Design and Characterization of a New Formulation for the Delivery of COVID-19-mRNA Vaccine to the Nasal Mucosa.

Chitosan, a natural polysaccharide derived from chitin, possesses biocompatibility, biodegradability, and mucoadhesive characteristics, making it an attractive material for the delivery of mRNA payloads to the nasal mucosa and promoting their uptake by target cells such as epithelial and immune cells (e.g., dendritic cells and macrophages). In this project, we aimed at developing novel lipid-based nanoformulations for mRNA delivery to counteract the pandemic caused by SARS-CoV-2 virus. The formulations achieved a mRNA encapsulation efficiency of ~80.2% with chitosan-lipid nanoparticles, as measured by the RiboGreen assay. Furthermore, the evaluation of SARS-CoV-2 Spike (S) receptor-binding domain (RBD) expression via ELISA for our vaccine formulations showed transfection levels in human embryonic kidney cells (HEK 293), lung carcinoma cells (A549), and dendritic cells (DC 2.4) equal to 9.9 ± 0.1 ng/mL (174.7 ± 1.1 fold change from untreated cells (UT)), 7.0 ± 0.2 ng/mL (128.1 ± 4.9 fold change from UT), and 0.9 ± 0.0 ng/mL (18.0 ± 0.1 fold change from UT), respectively. Our most promising vaccine formulation was also demonstrated to be amenable to lyophilization with minimal degradation of loaded mRNA, paving the way towards a more accessible and stable vaccine. Preliminary in vivo studies in mice were performed to assess the systemic and local immune responses. Nasal bronchoalveolar lavage fluid (BALF) wash showed that utilizing the optimized formulation resulted in local antibody concentrations and did not trigger any systemic antibody response. However, if further improved and developed, it could potentially contribute to the management of COVID-19 through nasopharyngeal immunization strategies.

Dengue and Zika RNA-RNA interactomes reveal pro- and anti-viral RNA in human cells.

BACKGROUND: Identifying host factors is key to understanding RNA virus pathogenicity. Besides proteins, RNAs can interact with virus genomes to impact replication. RESULTS: Here, we use proximity ligation sequencing to identify virus-host RNA interactions for four strains of Zika virus (ZIKV) and one strain of dengue virus (DENV-1) in human cells. We find hundreds of coding and non-coding RNAs that bind to DENV and ZIKV viruses. Host RNAs tend to bind to single-stranded regions along the virus genomes according to hybridization energetics. Compared to SARS-CoV-2 interactors, ZIKV-interacting host RNAs tend to be downregulated upon virus infection. Knockdown of several short non-coding RNAs, including miR19a-3p, and 7SK RNA results in a decrease in viral replication, suggesting that they act as virus-permissive factors. In addition, the 3'UTR of DYNLT1 mRNA acts as a virus-restrictive factor by binding to the conserved dumbbell region on DENV and ZIKV 3'UTR to decrease virus replication. We also identify a conserved set of host RNAs that interacts with DENV, ZIKV, and SARS-CoV-2, suggesting that these RNAs are broadly important for RNA virus infection. CONCLUSIONS: This study demonstrates that host RNAs can impact virus replication in permissive and restrictive ways, expanding our understanding of host factors and RNA-based gene regulation during viral pathogenesis.

Role of Alternative Splicing in Regulating Host Response to Viral Infection.

The importance of transcriptional regulation of host genes in innate immunity against viral infection has been widely recognized. More recently, post-transcriptional regulatory mechanisms have gained appreciation as an additional and important layer of regulation to fine-tune host immune responses. Here, we review the functional significance of alternative splicing in innate immune responses to viral infection. We describe how several central components of the Type I and III interferon pathways encode spliced isoforms to regulate IFN activation and function. Additionally, the functional roles of splicing factors and modulators in antiviral immunity are discussed. Lastly, we discuss how cell death pathways are regulated by alternative splicing as well as the potential role of this regulation on host immunity and viral infection. Altogether, these studies highlight the importance of RNA splicing in regulating host-virus interactions and suggest a role in downregulating antiviral innate immunity; this may be critical to prevent pathological inflammation.

The RNA binding protein Quaking represses splicing of the Fibronectin EDA exon and downregulates the interferon response.

Quaking (QKI) controls RNA metabolism in many biological processes including innate immunity, where its roles remain incompletely understood. To illuminate these roles, we performed genome scale transcriptome profiling in QKI knockout cells with or without poly(I:C) transfection, a double-stranded RNA analog that mimics viral infection. Analysis of RNA-sequencing data shows that QKI knockout upregulates genes induced by interferons, suggesting that QKI is an immune suppressor. Furthermore, differential splicing analysis shows that QKI primarily controls cassette exons, and among these events, we noted that QKI silences splicing of the extra domain A (EDA) exon in fibronectin (FN1) transcripts. QKI knockout results in elevated production and secretion of FN1-EDA protein, which is a known activator of interferons. Consistent with an upregulation of the interferon response in QKI knockout cells, our results show reduced production of dengue virus-2 and Japanese encephalitis virus in these cells. In conclusion, we demonstrate that QKI downregulates the interferon system and attenuates the antiviral state.

