Suppression of interferon α and γ response by Huwe1-mediated Miz1 degradation promotes SARS-CoV-2 replication.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been demonstrated to limit the host interferon response; however, the underlying mechanism remains unclear. Here, we found that SARS-CoV-2 infection upregulated the E3 ubiquitin ligase Huwe1, which in turn facilitated the degradation of the transcription factor Miz1. The degradation of Miz1 hampered interferon alpha and gamma responses, consequently fostering viral replication and impeding viral clearance. Conversely, silencing or inhibiting Huwe1 enhanced the interferon responses, effectively curbing viral replication. Consistently, overexpressing Miz1 augmented the interferon responses and limited viral replication, whereas silencing Miz1 had the opposite effect. Targeting Huwe1 or overexpressing Miz1 elicited transcriptomic alterations characterized by enriched functions associated with bolstered antiviral response and diminished virus replication. Further study revealed Miz1 exerted epigenetic control over the transcription of specific interferon signaling molecules, which acted as common upstream regulators responsible for the observed transcriptomic changes following Huwe1 or Miz1 targeting. These findings underscore the critical role of the Huwe1-Miz1 axis in governing the host antiviral response, with its dysregulation contributing to the impaired interferon response observed during COVID-19.

Time-dependent enhancement of mRNA vaccines by 4-1BB costimulation.

mRNA vaccines have demonstrated efficacy against COVID-19. However, concerns regarding waning immunity and breakthrough infections have motivated the development of next-generation vaccines with enhanced efficacy. In this study, we investigated the impact of 4-1BB costimulation on immune responses elicited by mRNA vaccines in mice. We first vaccinated mice with an mRNA vaccine encoding the SARS-CoV-2 spike antigen like the Moderna and Pfizer-BioNTech vaccines, followed by administration of 4-1BB costimulatory antibodies at various times post-vaccination. Administering 4-1BB costimulatory antibodies during the priming phase did not enhance immune responses. However, administering 4-1BB costimulatory antibodies after 96 hours elicited a significant improvement in CD8 T cell responses, leading to enhanced protection against breakthrough infections. A similar improvement in immune responses was observed with multiple mRNA vaccines, including vaccines against common cold coronavirus, human immunodeficiency virus (HIV), and arenavirus. These findings demonstrate a time-dependent effect by 4-1BB costimulation and provide insights for developing improved mRNA vaccines.

Caveolin-1 mediates blood-brain barrier permeability, neuroinflammation, and cognitive impairment in SARS-CoV-2 infection.

Blood-brain barrier (BBB) permeability can cause neuroinflammation and cognitive impairment. Caveolin-1 (Cav-1) critically regulates BBB permeability, but its influence on the BBB and consequent neurological outcomes in respiratory viral infections is unknown. We used Cav-1-deficient mice with genetically encoded fluorescent endothelial tight junctions to determine how Cav-1 influences BBB permeability, neuroinflammation, and cognitive impairment following respiratory infection with mouse adapted (MA10) SARS-CoV-2 as a model for COVID-19. We found that SARS-CoV-2 infection increased brain endothelial Cav-1 and increased transcellular BBB permeability to albumin, decreased paracellular BBB Claudin-5 tight junctions, and caused T lymphocyte infiltration in the hippocampus, a region important for learning and memory. Concordantly, we observed learning and memory deficits in SARS-CoV-2 infected mice. Importantly, genetic deficiency in Cav-1 attenuated transcellular BBB permeability and paracellular BBB tight junction losses, T lymphocyte infiltration, and gliosis induced by SARS-CoV-2 infection. Moreover, Cav-1 KO mice were protected from the learning and memory deficits caused by SARS-CoV-2 infection. These results establish the contribution of Cav-1 to BBB permeability and behavioral dysfunction induced by SARS-CoV-2 neuroinflammation.

Engineered Wnt7a ligands rescue blood-brain barrier and cognitive deficits in a COVID-19 mouse model.

