general did not affect USUV replication. Therefore, we decided to specifically analyze the role of the selective autophagy receptor sequestosome 1 (p62/SQSTM1), since we identified this protein as an important interaction partner of USUV NS4A. We found that p62 is involved in the degradation of USUV NS4A. In agreement with this, the knockdown of p62 enhanced replication of USUV in A549 cells. P62 thus plays an antiviral role during USUV infection, although this antiviral effect might also be related to its functions outside the autophagy pathway, such as modulation of the immune response. In conclusion, this study showed that USUV NS4A induces autophagy and is then targeted by p62 for degradation by the autophagic machinery, uncovering a new role of p62 in the antiviral defense against USUV.

Infectious bronchitis virus (IBV) is a coronavirus first isolated in the 1930s infecting chickens. IBV causes great economic losses to the global poultry industry, as it affects egg production and causes mortality by leaving the host susceptible to secondary bacterial infections. Even though vaccines are available, they are poorly cross-protective against new variants of the virus, which are always on the cusp of emerging. Effective antiviral therapies, or possibly the production of transgenic animals immune to IBV infection, are therefore sorely needed. As the papain-like protease (PLpro) of IBV has deubiquitinating activity besides its crucial ability to cleave the viral polyprotein, we have applied a novel strategy of selecting ubiquitin variants (UbVs) from a phage-displayed library that have high affinity to this viral protease. These UbVs were found to inhibit the deubiquitinating activity of PLpro and consequently obstruct the virus's ability to evade the innate immune response in the host cell. By obstructing the proteolytic activity of PLpro, these UbVs were seemingly able to inhibit viral infection as assessed using immunofluorescence microscopy. Whilst virus infection was detected in around 5% of UbV-expressing cells, the virus was present in around 30-40% of GFP (control)-expressing cells. This suggests that the expression of UbVs indeed seems to inhibit IBV infection, making UbVs a potentially potent and innovative antiviral strategy in the quest for control of IBV infections.

Background/Objectives: Understanding virulence determinants can inform safer and more efficacious live attenuated vaccine design. However, applying this knowledge across related viruses does not always result in conserved phenotypes from similar mutants. Methods: Using Usutu virus (USUV), an emerging orthoflavivirus spreading through Europe, we assessed whether the attenuating effect of the mutations described for related orthoflaviviruses is conserved. Candidate attenuating mutations were selected based on previous studies in other orthoflaviviruses and incorporated into USUV. Results: Nine variants, with mutations in the USUV envelope, non-structural (NS) proteins NS1, NS2A, or NS4B were stable and selected for further characterisation. The variants with an attenuating phenotype in cell culture were then compared to the wild-type virus in an Ifnar−/− mouse model. Mutations of the envelope glycosylation sites and glycosaminoglycan binding sites, which were recognised as more-conserved mechanisms of orthoflavivirus attenuation, were attenuating in USUV as well. However, not all the mutations explored in the USUV non-structural proteins exhibited an attenuated phenotype. Instead, the attenuation was either less pronounced, or there was no change in phenotype relative to the wild-type virus at all. Conclusions: In addition to improving our understanding of USUV virulence determinants, these results add to a growing body of literature highlighting the most promising mechanisms to target for the design of safe live attenuated vaccines against emerging orthoflaviviruses.

The coronavirus papain-like protease (PLpro) is crucial for viral replicase polyprotein processing. Additionally, PLpro can subvert host defense mechanisms by its deubiquitinating (DUB) and deISGylating activities. To elucidate the role of these activities during SARS-CoV-2 infection, we introduced mutations that disrupt binding of PLpro to ubiquitin or ISG15. We identified several mutations that strongly reduced DUB activity of PLpro, without affecting viral polyprotein processing. In contrast, mutations that abrogated deISGylating activity also hampered viral polyprotein processing and when introduced into the virus these mutants were not viable. SARS-CoV-2 mutants exhibiting reduced DUB activity elicited a stronger interferon response in human lung cells. In a mouse model of severe disease, disruption of PLpro DUB activity did not affect lethality, virus replication, or innate immune responses in the lungs. This suggests that the DUB activity of SARS-CoV-2 PLpro is dispensable for virus replication and does not affect innate immune responsesin vivo. Interestingly, the DUB mutant of SARS-CoV replicated to slightly lower titers in mice and elicited a diminished immune response early in infection, although lethality was unaffected. We previously showed that a MERS-CoV mutant deficient in DUB and deISGylating activity was strongly attenuated in mice. Here, we demonstrate that the role of PLpro DUB activity during infection can vary considerably between highly pathogenic coronaviruses. Therefore, careful considerations should be taken when developing pan-coronavirus antiviral strategies targeting PLpro.