The RNA binding protein Quaking represses host interferon response by downregulating MAVS.

Quaking (QKI) is an RNA-binding protein (RBP) involved in multiple aspects of RNA metabolism and many biological processes. Despite a known immune function in regulating monocyte differentiation and inflammatory responses, the degree to which QKI regulates the host interferon (IFN) response remains poorly characterized. Here we show that QKI ablation enhances poly(I:C) and viral infection-induced IFNß transcription. Characterization of IFN-related signalling cascades reveals that QKI knockout results in higher levels of IRF3 phosphorylation. Interestingly, complementation with QKI-5 isoform alone is sufficient to rescue this phenotype and reduce IRF3 phosphorylation. Further analysis shows that MAVS, but not RIG-I or MDA5, is robustly upregulated in the absence of QKI, suggesting that QKI downregulates MAVS and thus represses the host IFN response. As expected, MAVS depletion reduces IFNß activation and knockout of MAVS in the QKI knockout cells completely abolishes IFNß induction. Consistently, ectopic expression of RIG-I activates stronger IFNß induction via MAVS-IRF3 pathway in the absence of QKI. Collectively, these findings demonstrate a novel role for QKI in negatively regulating host IFN response by reducing MAVS levels.

Application of immobilized ATP to the study of NLRP inflammasomes.

The NLRP proteins are a subfamily of the NOD-like receptor (NLR) innate immune sensors that possess an ATP-binding NACHT domain. As the most well studied member, NLRP3 can initiate the assembly process of a multiprotein complex, termed the inflammasome, upon detection of a wide range of microbial products and endogenous danger signals and results in the activation of pro-caspase-1, a cysteine protease that regulates multiple host defense pathways including cytokine maturation. Dysregulated NLRP3 activation contributes to inflammation and the pathogenesis of several chronic diseases, and the ATP-binding properties of NLRPs are thought to be critical for inflammasome activation. In light of this, we examined the utility of immobilized ATP matrices in the study of NLRP inflammasomes. Using NLRP3 as the prototypical member of the family, P-linked ATP Sepharose was determined to be a highly-effective capture agent. In subsequent examinations, P-linked ATP Sepharose was used as an enrichment tool to enable the effective profiling of NLRP3-biomarker signatures with selected reaction monitoring-mass spectrometry (SRM-MS). Finally, ATP Sepharose was used in combination with a fluorescence-linked enzyme chemoproteomic strategy (FLECS) screen to identify potential competitive inhibitors of NLRP3. The identification of a novel benzo[d]imidazol-2-one inhibitor that specifically targets the ATP-binding and hydrolysis properties of the NLRP3 protein implies that ATP Sepharose and FLECS could be applied other NLRPs as well.

Biochemistry and Molecular Biology of Flaviviruses.

Flaviviruses, such as dengue, Japanese encephalitis, tick-borne encephalitis, West Nile, yellow fever, and Zika viruses, are critically important human pathogens that sicken a staggeringly high number of humans every year. Most of these pathogens are transmitted by mosquitos, and not surprisingly, as the earth warms and human populations grow and move, their geographic reach is increasing. Flaviviruses are simple RNA-protein machines that carry out protein synthesis, genome replication, and virion packaging in close association with cellular lipid membranes. In this review, we examine the molecular biology of flaviviruses touching on the structure and function of viral components and how these interact with host factors. The latter are functionally divided into pro-viral and antiviral factors, both of which, not surprisingly, include many RNA binding proteins. In the interface between the virus and the hosts we highlight the role of a noncoding RNA produced by flaviviruses to impair antiviral host immune responses. Throughout the review, we highlight areas of intense investigation, or a need for it, and potential targets and tools to consider in the important battle against pathogenic flaviviruses.

Identification and characterization of host proteins bound to dengue virus 3' UTR reveal an antiviral role for quaking proteins.