Respiratory infection with SARS-CoV-2 causes systemic vascular inflammation and cognitive impairment. We sought to identify the underlying mechanisms mediating cerebrovascular dysfunction and inflammation following mild respiratory SARS-CoV-2 infection. To this end, we performed unbiased transcriptional analysis to identify brain endothelial cell signalling pathways dysregulated by mouse adapted SARS-CoV-2 MA10 in aged immunocompetent C57Bl/6 mice in vivo. This analysis revealed significant suppression of Wnt/ß-catenin signalling, a critical regulator of blood-brain barrier (BBB) integrity. We therefore hypothesized that enhancing cerebrovascular Wnt/ß-catenin activity would offer protection against BBB permeability, neuroinflammation, and neurological signs in acute infection. Indeed, we found that delivery of cerebrovascular-targeted, engineered Wnt7a ligands protected BBB integrity, reduced T-cell infiltration of the brain, and reduced microglial activation in SARS-CoV-2 infection. Importantly, this strategy also mitigated SARS-CoV-2 induced deficits in the novel object recognition assay for learning and memory and the pole descent task for bradykinesia. These observations suggest that enhancement of Wnt/ß-catenin signalling or its downstream effectors could be potential interventional strategies for restoring cognitive health following viral infections.

An HTLV-1 envelope mRNA vaccine is immunogenic and protective in New Zealand rabbits.

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus responsible for adult T-cell leukemia/lymphoma, a severe and fatal CD4+ T-cell malignancy. Additionally, HTLV-1 can lead to a chronic progressive neurodegenerative disease known as HTLV-1-associated myelopathy/tropical spastic paraparesis. Unfortunately, the prognosis for HTLV-1-related diseases is generally poor, and effective treatment options are limited. In this study, we designed and synthesized a codon optimized HTLV-1 envelope (Env) mRNA encapsulated in a lipid nanoparticle (LNP) and evaluated its efficacy as a vaccine candidate in an established rabbit model of HTLV-1 infection and persistence. Immunization regimens included a prime/boost protocol using Env mRNA-LNP or control green fluorescent protein (GFP) mRNA-LNP. After immunization, rabbits were challenged by intravenous injection with irradiated HTLV-1 producing cells. Three rabbits were partially protected and three rabbits were completely protected against HTLV-1 challenge. These rabbits were then rechallenged 15 weeks later, and two rabbits maintained sterilizing immunity. In Env mRNA-LNP immunized rabbits, proviral load and viral gene expression were significantly lower. After viral challenge in the Env mRNA-LNP vaccinated rabbits, an increase in both CD4+/IFN-γ+ and CD8+/IFN-γ+ T-cells was detected when stimulating with overlapping Env peptides. Env mRNA-LNP elicited a detectable anti-Env antibody response after prime/boost vaccination in all animals and significantly higher levels of neutralizing antibody activity. Neutralizing antibody activity was correlated with a reduction in proviral load. These findings hold promise for the development of preventive strategies and therapeutic interventions against HTLV-1 infection and its associated diseases.IMPORTANCEmRNA vaccine technology has proven to be a viable approach for effectively triggering immune responses that protect against or limit viral infections and disease. In our study, we synthesized a codon optimized human T-cell leukemia virus type 1 (HTLV-1) envelope (Env) mRNA that can be delivered in a lipid nanoparticle (LNP) vaccine approach. The HTLV-1 Env mRNA-LNP produced protective immune responses against viral challenge in a preclinical rabbit model. HTLV-1 is primarily transmitted through direct cell-to-cell contact, and the protection offered by mRNA vaccines in our rabbit model could have significant implications for optimizing the development of other viral vaccine candidates. This is particularly important in addressing the challenge of enhancing protection against infections that rely on cell-to-cell transmission.

SARS-CoV-2 Bottlenecks and Tissue-Specific Adaptation in the Central Nervous System.