Vault RNAs (vtRNAs) are a family of four small non-coding RNAs (ncRNAs) that are ubiquitously expressed in many eukaryotes and that regulate multiple cellular pathways. Their expression is increased upon infection with various DNA and RNA viruses. This suggests they are either co-opted by the virus to aid replication or function as an antiviral restriction factor. However, their precise molecular function remains unclear. Here, we show that replication of picornaviruses, alphaviruses, and beta-coronaviruses broadly enhances vtRNA expression. We find that genetic loss of vtRNAs inhibits replication of Sindbis virus (SINV) and encephalomyocarditis virus (EMCV), independent of the antiviral type I interferon (IFN) response. A proteomic screen uncovered the vtRNA interactome and revealed that vtRNAs associate with RNA binding proteins ELAVL1 and hnRNP C in uninfected and infected cells. VtRNAs facilitate the translocation of ELAVL1 and hnRNP C from the nucleus to the cytoplasm in infected cells, an event that is required for efficient viral replication. Moreover, hnRNP C and ELAVL1 fail to associate with viral RNA in the cytosol of SINV-infected cells in the absence of vtRNAs. Together, our findings reveal a novel molecular mechanism by which vtRNAs exert proviral activity during the course of SINV and EMCV infection, which opens up new avenues for therapeutic targeting to fight infectious diseases. ### Competing Interest Statement The authors have declared no competing interest.

Mitochondrial antiviral signaling protein (MAVS) is a crucial signaling adaptor in the sensing of positive-sense RNA viruses and the subsequent induction of the innate immune response. Coronaviruses have evolved multiple mechanisms to evade this response, amongst others, through their main protease (Mpro), which is responsible for the proteolytic cleavage of the largest part of the viral replicase polyproteins pp1a and pp1ab. Additionally, it can cleave cellular substrates, such as innate immune signaling factors, to dampen the immune response. Here, we show that MAVS is cleaved in cells infected with Middle East respiratory syndrome coronavirus (MERS-CoV), but not in cells infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This cleavage was independent of cellular negative feedback mechanisms that regulate MAVS activation. Furthermore, MERS-CoV Mpro expression induced MAVS cleavage upon overexpression and suppressed the activation of the interferon-β (IFN-β) and nuclear factor-κB (NF-κB) response. We conclude that we have uncovered a novel mechanism by which MERS-CoV downregulates the innate immune response, which is not observed among other highly pathogenic coronaviruses.

Human coronavirus 229E (HCoV-229E) is associated with upper respiratory tract infections and generally causes mild respiratory symptoms. HCoV-229E infection can cause cell death, but the molecular pathways that lead to virus-induced cell death as well as the interplay between viral proteins and cellular cell death effectors remain poorly characterized for HCoV-229E. Studying how HCoV-229E and other common cold coronaviruses interact with and affect cell death pathways may help to understand its pathogenesis and compare it to that of highly pathogenic coronaviruses. Here, we report that the main protease (Mpro) of HCoV-229E can cleave gasdermin D (GSDMD) at two different sites (Q29 and Q193) within its active N-terminal domain to generate fragments that are now unable to cause pyroptosis, a form of lytic cell death normally executed by this protein. Despite GSDMD cleavage by HCoV-229E Mpro, we show that HCoV-229E infection still leads to lytic cell death. We demonstrate that during virus infection caspase-3 cleaves and activates gasdermin E (GSDME), another key executioner of pyroptosis. Accordingly, GSDME knockout cells show a significant decrease in lytic cell death upon virus infection. Finally, we show that HCoV-229E infection leads to increased lytic cell death levels in cells expressing a GSDMD mutant uncleavable by Mpro (GSDMD Q29A+Q193A). We conclude that GSDMD is inactivated by Mpro during HCoV-229E infection, preventing GSDMD-mediated cell death, and point to the caspase-3/GSDME axis as an important player in the execution of virus-induced cell death. In the context of similar reported findings for highly pathogenic coronaviruses, our results suggest that these mechanisms do not contribute to differences in pathogenicity among coronaviruses. Nonetheless, understanding the interactions of common cold-associated coronaviruses and their proteins with the programmed cell death machineries may lead to new clues for coronavirus control strategies.