The four dengue viruses (DENV1-4) are rapidly reemerging infectious RNA viruses. These positive-strand viral genomes contain structured 3' untranslated regions (UTRs) that interact with various host RNA binding proteins (RBPs). These RBPs are functionally important in viral replication, pathogenesis, and defense against host immune mechanisms. Here, we combined RNA chromatography and quantitative mass spectrometry to identify proteins interacting with DENV1-4 3' UTRs. As expected, RBPs displayed distinct binding specificity. Among them, we focused on quaking (QKI) because of its preference for the DENV4 3' UTR (DENV-4/SG/06K2270DK1/2005). RNA immunoprecipitation experiments demonstrated that QKI interacted with DENV4 genomes in infected cells. Moreover, QKI depletion enhanced infectious particle production of DENV4. On the contrary, QKI did not interact with DENV2 3' UTR, and DENV2 replication was not affected consistently by QKI depletion. Next, we mapped the QKI interaction site and identified a QKI response element (QRE) in DENV4 3' UTR. Interestingly, removal of QRE from DENV4 3' UTR abolished this interaction and increased DENV4 viral particle production. Introduction of the QRE to DENV2 3' UTR led to QKI binding and reduced DENV2 infectious particle production. Finally, reporter assays suggest that QKI reduced translation efficiency of viral RNA. Our work describes a novel function of QKI in restricting viral replication.

Vitamin D3 metabolites enhance the NLRP3-dependent secretion of IL-1ß from human THP-1 monocytic cells.

Vitamin D3 has emerged as an important regulator of the immune system. With metabolic enzymes for vitamin D3 activation and vitamin D receptors (VDR) now identified in a variety of immune cells, the active vitamin D3 metabolite 1,25(OH)2D3, is thought to possess immunomodulatory properties. We examined whether 1,25(OH)2D3 might also enhance the NLRP3-dependent release of mature IL-1ß from macrophages. PMA-differentiated THP-1 cells were stimulated with vitamin D3 metabolites and assessed for CYP27, CYP24, NLRP3, ASC, pro-caspase-1 expression by western blot and real-time qPCR as well as inflammasome activation with pro-inflammatory cytokine IL-1ß release measured by ELISA. Exposure to 1,25(OH)2D3 had no effect on the basal expression levels of VDR; however, CYP27A1 transcript was suppressed and CYP24A1 transcript was substantively elevated. Both 1,25(OH)2D3 - and 25(OH)D3 induced IL-1ß release from THP-1 cells, and these effects were blocked with application of the caspase-1 inhibitor YVAD and the NLRP3 inhibitors glyburide and Bay 11-7082. Interestingly, 1,25 (OH)2D3 exposure reduced NLRP3 protein expression but had no effect on ASC or pro-caspase-1 protein levels. The increase in mature IL-1ß elicited by 1,25(OH)2D3 was modest compared to that found for ATP or C. difficile toxins. However, co-treatment of THP-1 cells with ATP and 1,25(OH)2D3 resulted in more IL-1ß secretion than ATP or 1,25(OH)2D3 alone.

Distinct regions of NLRP1B are required to respond to anthrax lethal toxin and metabolic inhibition.

Pattern recognition receptors monitor for signs of infection or cellular dysfunction and respond to these events by initiating an immune response. NLRP1B is a receptor that upon activation recruits multiple copies of procaspase-1, which promotes cytokine processing and a proinflammatory form of cell death termed pyroptosis. NLRP1B detects anthrax lethal toxin when the toxin cleaves an amino-terminal fragment from the protein. In addition, NLRP1B is activated when cells are deprived of glucose or treated with metabolic inhibitors, but the mechanism by which the resulting reduction in cytosolic ATP is sensed by NLRP1B is unknown. Here, we addressed whether these two activating signals of NLRP1B converge on a common sensing system. We show that an NLRP1B mutant lacking the amino-terminal region exhibits some spontaneous activity and fails to be further activated by lethal toxin. This mutant was still activated in cells depleted of ATP, however, indicating that the amino-terminal region is not the sole sensing domain of NLRP1B. Mutagenesis of the leucine-rich repeat domain of NLRP1B provided evidence that this domain is involved in autoinhibition of the receptor, but none of the mutants tested was specifically defective at sensing activating signals. Comparison of two alleles of NLRP1B that differed in their response to metabolic inhibitors, but not to lethal toxin, led to the finding that a repeated sequence in the function to find domain (FIIND) that arose from exon duplication facilitated detection of ATP depletion. These results suggest that distinct regions of NLRP1B detect activating signals.

Biochemical and structural aspects of the ATP-binding domain in inflammasome-forming human NLRP proteins.

Nucleotide-binding domain and leucine-rich repeat-containing receptors (NLRs) regulate innate immunity by activating inflammatory responses in a variety of biological systems following the recognition of pathogen- or disease-associated molecular patterns. NLRs are characterized by a central nucleotide-binding and oligomerization (NACHT) domain found in P-loop NTPases. In this review, we detail the functional and structural properties of the NACHT domain of a subfamily of NLRs, the NLRPs (NLR containing a pyrin domain), based on previous studies, sequence analysis, homology modeling, and structure predictions. Several NLRPs have been found to regulate inflammatory responses through the assembly of oligomeric caspase 1-activating platforms known as inflammasomes, the 3-dimensional structure of the NLRP NACHT domain has still not been solved. Homology modeling suggests that sequence variability within the NACHT domains of different NLRP family members may alter the topology of the ATP-binding pocket. Based on this finding, we discuss the potential therapeutic prospects aligned with the NACHT domain and the development of selective inhibitors of inflammasome activity.