Severe COVID-19 and post-acute sequelae of SARS-CoV-2 infection are associated with neurological complications that may be linked to direct infection of the central nervous system (CNS), but the selective pressures ruling neuroinvasion are poorly defined. Here, we assessed SARS-CoV-2 evolution in the lung versus CNS of infected mice. Higher levels of viral diversity were observed in the CNS than the lung after intranasal challenge with a high frequency of mutations in the Spike furin cleavage site (FCS). Deletion of the FCS significantly attenuated virulence after intranasal challenge, with lower viral titers and decreased morbidity compared to the wild-type virus. Intracranial inoculation of the FCS-deleted virus, however, was sufficient to restore virulence. After intracranial inoculation, both viruses established infection in the lung, but this required reversion of the FCS deletion. Cumulatively, these data suggest a critical role for the FCS in determining SARS-CoV-2 tropism and compartmentalization with possible implications for the treatment of neuroinvasive COVID-19.

Caveolin-1 mediates neuroinflammation and cognitive impairment in SARS-CoV-2 infection.

Leukocyte infiltration of the CNS can contribute to neuroinflammation and cognitive impairment. Brain endothelial cells regulate adhesion, activation, and diapedesis of T cells across the blood-brain barrier (BBB) in inflammatory diseases. The integral membrane protein Caveolin-1 (Cav-1) critically regulates BBB permeability, but its influence on T cell CNS infiltration in respiratory viral infections is unknown. In this study, we sought to determine the role of Cav-1 at the BBB in neuroinflammation in a COVID-19 mouse model. We used mice genetically deficient in Cav-1 to test the role of this protein in T cell infiltration and cognitive impairment. We found that SARS-CoV-2 infection upregulated brain endothelial Cav-1. Moreover, SARS-CoV-2 infection increased brain endothelial cell vascular cell adhesion molecule-1 (VCAM-1) and CD3+ T cell infiltration of the hippocampus, a region important for short term learning and memory. Concordantly, we observed learning and memory deficits. Importantly, genetic deficiency in Cav-1 attenuated brain endothelial VCAM-1 expression and T cell infiltration in the hippocampus of mice with SARS-CoV-2 infection. Moreover, Cav-1 KO mice were protected from the learning and memory deficits caused by SARS-CoV-2 infection. These results indicate the importance of BBB permeability in COVID-19 neuroinflammation and suggest potential therapeutic value of targeting Cav-1 to improve disease outcomes.

Pre-existing immunity modulates responses to mRNA boosters.

mRNA vaccines are effective in preventing severe COVID-19, but breakthrough infections, emerging variants, and waning immunity warrant the use of boosters. Although mRNA boosters are being implemented, the extent to which pre-existing immunity influences the efficacy of boosters remains unclear. In a cohort of individuals primed with the mRNA-1273 or BNT162b2 vaccines, we report that lower antibody levels before boost are associated with higher fold-increase in antibody levels after boost, suggesting that pre-existing antibody modulates the immunogenicity of mRNA vaccines. Our studies in mice show that pre-existing antibodies accelerate the clearance of vaccine antigen via Fc-dependent mechanisms, limiting the amount of antigen available to prime B cell responses after mRNA boosters. These data demonstrate a "tug of war" between pre-existing antibody responses and de novo B cell responses following mRNA vaccination, and they suggest that transient downmodulation of antibody effector function may improve the efficacy of mRNA boosters.

Type I IFN stimulates lymph node stromal cells from adult and old mice during a West Nile virus infection.

Advanced age is a significant risk factor during viral infection due to an age-associated decline in the immune response. Older individuals are especially susceptible to severe neuroinvasive disease after West Nile virus (WNV) infection. Previous studies have characterized age-associated defects in hematopoietic immune cells during WNV infection that culminate in diminished antiviral immunity. Situated amongst immune cells in the draining lymph node (DLN) are structural networks of nonhematopoietic lymph node stromal cells (LNSCs). LNSCs are comprised of numerous, diverse subsets, with critical roles in the coordination of robust immune responses. The contributions of LNSCs to WNV immunity and immune senescence are unclear. Here, we examine LNSC responses to WNV within adult and old DLNs. Acute WNV infection triggered cellular infiltration and LNSC expansion in adults. Comparatively, aged DLNs exhibited diminished leukocyte accumulation, delayed LNSC expansion, and altered fibroblast and endothelial cell subset composition, signified by fewer LECs. We established an ex vivo culture system to probe LNSC function. Adult and old LNSCs both recognized an ongoing viral infection primarily through type I IFN signaling. Gene expression signatures were similar between adult and old LNSCs. Aged LNSCs were found to constitutively upregulate immediate early response genes. Collectively, these data suggest LNSCs uniquely respond to WNV infection. We are the first to report age-associated differences in LNSCs on the population and gene expression level during WNV infection. These changes may compromise antiviral immunity, leading to increased WNV disease in older individuals.