Several viruses are now known to code for deubiquitinating proteases in their genomes. Ubiquitination is an essential post-translational modification of cellular substrates involved in many processes in the cell, including in innate immune signalling. This post-translational modification is regulated by the ubiquitin conjugation machinery, as well as various host deubiquitinating enzymes. The conjugation of ubiquitin chains to several innate immune related factors is often needed to induce downstream signalling, shaping the antiviral response. Viral deubiquitinating proteins, besides often having a primary function in the viral replication cycle by cleaving the viral polyprotein, are also able to cleave ubiquitin chains from such host substrates, in that way exerting a function in innate immune evasion. The presence of viral deubiquitinating enzymes has been firmly established for numerous animal-infecting viruses, such as some well-researched and clinically important nidoviruses, and their presence has now been confirmed in several plant viruses as well. Viral proteases in general have long been highlighted as promising drug targets, with a current focus on small molecule inhibitors. In this review, we will discuss the range of viral deubiquitinating proteases known to date, summarise the various avenues explored to inhibit such proteases and discuss novel strategies and models intended to inhibit and study these specific viral enzymes.

The SARS-CoV-2 pandemic and the emergence of novel virus variants have had a dramatic impact on public health and the world economy, underscoring the need for detailed studies that explore the high efficacy of additional vaccines in animal models. In this study, we confirm the pathogenicity of the SARS-CoV-2/Leiden_008 isolate (GenBank accession number MT705206.1) in K18-hACE2 transgenic mice. Using this isolate, we show that a vaccine consisting of capsid virus-like particles (cVLPs) displaying the receptor-binding domain (RBD) of SARS-CoV-2 (Wuhan strain) induces strong neutralizing antibody responses and sterilizing immunity in K18-hACE2 mice. Furthermore, we demonstrate that vaccination with the RBD-cVLP vaccine protects mice from both a lethal infection and symptomatic disease. Our data also indicate that immunization significantly reduces inflammation and lung pathology associated with severe disease in mice. Additionally, we show that the survival of naïve animals significantly increases when sera from animals vaccinated with RBD-cVLP are passively transferred, prior to a lethal virus dose. Finally, the RBD-cVLP vaccine has a similar antigen composition to the clinical ABNCOV2 vaccine, which has shown non-inferiority to the Comirnaty mRNA vaccine in phase I-III trials. Therefore, our study provides evidence that this vaccine design is highly immunogenic and confers full protection against severe disease in mice.

Objectives: The aim of this study was to assess the safety and immunogenicity of a dose -sparing fractional intradermal (ID) booster strategy with the mRNA-1273 COVID-19 vaccine. Methods: COVID-19 naive adults aged 18-30 years were recruited from a previous study on primary vaccination regimens that compared 20 mu g ID vaccinations with 100 mu g intramuscular (IM) vaccinations with mRNA-1273 as the primary vaccination series. Participants previously immunized with ID regimens were randomly assigned (1:1) to receive a fractional ID booster dose (20 mu g) or the standard-of-care intramuscular (IM) booster dose (50 mu g) of the mRNA-1273 vaccine, 6 months after completing their primary series (ID -ID and ID-IM group, respectively). Participants that had received a full dose IM regimen as the primary series, received the IM standard-of-care booster dose (IM-IM group). In addition, COVID-19 naive individuals aged 18-40 years who had received an IM mRNA vaccine as the primary series were recruited from the general population to receive a fractional ID booster dose (IM-ID group). Immunogenicity was assessed using IgG anti -spike antibody responses and neutralizing capacity against SARS-CoV-2. Cellular immune responses were measured in a sub-group. Safety and tolerability were monitored. Results: In January 2022,129 participants were included in the study. Fractional ID boosting was safe and well tolerated, with fewer systemic adverse events compared with IM boosting. At day 28 post -booster, anti -spike S1 IgG geometric mean concentrations were 9106 (95% CI, 7150-11 597) binding antibody units (BAU)/mL in the IM-IM group and 4357 (3003-6322) BAU/mL; 6629 (4913-8946) BAU/mL; and 5264 (4032-6873) BAU/mL in the ID-IM, ID -ID, and IM-ID groups, respectively. Discussion: Intradermal boosting provides robust immune responses and is a viable dose -sparing strategy for mRNA COVID-19 vaccines. The favourable side-effect pro file supports its potential to reduce vaccine hesitancy. Fractional dosing strategies should be considered early in the clinical development of future mRNA vaccines to enhance vaccine availability and pandemic preparedness. Geert V.T. Roozen, dlin Microbiol Infect 2024;30:930 (c) 2024 The Authors. Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious Diseases. This is an open access article under the CC BY license (http://creativecommons.org/ licenses/by/4.0/).