Activation of the Nlrp1b inflammasome by reduction of cytosolic ATP.

The efficacy of the innate immune system depends on its ability to mount an appropriate response to diverse infections and damaging agents. Key components of this system are pattern recognition receptors that detect pathogen-associated and damage-associated molecular patterns (PAMPs and DAMPs). Nlrp1b is a pattern recognition receptor that forms a caspase-1 activation platform, known as an inflammasome, upon sensing the proteolytic activity of anthrax lethal toxin. The activation of caspase-1 leads to the release of proinflammatory cytokines that aid in the clearance of the anthrax infection. Here, we demonstrate that Nlrp1b also becomes activated in cells that are subjected to energy stress caused by metabolic inhibitors or by nutrient deprivation. Glucose starvation and hypoxia were used to correlate the level of cytosolic ATP to the degree of inflammasome activation. Because lowering the ratio of cytosolic ATP to AMP activates the main cellular energy sensor, AMP-activated protein kinase (AMPK), we assessed whether AMPK promoted inflammasome activity by using a combination of small interfering RNA (siRNA) and transfection of a dominant negative AMPK subunit. We found that AMPK promoted inflammasome activity, but activation of AMPK in the absence of ATP depletion was not sufficient for caspase-1-mediated pro-interleukin 1ß (pro-IL-1ß) processing. Finally, we found that mutation of the ATP-binding motif of Nlrp1b caused constitutive activation, suggesting that ATP might inhibit the Nlrp1b inflammasome instead of being required for its assembly.

An anthrax lethal factor mutant that is defective at causing pyroptosis retains proapoptotic activity.

Anthrax lethal toxin triggers death in some cell types, such as macrophages, and causes a variety of cellular dysfunctions in others. Collectively, these effects dampen the innate and adaptive immune systems to allow Bacillus anthracis to survive and proliferate in the mammalian host. The diverse effects caused by the toxin have in part been attributed to its interference with signaling pathways in target cells. Lethal factor (LF) is the proteolytic component of the toxin, and cleaves six members of the mitogen-activated protein kinase kinase family after being delivered to the cytosol by the cell-binding component of the toxin, protective antigen. The effect of cleaving these mitogen-activated protein kinase kinases is to interfere with extracellular signal-related kinase (ERK), p38 and c-Jun N-terminal kinase signaling. Here, we characterized an LF mutant, LF-K518E/E682G, that was defective at causing pyroptosis in RAW 264.7 cells and at activating the Nlrp1b inflammasome in a heterologous expression system. LF-K518E/E682G did not exhibit an overall impairment of function, however, because it was able to downregulate the ERK pathway, but not the p38 or c-Jun N-terminal kinase pathways. Furthermore, LF-K518E/E682G efficiently killed melanoma cells, which were shown previously to undergo apoptosis in response to lethal toxin or to pharmacological inhibition of the ERK pathway. Our results suggest that LF-K518E/E682G is defective at cleaving a substrate involved in the activation of the Nlrp1b inflammasome.

Expression of Nlrp1b inflammasome components in human fibroblasts confers susceptibility to anthrax lethal toxin.

Anthrax lethal toxin causes macrophages and dendritic cells from some mouse strains to undergo caspase-1-dependent cell death. Central to this process is the NOD-like receptor Nlrp1b (Nalp1b), which detects intoxication and then self-associates to form a complex, termed an inflammasome, that is capable of activating the procaspase-1 zymogen. The nature of the signal detected directly by Nlrp1b is not known, and the mechanisms of inflammasome assembly are poorly understood. Here, we demonstrate that transfection of human fibroblasts with plasmids encoding murine Nlrp1b and procaspase-1 was sufficient to confer susceptibility to lethal toxin-mediated death on the cells. As has been observed in murine macrophages, the enzymatic activities of lethal toxin and the proteasome were both required for activation of the Nlrp1b inflammasome and this activation led to prointerleukin-1 beta processing. Release of interleukin-1beta from cells was not dependent on cell lysis, as its secretion was not affected by an osmoprotectant that prevented the appearance of lactate dehydrogenase in the culture medium. We generated constitutively active mutants of Nlrp1b by making amino-terminal deletions to the protein and observed that the ability to activate procaspase-1 was dependent on the CARD domain, which bound procaspase-1, and a region adjacent to the CARD domain that promoted self-association. Our results demonstrate that lethal toxin can activate Nlrp1b in a nonmyeloid cell line and are consistent with work that suggests that activation induces proximity of procaspase-1.