Type I IFN stimulates lymph node stromal cells from adult and old mice during a West Nile virus infection.

Advanced age is a significant risk factor during viral infection due to an age-associated decline in the immune response. Older individuals are especially susceptible to severe neuroinvasive disease after West Nile virus (WNV) infection. Previous studies have characterized age-associated defects in hematopoietic immune cells during WNV infection that culminate in diminished antiviral immunity. Situated amongst immune cells in the draining lymph node (DLN) are structural networks of nonhematopoietic lymph node stromal cells (LNSCs). LNSCs are comprised of numerous, diverse subsets, with critical roles in the coordination of robust immune responses. The contributions of LNSCs to WNV immunity and immune senescence are unclear. Here, we examine LNSC responses to WNV within adult and old DLNs. Acute WNV infection triggered cellular infiltration and LNSC expansion in adult. Comparatively, aged DLNs exhibited diminished leukocyte accumulation, delayed LNSC expansion, and altered fibroblast and endothelial cell subset composition, signified by fewer LECs. We established an ex vivo culture system to probe LNSC function. Adult and old LNSCs both recognized an ongoing viral infection primarily through type I IFN signaling. Gene expression signatures were similar between adult and old LNSCs. Aged LNSCs were found to constitutively upregulate immediate early response genes. Collectively, these data suggest LNSCs uniquely respond to WNV infection. We are the first to report age-associated differences in LNSCs on the population- and gene expression-level during WNV infection. These changes may compromise antiviral immunity, leading to increased WNV disease in older individuals.

Host microRNAs exhibit differential propensity to interact with SARS-CoV-2 and variants of concern.

A significant number of SARS-CoV-2-infected individuals naturally overcome viral infection, suggesting the existence of a potent endogenous antiviral mechanism. As an innate defense mechanism, microRNA (miRNA) pathways in mammals have evolved to restrict viruses, besides regulating endogenous mRNAs. In this study, we systematically examined the complete repertoire of human miRNAs for potential binding sites on SARS-CoV-2 Wuhan-Hu-1, Beta, Delta, and Omicron. Human miRNA and viral genome interaction were analyzed using RNAhybrid 2.2 with stringent parameters to identify highly bonafide miRNA targets. Using publicly available data, we filtered for miRNAs expressed in lung epithelial cells/tissue and oral keratinocytes, concentrating on the miRNAs that target SARS-CoV-2 S protein mRNAs. Our results show a significant loss of human miRNA and SARS-CoV-2 interactions in Omicron (130 miRNAs) compared to Wuhan-Hu-1 (271 miRNAs), Beta (279 miRNAs), and Delta (275 miRNAs). In particular, hsa-miR-3150b-3p and hsa-miR-4784 show binding affinity for S protein of Wuhan strain but not Beta, Delta, and Omicron. Loss of miRNA binding sites on N protein was also observed for Omicron. Through Ingenuity Pathway Analysis (IPA), we examined the experimentally validated and highly predicted functional role of these miRNAs. We found that hsa-miR-3150b-3p and hsa-miR-4784 have several experimentally validated or highly predicted target genes in the Toll-like receptor, IL-17, Th1, Th2, interferon, and coronavirus pathogenesis pathways. Focusing on the coronavirus pathogenesis pathway, we found that hsa-miR-3150b-3p and hsa-miR-4784 are highly predicted to target MAPK13. Exploring miRNAs to manipulate viral genome/gene expression can provide a promising strategy with successful outcomes by targeting specific VOCs.