Usutu virus (USUV) is an emerging flavivirus that can infect birds and mammals. In humans, in severe cases, it may cause neuroinvasive disease. The innate immune system, and in particular the interferon response, functions as the important first line of defense against invading pathogens such as USUV. Many, if not all, viruses have developed mechanisms to suppress and/or evade the interferon response in order to facilitate their replication. The ability of USUV to antagonize the interferon response has so far remained largely unexplored. Using dual-luciferase reporter assays we observed that multiple of the USUV nonstructural (NS) proteins were involved in suppressing IFN-β production and signaling. In particular NS4A was very effective at suppressing IFN-β production. We found that NS4A interacted with the mitochondrial antiviral signaling protein (MAVS) and thereby blocked its interaction with melanoma differentiation-associated protein 5 (MDA5), resulting in reduced IFN-β production. The TM1 domain of NS4A was found to be essential for binding to MAVS. By screening a panel of flavivirus NS4A proteins we found that the interaction of NS4A with MAVS is conserved among flaviviruses. The increased understanding of the role of NS4A in flavivirus immune evasion could aid the development of vaccines and therapeutic strategies.

The interferon-stimulated gene 15 (ISG15) is a ubiquitin-like modifier induced by type I Interferon (IFN-I) and plays a crucial role in the innate immune response against viral infections. ISG15 is conjugated to target proteins by an enzymatic cascade through a process called ISGylation. While ubiquitin-specific protease 18 (USP18) is a well-defined deISGylase counteracting ISG15 conjugation, ISG15 cross-reactive deubiquitylating enzymes (DUBs) have also been reported. Our study reports USP24 as a novel ISG15 cross-reactive DUB identified through activity-based protein profiling (ABPP). We demonstrate that recombinant USP24 processed pro-ISG15 and ISG15-linked synthetic substrates in vitro . Moreover, the depletion of USP24 significantly increased the accumulation of ISG15 conjugates upon IFN-β stimulation. An extensive proteomic analysis of the USP24-dependent ISGylome, integrating total proteome, GG-peptidome, and ISG15 interactome data, identified the helicase Moloney leukemia virus 10 (MOV10) as a specific target of USP24 for deISGylation. Further validation in cells revealed that ISGylated MOV10 enhances IFN-β production/secretion, whereas USP24 deISGylates MOV10 to negatively regulate the innate immune response. This study showcases USP24’s novel roles in modulating ISGylation and modulation of the IFN-I-dependent immune responses, with potential therapeutic implications in infectious diseases, cancer, autoimmunity, and neuroinflammation. ### Competing Interest Statement The authors have declared no competing interest.

The spike protein (S) of SARS-CoV-2 induces neutralizing antibodies and is the key component of current COVID-19 vaccines. The most efficacious COVID-19 vaccines are genetically-encoded spikes with a double proline substitution in the hinge region to stabilize S in the prefusion conformation (S-2P). A subunit vaccine can be a valuable addition to mRNA and viral vector-based vaccines but requires high stability of spike. In addition, further stabilization of the prefusion conformation of spike might improve immunogenicity. To test this, five spike proteins were designed and characterized, ranging from low to high stability. The immunogenicity of these proteins was assessed in mice, demonstrating that a spike (S-closed-2) with a high melting temperature, which still allowed ACE2 binding, induced the highest neutralization titers against homologous and heterologous strains (up to 16-fold higher than the least stabilized spike). In contrast, the most stable spike variant (S-locked), in which the receptor binding domains (RBDs) were locked in a closed conformation and thus not able to breathe, induced relatively low neutralizing antibody titers against heterologous strains. These data demonstrate that S protein stabilization with RBDs exposing highly conserved epitopes may be needed to increase the immunogenicity of spike proteins for future COVID-19 vaccines.