Improved control of SARS-CoV-2 by treatment with a nucleocapsid-specific monoclonal antibody.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein is the main antigen in all approved COVID-19 vaccines and is also the only target for monoclonal antibody (mAb) therapies. Immune responses to other viral antigens are generated after SARS-CoV-2 infection, but their contribution to the antiviral response remains unclear. Here, we interrogated whether nucleocapsid-specific antibodies can improve protection against SARS-CoV-2. We first immunized mice with a nucleocapsid-based vaccine and then transferred sera from these mice into naive mice, followed by challenge with SARS-CoV-2. We show that mice that received nucleocapsid-specific sera or a nucleocapsid-specific mAb exhibited enhanced control of SARS-CoV-2. Nucleocapsid-specific antibodies elicited NK-mediated, antibody-dependent cellular cytotoxicity (ADCC) against infected cells. To our knowledge, these findings provide the first demonstration in the coronavirus literature that antibody responses specific to the nucleocapsid protein can improve viral clearance, providing a rationale for the clinical evaluation of nucleocapsid-based mAb therapies to treat COVID-19.

A Broad-Based Characterization of a Cell-Penetrating, Single Domain Camelid Bi-Specific Antibody Monomer That Targets STAT3 and KRAS Dependent Cancers.

STAT3 and KRAS regulate cell proliferation, survival, apoptosis, cell migration, and angiogenesis. Aberrant expression of STAT3 and mutant active forms of KRAS have been well-established in the induction and maintenance of multiple cancers. STAT3 and KRAS mutant proteins have been considered anti-cancer targets; however, they are also considered to be clinically "undruggable" intracellular molecules, except for KRAS(G12C). Here we report a first-in-class molecule, a novel, single domain camelid VHH antibody (15 kDa), SBT-100, that binds to both STAT3 and KRAS and can penetrate the tumor cell membrane, and significantly inhibit cancer cell growth. Additionally, SBT-100 inhibits KRAS GTPase activity and downstream phosphorylation of ERK in vitro. In addition, SBT-100 inhibits the growth of multiple human cancers in vitro and in vivo. These results demonstrate the feasibility of targeting hard-to-reach aberrant intracellular transcription factors and signaling proteins simultaneously with one VHH to improve cancer therapies.

The amino acid sensor GCN2 suppresses terminal oligopyrimidine (TOP) mRNA translation via La-related protein 1 (LARP1).

La-related protein 1 (LARP1) has been identified as a key translational inhibitor of terminal oligopyrimidine (TOP) mRNAs downstream of the nutrient sensing protein kinase complex, mTORC1. LARP1 exerts this inhibitory effect on TOP mRNA translation by binding to the mRNA cap and the adjacent 5'TOP motif, resulting in the displacement of the cap-binding protein eIF4E from TOP mRNAs. However, the involvement of additional signaling pathway in regulating LARP1-mediated inhibition of TOP mRNA translation is largely unexplored. In the present study, we identify a second nutrient sensing kinase GCN2 that converges on LARP1 to control TOP mRNA translation. Using chromatin-immunoprecipitation followed by massive parallel sequencing (ChIP-seq) analysis of activating transcription factor 4 (ATF4), an effector of GCN2 in nutrient stress conditions, in WT and GCN2 KO mouse embryonic fibroblasts, we determined that LARP1 is a GCN2-dependent transcriptional target of ATF4. Moreover, we identified GCN1, a GCN2 activator, participates in a complex with LARP1 on stalled ribosomes, suggesting a role for GCN1 in LARP1-mediated translation inhibition in response to ribosome stalling. Therefore, our data suggest that the GCN2 pathway controls LARP1 activity via two mechanisms: ATF4-dependent transcriptional induction of LARP1 mRNA and GCN1-mediated recruitment of LARP1 to stalled ribosomes.

Pre-existing immunity modulates responses to mRNA boosters.

mRNA vaccines have shown high efficacy in preventing severe COVID-19, but breakthrough infections, emerging variants and waning antibody levels have warranted the use of boosters. Although mRNA boosters have been widely implemented, the extent to which pre-existing immunity influences the efficacy of boosters remains unclear. In a cohort of individuals primed with the mRNA-1273 or BNT162b2 vaccines, we observed that lower antibody levels before boost were associated with higher fold-increase in antibody levels after boost, suggesting that pre-existing antibody modulates the boosting capacity of mRNA vaccines. Mechanistic studies in mice show that pre-existing antibodies significantly limit antigen expression and priming of B cell responses after mRNA vaccination. Furthermore, we demonstrate that the relative superiority of an updated Omicron vaccine over the original vaccine is critically dependent on the serostatus of the host. These data demonstrate that pre-existing immunity dictates responses to mRNA vaccination, elucidating specific circumstances when updated SARS-CoV-2 vaccines confer superior protection to original vaccines.

Cross-protective immunity following coronavirus vaccination and coronavirus infection.

Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines have shown efficacy against SARS-CoV-2, it is unknown if coronavirus vaccines can also protect against other coronaviruses that may infect humans in the future. Here, we show that coronavirus vaccines elicited cross-protective immune responses against heterologous coronaviruses. In particular, we show that a severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) vaccine developed in 2004 and known to protect against SARS-CoV-1 conferred robust heterologous protection against SARS-CoV-2 in mice. Similarly, prior coronavirus infections conferred heterologous protection against distinct coronaviruses. Cross-reactive immunity was also reported in patients with coronavirus disease 2019 (COVID-19) and in individuals who received SARS-CoV-2 vaccines, and transfer of plasma from these individuals into mice improved protection against coronavirus challenges. These findings provide the first demonstration to our knowledge that coronavirus vaccines (and prior coronavirus infections) can confer broad protection against heterologous coronaviruses and establish a rationale for universal coronavirus vaccines.

Interleukin-1RA Mitigates SARS-CoV-2-Induced Inflammatory Lung Vascular Leakage and Mortality in Humanized K18-hACE-2 Mice.

[Figure: see text].

Combining spike- and nucleocapsid-based vaccines improves distal control of SARS-CoV-2.

SARS-CoV-2 infection causes respiratory insufficiency and neurological manifestations, including loss of smell and psychiatric disorders, and can be fatal. Most vaccines are based on the spike antigen alone, and although they have shown efficacy at preventing severe disease and death, they do not always confer sterilizing immunity. Here, we interrogate whether SARS-CoV-2 vaccines could be improved by incorporating nucleocapsid as an antigen. We show that, after 72 h of challenge, a spike-based vaccine confers acute protection in the lung, but not in the brain. However, combining a spike-based vaccine with a nucleocapsid-based vaccine confers acute protection in both the lung and brain. These findings suggest that nucleocapsid-specific immunity can improve the distal control of SARS-CoV-2, warranting the inclusion of nucleocapsid in next-generation COVID-19 vaccines.

Signal-regulatory protein alpha is an anti-viral entry factor targeting viruses using endocytic pathways.

Signal-regulatory protein alpha (SIRPA) is a well-known inhibitor of phagocytosis when it complexes with CD47 expressed on target cells. Here we show that SIRPA decreased in vitro infection by a number of pathogenic viruses, including New World and Old World arenaviruses, Zika virus, vesicular stomatitis virus and pseudoviruses bearing the Machupo virus, Ebola virus and SARS-CoV-2 glycoproteins, but not HSV-1, MLV or mNoV. Moreover, mice with targeted mutation of the Sirpa gene that renders it non-functional were more susceptible to infection with the New World arenaviruses Junín virus vaccine strain Candid 1 and Tacaribe virus, but not MLV or mNoV. All SIRPA-inhibited viruses have in common the requirement for trafficking to a low pH endosomal compartment. This was clearly demonstrated with SARS-CoV-2 pseudovirus, which was only inhibited by SIRPA in cells in which it required trafficking to the endosome. Similar to its role in phagocytosis inhibition, SIRPA decreased virus internalization but not binding to cell surface receptors. We also found that increasing SIRPA levels via treatment with IL-4 led to even greater anti-viral activity. These data suggest that enhancing SIRPA's activity could be a target for anti-viral therapies.

Tonsil organoids: peering down the throat of human immunity.

Animal studies and explant cultures of human lymphoid tissues do not reliably model human vaccine responses. A remarkable strategy for reassociation of human tonsillar cells in ex vivo culture leads to organoid formation and provides an exciting new tool to probe human humoral immune responses to infection.